ORIGINAL_ARTICLE
Expression, Purification and Characterization of Human Recombinant Galectin 3 in Pichia pastoris
Background: Over the past century, the areas of genomics, proteomics and lipids have captured the attention of investigators worldwide. Carbohydrates, have recently received increased attention through the expanding field of glycobiology; probably because they are very complex and not encoded in the genome. Objectives: The purpose of this study was to express and purify recombinant human galectin 3via the Pichiapastoris expression system. Materials and Methods:cDNA of human galectin 3 gene was amplified with specific primers and cloned into a pcDNA3.1 vector with His-tag for easier purification using Ni2andchromatography. Furthermore, galectin 3was purified to homogeneity and confirmed using SDS-PAGE and western blotting. Results:The protein band corresponding to 29 kDa was excised from the gels, digested with trypsin and processed for mass spectrometric analysis by Matrix Assisted Laser Desorption/Ionization- Time of Flight Mass Spectroscopy (MALDI-TOF MS), using a Reflex III instrument. Conclusions:Tryptic digest analysis clearly revealed that the purified protein was indeed galectin 3. Similarly, the biological activity of recombinant galectin 3 was confirmed using the hemagglutination inhibition assay.
https://www.ijbiotech.com/article_7257_9cfff19709b6aa689d9bd3d146f35284.pdf
2014-04-01
3
8
10.5812/ijb.17330
Pichiapastoris
Galectin 3
Lgals3
Glycomics
Hemagglutination
Praveen
Kumar Vemuri
1
Department of Biotechnology, K L University, Guntur District, Andhra Pradesh, India
LEAD_AUTHOR
Suryanarayana
Veeravalli
2
Department of Applied Biology, Jigjiga Univeristy, Ethiopia
AUTHOR
1. Barondes SH, Castronovo V, Cooper DNW, Cummings RD, Drickamer K, Felzi T, et al. Galectins: A family of animal β-galactoside-binding lectins. Cell. 1994;76(4):597-8.
1
2. Anatole AK, Peter GT. Galectins in Disease and Potential Therapeutic Approaches. ACS Symposium Series. U S: American Chemical Society; 2012 p. 3-43.
2
3. Inohara H, Raz A. Functional evidence that cell surface galectin 3 mediates homotypic cell adhesion. Cancer Res. 1995;55(15):3267-71.
3
4. Kuwabara I, Liu FT. Galectin 3 promotes adhesion of human neutrophils to laminin. J Immunol. 1996;156(10):3939-44.
4
5. Liu FT, Hsu DK, Zuberi RI, Kuwabara I, Chi EY, Henderson WR, Jr. Expression and function of galectin 3, a beta-galactoside-binding lectin, in human monocytes and macrophages. Am J Pathol. 1995;147(4):1016-28.
5
6. Sato S, Hughes RC. Binding specificity of a baby hamster kidney lectin for H type I and II chains, polylactosamine glycans, and appropriately glycosylated forms of laminin and fibronectin. J Biol Chem. 1992;267(10):6983-90.
6
7. Yamaoka A, Kuwabara I, Frigeri LG, Liu FT. A human lectin, galectin 3 (epsilon bp/Mac-2), stimulates superoxide production by neutrophils. J Immunol. 1995;154(7):3479-87.
7
8. Akahani S, Nangia-Makker P, Inohara H, Kim HR, Raz A. Galectin 3: a novel antiapoptotic molecule with a functional BH1 (NWGR) domain of Bcl-2 family. Cancer Res. 1997;57(23):5272-6.
8
9. Cumpstey I, Sundin A, Leffler H, Nilsson UJ. C2-symmetrical thiodigalactoside bis-benzamido derivatives as high-affinity inhibitors of galectin 3: efficient lectin inhibition through double arginine-arene interactions. Angew Chem Int Ed Engl. 2005;44(32):5110-2.
9
10. Pieters RJ. Inhibition and detection of galectins. Chembiochem. 2006;7(5):721-8.
10
11. Cregg JM, Cereghino JL, Shi J, Higgins DR. Recombinant Protein Expression in Pichia pastoris. Molecular Biotechnology. 2000;16(1):23-52.
11
12. Stadlmayr G, Mecklenbrauker A, Rothmuller M, Maurer M, Sauer M, Mattanovich D, et al. Identification and characterisation of novel Pichia pastoris promoters for heterologous protein production. J Biotechnol. 2010;150(4):519-29.
12
13. Hamilton SR, Bobrowicz P, Bobrowicz B, Davidson RC, Li H, Mitchell T, et al. Production of complex human glycoproteins in yeast. Science. 2003;301(5637):1244-6.
13
14. Iglesias MM, Rabinovich GA, Ambrosio AL, Castagna LF, Sotomayor CE, Wolfenstein-Todel C. Purification of galectin 3 from ovine placenta: Developmentally regulated expression and immunological relevance. Glycobiology. 1998;8(1):59-65.
14
15. Hellman U, Wernstedt C, Gonez J, Heldin CH. Improvement of an "In-Gel" digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal Biochem. 1995;224(1):451-5.
15
16. Erdjument-Bromage H, Lui M, Lacomis L, Grewal A, Annan RS, McNulty DE, et al. Examination of micro-tip reversed-phase liquid chromatographic extraction of peptide pools for mass spectrometric analysis. Journal of Chromatography A. 1998;826(2):167-81.
16
17. Mann M, Hojrup P, Roepstorff P. Use of mass spectrometric molecular weight information to identify proteins in sequence databases. Biol Mass Spectrom. 1993;22(6):338-45.
17
ORIGINAL_ARTICLE
Cloning and Expression of the Variable Regions of Anti-EGFR Monoclonal Antibody in E. coli for Production of a Single Chain Antibody
Background:Epidermal growth factor receptor (EGFR) overexpression is a characteristic of several malignancies and could be considered as an excellent target for designing specific inhibitors such as anti-EGFR monoclonal antibodies for cancer therapy. Drawbacks exerted by large sizes of full-length antibodies have lead to the development of single chain antibodies, which benefit from having smaller sizes and short circulation half-lives. Objectives:The aim of this study was cloning, expression and purification of variable regions of anti-EGFR monoclonal antibody in E. coli for production of single chain antibodies. Materials and Methods:The RNA, extracted from the C225 hybridoma cells, was reverse transcribed into cDNA and used for PCR amplification of genes encoding light and heavy chains from the variable regions. The PCR products were cloned and expressed in E. coli BL21 for production of a single chain antibody. The expressed protein was analyzed by SDS-PAGE and purified by Ni-NTA affinity chromatography. The reactivity of purified C225-scFv with EGFR-expressing A431 tumor cell line was tested by western blotting and enzyme-linked immunosorbent assays. Results:The results indicated that C225-scFv was highly expressed in E. coli and appeared as a protein with a mass of 27 kDa in the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the induced cell lysate. Reactivity analysis of the purified C225-scFv with A431 tumor cell line by western blotting and enzyme linked immune sorbant assay (ELISA) revealed high binding affinity of the recombinant C225-scFv to the target cells. Conclusions:The results of this study indicated that C225-scFv is capable of binding to EGFR and could be considered as a useful tool for diagnosis and treatment of EGFR-overexpressing tumor cells.
https://www.ijbiotech.com/article_7251_a16219ecffaaa4edb07a22d0174b84be.pdf
2014-04-01
9
14
10.5812/ijb.17522
Single-Chain Antibody
EGFR
C225
Farzaneh
Jalalypour
1
Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, I.R. IRAN
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, I.R. IRAN
AUTHOR
Safar
Farajnia
farajnia@gmail.com
2
Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, I.R. IRAN
LEAD_AUTHOR
Fatemeh
Mahmoudi
3
Department of Cellular and Molecular Biology, Azarbaijan Shahid Madani University, Tabriz, I.R. IRAN
AUTHOR
Behzad
Baradaran
4
Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, I.R. IRAN
AUTHOR
Davoud
Farajzadeh
farajzadeh_d@yahoo.com
5
Department of Cellular and Molecular Biology, Azarbaijan Shahid Madani University, Tabriz, I.R. IRAN
AUTHOR
Leila
Rahbarnia
6
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, I.R. IRAN
AUTHOR
Jafar
Majidi
7
Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, I.R. IRAN
AUTHOR
1. Arkhipov A, Shan Y, Das R, Endres NF, Eastwood MP, Wemmer DE, et al. Architecture and membrane interactions of the EGF receptor. Cell. 2013;152(3):557-69.
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2. Schneider MR, Wolf E. The epidermal growth factor receptor ligands at a glance. J Cell Physiol. 2009;218(3):460-6.
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5. Mamot C, Drummond DC, Greiser U, Hong K, Kirpotin DB, Marks JD, et al. Epidermal growth factor receptor (EGFR)-targeted immunoliposomes mediate specific and efficient drug delivery to EGFR- and EGFRvIII-overexpressing tumor cells. Cancer Res. 2003;63(12):3154-61.
5
6. Najar AG, Pashaei-Asl R, Omidi Y, Farajnia S, Nourazarian AR. EGFR antisense oligonucleotides encapsulated with nanoparticles decrease EGFR, MAPK1 and STAT5 expression in a human colon cancer cell line. Asian Pac J Cancer Prev. 2013;14(1):495-8.
6
7. Salehi E, Farajnia S, Parivar K, Baradaran B, Majidi J, Omidi Y, et al. Recombinant expression and purification of L2 domain of human epidermal growth factor receptor. Afr J Biotechno. 2013;9(33):5292-6.
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10. Patel D, Lahiji A, Patel S, Franklin M, Jimenez X, Hicklin DJ, et al. Monoclonal antibody cetuximab binds to and down-regulates constitutively activated epidermal growth factor receptor vIII on the cell surface. Anticancer Res. 2007;27(5A):3355-66.
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15. Wang SH, Zhang JB, Zhang ZP, Zhou YF, Yang RF, Chen J, et al. Construction of single chain variable fragment (ScFv) and BiscFv-alkaline phosphatase fusion protein for detection of Bacillus anthracis. Anal Chem. 2006;78(4):997-1004.
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16. Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M. scFv antibody: principles and clinical application. Clin Dev Immunol. 2012;2012:980250.
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17. Kim SJ, Park Y, Hong HJ. Antibody engineering for the development of therapeutic antibodies. Mol Cells. 2005;20(1):17-29.
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18. Koo K, Foegeding PM, Swaisgood HE. Construction and expression of a bifunctional single-chain antibody against Bacillus cereus p6ores. Appl Environ Microbiol. 1998;64(7):2490-6.
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19. Hudson PJ, Kortt AA. High avidity scFv multimers; diabodies and triabodies. J Immunol Methods. 1999;231(1-2):177-89.
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20. Luo D, Mah N, Krantz M, Wilde K, Wishart D, Zhang Y, et al. Vl-linker-Vh orientation-dependent expression of single chain Fv-containing an engineered disulfide-stabilized bond in the framework regions. J Biochem. 1995;118(4):825-31.
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21. Natarajan A, Xiong CY, Gruettner C, DeNardo GL, DeNardo SJ. Development of multivalent radioimmunonanoparticles for cancer imaging and therapy. Cancer Biother Radiopharm. 2008;23(1):82-91.
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22. Holliger P, Hudson PJ. Engineered antibody fragments and the rise of single domains. Nat Biotechnol. 2005;23(9):1126-36.
22
23. Safdari Y, Farajnia S, Asgharzadeh M, Omidfar K, Khalili M. humMR1, a highly specific humanized single chain antibody for targeting EGFRvIII. Int Immunopharmacol. 2014;18(2):304-10.
23
24. Safdari Y, Farajnia S, Asgharzadeh M, Khalili M. Antibody humanization methods - a review and update. Biotechnol Genet Eng Rev. 2013;29(2):175-86.
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25
26. Nourazarian AR, Najar AG, Farajnia S, Khosroushahi AY, Pashaei-Asl R, Omidi Y. Combined EGFR and c-Src antisense oligodeoxynucleotides encapsulated with PAMAM Denderimers inhibit HT-29 colon cancer cell proliferation. Asian Pac J Cancer Prev. 2012;13(9):4751-6.
26
27. Nicholson RI, Gee JM, Harper ME. EGFR and cancer prognosis. Eur J Cancer. 2001;37 Suppl 4:S9-15.
27
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29. Saridaki Z, Georgoulias V, Souglakos J. Mechanisms of resistance to anti-EGFR monoclonal antibody treatment in metastatic colorectal cancer. World J Gastroenterol. 2010;16(10):1177-87.
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30
31. Reeves TD, Hill EG, Armeson KE, Gillespie MB. Cetuximab therapy for head and neck squamous cell carcinoma: a systematic review of the data. Otolaryngol Head Neck Surg. 2011;144(5):676-84.
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34. Zhao JX, Yang L, Gu ZN, Chen HQ, Tian FW, Chen YQ, et al. Stabilization of the single-chain fragment variable by an interdomain disulfide bond and its effect on antibody affinity. Int J Mol Sci. 2010;12(1):1-11.
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35. Kairemo KJ. Radioimmunotherapy of solid cancers: A review. Acta Oncol. 1996;35(3):343-55.
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36. Miethe S, Meyer T, Wohl-Bruhn S, Frenzel A, Schirrmann T, Dubel S, et al. Production of single chain fragment variable (scFv) antibodies in Escherichia coli using the LEX bioreactor. J Biotechnol. 2013;163(2):105-11.
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37. Guglielmi L, Martineau P. Expression of single-chain Fv fragments in E. coli cytoplasm. Methods Mol Biol. 2009;562:215-24.
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38. Schmidt M, Maurer-Gebhard M, Groner B, Kohler G, Brochmann-Santos G, Wels W. Suppression of metastasis formation by a recombinant single chain antibody-toxin targeted to full-length and oncogenic variant EGF receptors. Oncogene. 1999;18(9):1711-21.
38
39. Cho WK, Sohn U, Kwak JW. Production and in vitro refolding of a single-chain antibody specific for human plasma apolipoprotein A-I. J Biotechnol. 2000;77(2-3):169-78.
39
ORIGINAL_ARTICLE
Simultaneous Camouflage of Major and Minor Antigens on Red Blood Cell Surface With Activated mPEGs
Background: Host immune system response against blood group antigens is a major problem in blood transfusions, especially for thalassemic patients. Thus, an approach was proposed coating the red blood cell (RBC) surface by polyethylene glycol. Objectives: This study aimed to obtain the optimal simultaneous camouflge of the major and minor antigens by activated methoxy polyethylene glycol (mPEG) with succinimidyl valerate (SVA) and succinimidyl carbonate (SC), separately. Materials and Methods: The degree of RBC agglutination by antibodies against the major and minor blood groups was used as a surrogate measurement for quantitative assessment of the effctiveness of the surface coating. Also, the RBC morphology was assessed using scanning electron microscope (SEM). In addition, to evaluate the host immune system response, the PEGylated RBCs were transferred between two diffrent mouse strains. Results: Statistical analysis of the results demonstrated that the optimal reaction conditions for simultaneous coating of the antigens by mPEG-SVA and mPEG-SC are as mPEG20 in the polymer mixture, 91.2 and 90.0%, and polymer concentration, 17.21 and 19.80 mg.mL-1, respectively. However, according to the SEM results, the maximum polymer concentration of 14.5 mg.mL-1 was suggested as the best condition for mPEG-SVA modifid human RBCs. Conclusions: It is concluded that the membrane PEGylation camouflges the blood group antigens. This effct is observed signifiantly for non-ABO/Rh(D) antigens. Also, it is found that the mPEG-SVA provide better coverage than mPEG-SC. The results of in vivo analysis showed that the immune reactions against PEGylated RBCs were considerably reduced, so that the levels of the relevant biochemical parameters in serum were similar to those of the normal hosts 24 hours after transfusion.
https://www.ijbiotech.com/article_7260_6a688c66a63f47c2b7c067dd7a1f8310.pdf
2014-04-01
15
25
10.5812/ijb.17776
Red blood cells
Methoxy Polyethylene Glycol
Carbonates
transfusion
Zahra
Gholami
1
Faculty of Chemical Engineering, Tarbiat Modares University, Tehran, I.R. IRAN
AUTHOR
Sameereh
Hashemi Najafabadi
2
Faculty of Chemical Engineering, Tarbiat Modares University, Tehran, I.R. IRAN
LEAD_AUTHOR
Masoud
Soleimani
soleim_m@modares.ac.ir
3
Faculty of Medical Sciences, Tarbiat Modares University, Tehran, I.R. IRAN
AUTHOR
1. Tan Y, Qiu Y, Xu H, Ji S, Li S, Gong F, et al. Decreased immunorejection in unmatched blood transfusions by attachment of methoxypolyethylene glycol on human red blood cells and the effct on D
1
antigen. Transfusion. 2006;46(12):2122–7.
2
2. Scott MD, Murad KL. Cellular camouflge: fooling the immune system with polymers. Curr Pharm Des. 1998;4(6):423–38.
3
3. Castro O, Sandler SG, Houston-Yu P, Rana S. Predicting the effct of
4
transfusing only phenotype-matched RBCs to patients with sickle
5
cell disease: theoretical and practical implications. Transfusion.
6
2002;42(6):684–90.
7
4. Vichinsky EP, Earles A, Johnson RA, Hoag MS, Williams A, Lubin B.
8
Alloimmunization in sickle cell anemia and transfusion of racially unmatched blood. N Engl J Med. 1990;322(23):1617–21.
9
5. Wang D, Kyluik DL, Murad KL, Toyofuku WM, Scott MD. Polymermediated immunocamouflge of red blood cells: effcts of polymer size on antigenic and immunogenic recognition of allogeneic donor blood cells. Sci China Life Sci. 2011;54(7):589–98.
10
6. Blackall DP, Armstrong JK, Meiselman HJ, Fisher TC. Polyethylene
11
glycol-coated red blood cells fail to bind glycophorin A-specifi
12
antibodies and are impervious to invasion by the Plasmodium
13
falciparum malaria parasite. Blood. 2001;97(2):551–6.
14
7. Murad KL, Mahany KL, Brugnara C, Kuypers FA, Eaton JW, Scott MD.
15
Structural and functional consequences of antigenic modulation of red blood cells with methoxypoly(ethylene glycol). Blood.
16
1999;93(6):2121–7.
17
8. Le Y, Scott MD. Immunocamouflge: the biophysical basis of
18
immunoprotection by grafted methoxypoly(ethylene glycol)
19
(mPEG). Acta Biomater. 2010;6(7):2631–41.
20
9. Sezer A, Yağc A. Overview of peptide and protein PEGylation: properties and general strategies. Acta Pharm Sci. 2010;52:377–89.
21
10. Bradley AJ, Test ST, Murad KL, Mitsuyoshi J, Scott MD. Interactions
22
of IgM ABO antibodies and complement with methoxy-PEG-modifid human RBCs. Transfusion. 2001;41(10):1225–33.
23
11. Bradley AJ, Scott MD. Immune complex binding by immunocamouflged [poly(ethylene glycol)-grafted] erythrocytes. Am J Hematol. 2007;82(11):970–5.
24
12. Scott MD, Chen AM. Beyond the red cell: pegylation of other blood
25
cells and tissues. Transfus Clin Biol. 2004;11(1):40–6.
26
13. Armstrong JK, Meiselman HJ, Fisher TC. Covalent binding of
27
poly(ethylene glycol) (PEG) to the surface of red blood cells inhibits aggregation and reduces low shear blood viscosity. Am J Hematol. 1997;56(1):26–8.
28
14. Murad KL, Gosselin EJ, Eaton JW, Scott MD. Stealth cells: prevention
29
of major histocompatibility complex class II-mediated T-cell activation by cell surface modifiation. Blood. 1999;94(6):2135–41.
30
15. Chen AM, Scott MD. Immunocamouflge: prevention of transfusion-induced graft-versus-host disease via polymer grafting of donor cells. J Biomed Mater Res A. 2003;67(2):626–36.
31
16. Hashemi-Najafabadi S, Vasheghani-Farahani E, Shojaosadati SA,
32
Rasaee MJ, Armstrong JK, Moin M, et al. A method to optimize PEGcoating of red blood cells. Bioconjug Chem. 2006;17(5):1288–93.
33
17. Sarvi F, Vasheghani Farahani E, Shojaosadati SA, Hashemi Najafabadi S, Moin M, Pourpak Z. Surface treatment of red blood cells
34
with monomethoxypoly(ethylene glycol) activated by succinimidyl carbonate. Iran J Polym. 2006;15:525–34.
35
18. Miron T, Wilchek M. A simplifid method for the preparation of
36
succinimidyl carbonate polyethylene glycol for coupling to proteins. Bioconjug Chem. 1993;4(6):568–9.
37
19. Bradley AJ, Murad KL, Regan KL, Scott MD. Biophysical consequences of linker chemistry and polymer size on stealth erythrocytes:
38
size does matter. Biochim Biophys Acta. 2002;1561(2):147–58.
39
20. Kayden HJ, Bessis M. Morphology of normal erythrocyte and acanthocyte using Nomarski optics and the scanning electron microscope. Blood. 1970;35(4):427–36.
40
21. Prakash O, Talat M, Hasan SH, Pandey RK. Factorial design for the
41
optimization of enzymatic detection of cadmium in aqueous solution using immobilized urease from vegetable waste. Bioresour
42
Technol. 2008;99(16):7565–72.
43
22. Poraicu D, Sandor S, Menessy I. Decrease of red blood cell fiterability seen in intensive care. II. Red blood cell crenelation "in vivo"
44
as morphological evidence of increased red blood cell viscosity in
45
low flw states. Resuscitation. 1983;10(4):305–16.
46
23. Bradley AJ, Scott MD. Separation and purifiation of
47
methoxypoly(ethylene glycol) grafted red blood cells via twophase partitioning. J Chromatogr B Analyt Technol Biomed Life Sci.
48
2004;807(1):163–8.
49
24. Zalipsky S, Seltzer R, Menon-Rudolph S. Evaluation of a new reagent for covalent attachment of polyethylene glycol to proteins.
50
Biotechnol Appl Biochem. 1992;15(1):100–14.
51
25. Scott MD, Murad KL, Koumpouras F, Talbot M, Eaton JW. Chemical
52
camouflge of antigenic determinants: stealth erythrocytes. Proc
53
Natl Acad Sci U S A. 1997;94(14):7566–71.
54
26. Neu B, Armstrong JK, Fisher TC, Meiselman HJ. Surface characterization of poly(ethylene glycol) coated human red blood cells by
55
particle electrophoresis. Biorheology. 2003;40(4):477–87.
56
27. Fisher TC. PEG-coated red blood cells-simplifying blood transfusion in the new millennium? Immunohematology. 2000;16(1):37–
57
ORIGINAL_ARTICLE
Enhancement of Trichoderma Harzianum Activity Against Sclerotinia sclerotiorum by Overexpression of Chit42
Backgoround: Plant diseases, caused by a wide range of phytopathogenic fungi, could be managed using of Trichoderma sp, as a biocontrol agent. Cell wall degrading enzymes like chitinase from T. harzianum are important means for fungal pathogen inhibition. Overexpression of these chitinase enzymes can improve the antagonistic potential of Trichoderma sp. strains. Objectives: This study aimed to produce a new enhanced biocontrol system of Trichoderma harzianum, overexpressing chit42 gene. The improved T. harzianum could be an appropriate biocontrol agent for controlling the stem rot disease of canola caused by Sclerotinia sclerotiorum. Materials and Methods: T. harzianum protoplast cotransformation was carried out by pLMRS3-Chit42 and p3SR2 plasmids. The transformants were selected based on their growth on colloidal chitin containing medium. The improvement of transformants was investigated by quantification of mRNA using real-time quantitative polymerase chain reaction (RT-PCR) and measurement of chitinase activity in the medium containing colloidal chitin as the carbon source. Furthermore, the antagonistic activity of transformants against S. sclerotiorum was assessed by dual culture experiments. Results: The overexpressing transformants of Chit42 displayed higher levels of chitinase activity to inhibit S. sclerotiorum growth compared with the wild type. The results indicated that the value of the chitinase activity (126.42 + 0.07 U/mL) of Chit42-9 increased 64.17 fold. Transcriptomic analysis demonstrated that Chit42-9 transformant expressed 5.2 fold of Chit42 transcript as compared with the parent strain. Biocontrol inhibition of this transformant was 4.98-fold more compared with the non-transformant type. Conclusion: The improved Chit42-9 transformant can be used for biocontrolling S. sclerotiorum, cause of stem rot disease in canola.
https://www.ijbiotech.com/article_7253_384a1c2597ff2f0a3301bdfa589eb6f7.pdf
2014-04-01
26
31
10.5812/ijb.13869
Trichoderma
Sclerotinia sclerotiorum
Chitinase
Real-Time Polymerase Chain Reaction
Mojegan
Kowsari
1
National Institute of Genetic Engineering and Biotechnology, Tehran, I.R. IRAN
and
Agricultural Biotechnology Research Institute of Iran, Karaj, I.R. IRAN
AUTHOR
Mohammad Reza
Zamani
zamani@nigeb.ac.ir
2
National Institute of Genetic Engineering and Biotechnology, Tehran, I.R. IRAN
LEAD_AUTHOR
Mostafa
Motallebi
motalebi@nigeb.ac.ir
3
National Institute of Genetic Engineering and Biotechnology, Tehran, I.R. IRAN
AUTHOR
1. Woo SL, Scala F, Ruocco M, Lorito M. The Molecular Biology of the Interactions Between Trichoderma spp., Phytopathogenic Fungi, and Plants. Phytopathology. 2006;96(2):181-5.
1
2. Carreras VN, Sanchez AJA, Herrera EAH. Trichoderma: sensing the environment for survival and dispersal. Microbiology. 2011;158(1):3-16.
2
3. Vinale F, Sivasithamparam K, Ghisalberti EL, Marra R, Woo SL, Lorito M. Trichoderma–plant–pathogen interactions. Soil Biology and Biochemistry. 2008;40(1):1-10.
3
4. Harman GE, Howell CR, Viterbo A, Chet I, Lorito M. Trichoderma species--opportunistic, avirulent plant symbionts. Nat Rev Microbiol. 2004;2(1):43-56.
4
5. Lorito M. Chitinolytic Enzymes Produced byTrichoderma harzianum: Antifungal Activity of Purified Endochitinase and Chitobiosidase. Phytopathology. 1993;83(3):302.
5
6. Kubicek CP, Mach RL, Peterbauer CK, Lorito M. Trichoderma. From genes to biocontrol [plant diseases]. Journal of Plant Pathology. 2001;83:11-23.
6
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37
ORIGINAL_ARTICLE
Molecular Characterization and Antimicrobial Resistance of Uropathogenic Escherichia coli
Background:Urinary Tract Infections (UTIs) are the most common infectious diseases in childhood. The Uropathogenic Escherichia coli (UPEC) strains account for as much as 80% of UTIs. Objective:From a clinical perspective, it is important to know which virulence factors and antibiotic resistance properties are present in UPEC strains in pediatrics. Therefore, this study was carried out to investigate the molecular characterization and antimicrobial resistance of UPEC strains isolated from hospitalized patients in pediatric ward of Baqiyatallah Hospital in Tehran. Patients and Methods:One hundred and twenty-one urine specimens were collected from the patients infected with UTIs (51 boys and 70 girls). The urine samples were cultured immediately, and those with E. coli-positive were analyzed for the presence of antibiotic resistance genes and bacterial virulence factors using Polymerase Chain Reaction (PCR). Also, antimicrobial susceptibility testing was performed using disk diffusion methodology with Mueller–Hinton agar according to the instruction of Clinical Laboratory and Standard Institute. Results:Nineteen out of 51 (37.25%) urine samples from boys and 47 out of 70 (67.14%) urine samples from girls harbored E. coli. A significant difference was found between the frequency of UPEC strains in boys and girls (P <.05). High resistance levels to tetracycline (69.6%), ampicillin (69.6%) and norfloxacin (63.6%) were also observed. Totally, 1.66% of tested strains were resistant to more than 8 antibiotics. The incidence of genes encoding resistance against gentamicin (aac (3)-IV), sulfonamide (sul1), beta-lactams (blaSHV and CITM), tetracycline (tetA and tetB), trimethoprim (dfrA1), and quinolones (qnr) were 25.7%, 22.7%, 83.2%, 71.1%, 19.6% and 21.2%, respectively. The most commonly detected virulence factors were fim (71.2%), set-1 (66.6%), iha (62.1), papGI (59%), usp (56%) and sen (22.7%). Conclusion:Resistant strains of uropathogenic E. coli had the lower incidence of uropathogenic virulence factors. We suggested prescription of imipenem and amikacin to treat pediatric patients infected with UTIs.
https://www.ijbiotech.com/article_7246_c39939315203d7b298c096899006dbb7.pdf
2014-04-01
32
40
10.5812/ijb.16833
Uropathogenic Escherichia coli
Virulence factors
Antimicrobial
Pediatrics
Iran
Fatemeh
Mashayekhi
1
School of Nursing and Midwifery, Jiroft University of Medical Sciences, Jiroft, I.R. IRAN
AUTHOR
Mandana
Moghny
2
Department of Clinical Pathology, Shahrekod University of Medical Science, Shahrekord, I.R. IRAN
AUTHOR
Motahare
Faramarzpoor
3
School of Nursing and Midwifery, Jiroft University of Medical Sciences, Jiroft, I.R. IRAN
AUTHOR
Emad
Yahaghi
4
Baqiyatallah University of Medical Sciences, Tehran, I.R. IRAN
AUTHOR
Ebrahim
Khodaverdi Darian
5
Young Researchers and Elite Club, Karaj Branch, Islamic Azad University, Karaj, I.R. IRAN
AUTHOR
Vahideh
Tarhriz
6
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences,Tabriz, I.R. IRAN
AUTHOR
Banafsheh
Dormanesh
7
Department of Pediatric Nephrology, AJA University of Medical Sciences, Tehran, I.R. IRAN
LEAD_AUTHOR
1. Schlager T. Urinary Tract Infections in Children Younger Than 5 Years of Age. Paediatr Drug. 2001;3(3):219-27.
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2. Riccabona M. Urinary tract infections in children. Curr Opin Urol. 2003;13(1):59-62.
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3. Momtaz H, Karimian A, Madani M, Safarpoor Dehkordi F, Ranjbar R, Sarshar M, et al. Uropathogenic Escherichia coli in Iran: serogroup distributions, virulence factors and antimicrobial resistance properties. Ann Clin Microbiol Antimicrob. 2013;12:8.
3
4. Soto SM, Guiral E, Bosch J, Vila J. Prevalence of the set-1B and astA genes encoding enterotoxins in uropathogenic Escherichia coli clinical isolates. Microb Pathog. 2009;47(6):305-7.
4
5. Bauer RJ, Zhang L, Foxman B, Siitonen A, Jantunen ME, Saxen H, et al. Molecular epidemiology of 3 putative virulence genes for Escherichia coli urinary tract infection-usp, iha, and iroN(E. coli). J Infect Dis. 2002;185(10):1521-4.
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6. Lutter SA, Currie ML, Mitz LB, Greenbaum LA. Antibiotic resistance patterns in children hospitalized for urinary tract infections. Arch Pediatr Adolesc Med. 2005;159(10):924-8.
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7. Ilic T, Gracan S, Arapovic A, Capkun V, Subat-Dezulovic M, Saraga M. Changes in bacterial resistance patterns in children with urinary tract infections on antimicrobial prophylaxis at University Hospital in Split. Med Sci Monit. 2011;17(7):CR355-61.
7
8. Aghamahdi F, Hashemian H, Shafiei M, Akbarian Z, Rostam Nejad M, Fallah Karkan M. Etiologies and Antibiotic Resistance Patterns in Infants With Urinary Tract Infections Hospitalized in Children Medical Center, Rasht, Iran. Iranian Journal of NeonatologyIJN. 2013;4(2):21-5.
8
9. Khoshbakht R, Salimi A, Aski HS, Keshavarzi H. Antibiotic Susceptibility of Bacterial Strains Isolated From Urinary Tract Infections in Karaj, Iran. Iran Jundishapur J Microbiol. 2013;6(1).
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10. Ghadiri H, Vaez H, Khosravi S, Soleymani E. The antibiotic resistance profiles of bacterial strains isolated from patients with hospital-acquired bloodstream and urinary tract infections. Crit Care Res Pract. 2012;2012:890797.
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16. Johnson JR, O'Bryan TT, Low DA, Ling G, Delavari P, Fasching C, et al. Evidence of commonality between canine and human extraintestinal pathogenic Escherichia coli strains that express papG allele III. Infect Immun. 2000;68(6):3327-36.
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17. Struve C, Krogfelt KA. In vivo detection of Escherichia coli type 1 fimbrial expression and phase variation during experimental urinary tract infection. Microbiology. 1999;145 ( Pt 10):2683-90.
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18. Kanamaru S, Kurazono H, Ishitoya S, Terai A, Habuchi T, Nakano M, et al. Distribution and genetic association of putative uropathogenic virulence factors iroN, iha, kpsMT, ompT and usp in Escherichia coli isolated from urinary tract infections in Japan. J Urol. 2003;170(6 Pt 1):2490-3.
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19. Johnson JR, Brown JJ. A novel multiply primed polymerase chain reaction assay for identification of variant papG genes encoding the Gal(alpha 1-4)Gal-binding PapG adhesins of Escherichia coli. J Infect Dis. 1996;173(4):920-6.
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20. Tivendale L, Scott J, Ternan A. Pressure support and elevation following the removal of a radial artery for coronary artery bypass grafting. Aust Crit Care. 2000;13(4):153-8.
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21. Van TT, Chin J, Chapman T, Tran LT, Coloe PJ. Safety of raw meat and shellfish in Vietnam: an analysis of Escherichia coli isolations for antibiotic resistance and virulence genes. Int J Food Microbiol. 2008;124(3):217-23.
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22. Mammeri H, Van De Loo M, Poirel L, Martinez-Martinez L, Nordmann P. Emergence of plasmid-mediated quinolone resistance in Escherichia coli in Europe. Antimicrob Agents Chemother. 2005;49(1):71-6.
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23. Toro CS, Farfan M, Contreras I, Flores O, Navarro N, Mora GC, et al. Genetic analysis of antibiotic-resistance determinants in multidrug-resistant Shigella strains isolated from Chilean children. Epidemiol Infect. 2005;133(1):81-6.
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24. Randall LP, Cooles SW, Osborn MK, Piddock LJ, Woodward MJ. Antibiotic resistance genes, integrons and multiple antibiotic resistance in thirty-five serotypes of Salmonella enterica isolated from humans and animals in the UK. J Antimicrob Chemother. 2004;53(2):208-16.
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25. Bien J, Sokolova O, Bozko P. Role of Uropathogenic Escherichia coli Virulence Factors in Development of Urinary Tract Infection and Kidney Damage. Int J Nephrol. 2012;2012:681473.
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26. Oelschlaeger TA, Dobrindt U, Hacker J. Virulence factors of uropathogens. Curr Opin Urol. 2002;12(1):33-8.
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27. Jadhav S, Hussain A, Devi S, Kumar A, Parveen S, Gandham N, et al. Virulence characteristics and genetic affinities of multiple drug resistant uropathogenic Escherichia coli from a semi urban locality in India. PLoS One. 2011;6(3):e18063.
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28. Vollmerhausen TL, Ramos NL, Gundogdu A, Robinson W, Brauner A, Katouli M. Population structure and uropathogenic virulence-associated genes of faecal Escherichia coli from healthy young and elderly adults. J Med Microbiol. 2011;60(Pt 5):574-81.
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29. Chang SL, Shortliffe LD. Pediatric urinary tract infections. Pediatr Clin North Am. 2006;53(3):379-400, vi.
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31. Farshad S, Ranjbar R, Japoni A, Hosseini M, Anvarinejad M, Mohammadzadegan R. Microbial susceptibility, virulence factors, and plasmid profiles of uropathogenic Escherichia coli strains isolated from children in Jahrom, Iran. Arch Iran Med. 2012;15(5):312-6.
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32. Mandal J, Acharya NS, Buddhapriya D, Parija SC. Antibiotic resistance pattern among common bacterial uropathogens with a special reference to ciprofloxacin resistant Escherichia coli. Indian J Med Res. 2012;136(5):842-9.
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33. Japoni A, Gudarzi M, Farshad S, Basiri E, Ziyaeyan M, Alborzi A, et al. Assay for integrons and pattern of antibiotic resistance in clinical Escherichia coli strains by PCR-RFLP in Southern Iran. Jpn J Infect Dis. 2008;61(1):85-8.
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34. Fallah F, Behzadnia H, Moradi A, Eslami G, Sharifian M, Tabatabaei SR, et al. Antimicrobial resistance pattern in urinary tract infections in children on continuous ambulatory peritoneal dialysis. Iran J Clin Infect Dis. 2008;3(3).
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35. Farshad S, Emamghoraishi F, Japoni A. Association of Virulent Genes hly, sfa, cnf-1 and pap with Antibiotic Sensitivity in Escherichia coli Strains Isolated from Children with Community-Acquired UTI. Iran Red Crescent Med J. 2010;12(1):33-7.
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36. Rijavec M, Muller-Premru M, Zakotnik B, Zgur-Bertok D. Virulence factors and biofilm production among Escherichia coli strains causing bacteraemia of urinary tract origin. J Med Microbiol. 2008;57(Pt 11):1329-34.
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37. Mokracka J, Koczura R, Jablonska L, Kaznowski A. Phylogenetic groups, virulence genes and quinolone resistance of integron-bearing Escherichia coli strains isolated from a wastewater treatment plant. Antonie Van Leeuwenhoek. 2011;99(4):817-24.
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38. Gundogdu A, Long YB, Vollmerhausen TL, Katouli M. Antimicrobial resistance and distribution of sul genes and integron-associated intI genes among uropathogenic Escherichia coli in Queensland, Australia. J Med Microbiol. 2011;60(Pt 11):1633-42.
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39. Arabi S. The Common Fimbarie genotyping in Uropathogenic Escherichia coli Shifteh Arabi, Fatemeh Tohidi, Sobgol Naderi, Ali Nazemi*, Mostafa Jafarpour, Rozbeh Naghshbandi. Ann Biol Res.
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40
41. Qin X, Hu F, Wu S, Ye X, Zhu D, Zhang Y, et al. Comparison of adhesin genes and antimicrobial susceptibilities between uropathogenic and intestinal commensal Escherichia coli strains. PLoS One. 2013;8(4):e61169.
41
ORIGINAL_ARTICLE
Molecular Phylogeny of the Genus Lathyrus (Fabaceae-Fabeae) Based on cpDNA matK Sequence in Iran
Background: More than 60 species of the genus Lathyrus are distributed in Southwest Asia. It is the second largest genus of the tribe Fabeae, after Vicia, in the region (and in Iran with 23 species). In the regional Flora (Flora of Turkey, FloraIranicaand flora of Iran), the genus has been divided into 9-10 sections. Here we analyzed the phylogeny of Lathyrus and its relationship with Pisum based on plastid gene matK sequences. Objectives:The present study utilized several approaches including maximum parsimony, Bayesian and maximum likelihood methods to evaluate the monophyly and relationship within the genus Lathyrus, both at the sectional level and species level, mainly based on the taxa growing in Iran. Materials and Methods:A total of 52 accessions, representing 38 species of Lathyrus, three species of Pisum and four species of Vicia and Lens as out-groups, were analyzed for reconstructing the phylogenetic relationship using chloroplast gene matK sequences. Maximum parsimony, Bayesian and maximum likelihood methods were used to construct phylogenetic trees. Results:The present study indicated that Pisum was nested among Lathyrus species. Two members of the Lathyrus section, Clymenum (Lathyrus ochrus and L. Clymenum) with Pisum, formed a weakly supported clade as sister to the larger polytomy comprising the remainder of the Lathyrus species. Several sections of Lathyrus including Lathyrostylis, Lathyrus and Clymenum were monophyletic. Lathyrus roseus (of the monotypic section Orobon) were nested among the members of section Lathyrus. The newly taxon described species L. alamutensis, endemic to Iran, were nested among other species of Lathyrostylis. Linearicarpus, Orobus and Pratensis were not monophyletic sections. Pratensis and the monotypic section Aphaca were the closest taxa. In our analysis, L. Pratensis formed a sister group relationship with the Aphaca clade, not its own section. Conclusions:Shimodaira-Hasegawa (SH) test of the matK dataset showed that all analyzed Lathyrus species formed their own clade and Pisum was sister to them. Furthermore, when we removed the two above-mentioned Lathyrus species, the analysis retrieved Pisum, as a well-supported clade being sister to the Lathyrus calde.
https://www.ijbiotech.com/article_7254_59f6114cb7abeee21e3c20d2bcadeedf.pdf
2014-04-01
41
48
10.5812/ijb.10315
Fabaceae
Lathyrus
Tribe Fabeae
Roghayeh
Oskoueiyan
1
Department of Biology, Ayatollah Amoli Branch, Islamic Azad University, Amol, I.R. IRAN
LEAD_AUTHOR
Shahrokh
Kazempour Osaloo
2
Department of Plant Biology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, I.R. IRAN
AUTHOR
Atefeh
Amirahmadi
3
Department of Biology, Damghan University, Damghan, I.R. IRAN
AUTHOR
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34
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35
ORIGINAL_ARTICLE
Optimization of Dynamic Binding Capacity of Anion Exchange Chromatography Media for Recombinant Erythropoietin Purification
Background:The dynamic binding capacity (DBC) of a chromatography matrix in protein purification is the amount of the total protein absorbed into the matrix, before occurrence of a significant break in the breakthrough curve. Optimization of the process criteria for maximum DBC avoids extra process scale-up and reduces the processing time, costs and protein loss. Taguchi method is a simple useful tool in experimental design to estimate the optimal condition with minimum experiments. Objectives:In this research, linear flow rate, pH and protein concentration of the feed were checked according to an L9 orthogonal Taguchi array, to estimate the best conditions for maximum DBC of Q-sepharose fast flow (QSFF) resin in recombinant human erythropoietin purification process. Materials and Methods:A crud sample containing human recombinant erythropoietin was harvested from a cell culture of Chinese hamster ovary (CHO) cell line. Desalted harvests with different total protein concentrations (30, 40 and 50 µg.mL-1) and pH values (5, 6 and 7) were loaded into a packed column of QSFFwith different linear flow rates (60, 120 and 280 cm.h-1) up to 10% of the breakthrough curve. The total protein loading to the column was checked by UV absorbance and Lowry method, and erythropoietin concentration was measured by ELISA. Analysis of variance (ANOVA) was applied to determine the optimum condition. Results:Finally, total protein concentration of 50 µg.mL-1, pH of 5 and flow rate of 120 cm.h-1, were anticipated as the optimal process conditions with 5.85 mg.mL-1of resin as the dynamic binding capacity. Conclusions:Experiments with anticipated optimal criteria were performed three times and no significant difference was observed (p = 0.136, and 6.06 mg/mL as the average dynamic binding capacity).
https://www.ijbiotech.com/article_7256_f075b0d6071e0a91b3adb53c7419e25b.pdf
2014-04-01
49
55
10.5812/ijb.17352
Binding Capacity
Methods
Ion Exchange Chromatography
Recombinant Human Erythropoietin
Mina
Sepahi
1
Recombinant Biopharmaceutical Production Department, Research and Production Complex, Pasteur Institute of Iran, Karaj, I.R. IRAN
LEAD_AUTHOR
Hooman
Kaghazian
2
Recombinant Biopharmaceutical Production Department, Research and Production Complex, Pasteur Institute of Iran, Karaj, I.R. IRAN
AUTHOR
Mina
Payravi Sereshkeh
3
Recombinant Biopharmaceutical Production Department, Research and Production Complex, Pasteur Institute of Iran, Karaj, I.R. IRAN
AUTHOR
Tahereh
Sadeghcheh
4
Quality Control Department, Research and Production Complex, Pasteur Institute of Iran, Karaj, I.R. IRAN
AUTHOR
Shahin
Hadadian
5
Quality Control Department, Research and Production Complex, Pasteur Institute of Iran, Karaj, I.R. IRAN
AUTHOR
Mohammad Reza
Jebeli
6
Recombinant Biopharmaceutical Production Department, Research and Production Complex, Pasteur Institute of Iran, Karaj, I.R. IRAN
AUTHOR
Fereshteh
Yavari
7
Science and Research Branch, Islamic Azad University, Tehran, I.R. IRAN
AUTHOR
1. Determination of Dynamic Binding Capacity for ProSep-vA Media. Millipore Tech Pub. 2005;TB1175EN00.
1
2. Bergander T, Nilsson-Valimaa K, Oberg K, Lacki KM. High-throughput process development: determination of dynamic binding capacity using microtiter filter plates filled with chromatography resin. Biotechnol Prog. 2008;24(3):632-9.
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3. LeVan M, Carta G, Yon C. Adsorption and ion exchange. In: DW G, editor. Perry's Chemical engineers Handbook. New York: McGraw-Hill; 1997.
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4. Mönster A, Villain L, Scheper T, Beutel S. One-step-purification of penicillin G amidase from cell lysate using ion-exchange membrane adsorbers. J Memb Sci. 2013;444:359-64.
4
5. Pabst TM, Suda EJ, Thomas KE, Mensah P, Ramasubramanyan N, Gustafson ME, et al. Binding and elution behavior of proteins on strong cation exchangers. J Chromatogr A. 2009;1216(45):7950-6.
5
6. Shapiro MS, Haswell SJ, Lye GJ, Bracewell DG. Design and characterization of a microfluidic packed bed system for protein breakthrough and dynamic binding capacity determination. Biotechnol Prog. 2009;25(1):277-85.
6
7. Staby A, Jensen IH. Comparison of chromatographic ion-exchange resins. II. More strong anion-exchange resins. J Chromatogr A. 2001;908(1-2):149-61.
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8. Ion Exchange Chromatography: Principles and Methods. 3 ed. Editor, editor^editors.: Pharmacia LKB Biotechnology; 1991.
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9. Hofer S, Ronacher A, Horak J, Graalfs H, Lindner W. Static and dynamic binding capacities of human immunoglobulin G on polymethacrylate based mixed-modal, thiophilic and hydrophobic cation exchangers. J Chromatogr A. 2011;1218(49):8925-36.
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10. Ahamed T. High - Throughput Technologies for Bioseparation Process Development. Editor, editor^editors. Netherland: Delft university of Technology; 2008.
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11. Carta G. Predicting protein dynamic binding capacity from batch adsorption tests. Biotechnol J. 2012;7(10):1216-20.
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12. Sofer GK, Hagel L. Handbook of process chromatography: a guide to optimization, scale up, and validation, Volume 1. illustrated ed. Editor, editor^editors.: Academic Press; 1997.
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13. Bhambure R, Kumar K, Rathore AS. High-throughput process development for biopharmaceutical drug substances. Trends Biotechnol. 2011;29(3):127-35.
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14. Shukla AA, Etzel MR, Gadam S. Process scale bioseparations for the biopharmaceutical industry. Springer. 2007.
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15. Sofer G, Chirica LC. Downstream Processing: Improving Productivity in Downstream Processing. Biopharm international. 2006;19(11).
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16. Faude A, Zacher D, Muller E, Bottinger H. Fast determination of conditions for maximum dynamic capacity in cation-exchange chromatography of human monoclonal antibodies. J Chromatogr A. 2007;1161(1-2):29-35.
16
17. Hahn R, Schulz PM, Schaupp C, Jungbauer A. Bovine whey fractionation based on cation-exchange chromatography. J Chromatogr A. 1998;795(2):277-87.
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18. Harinarayan C, Mueller J, Ljunglof A, Fahrner R, Van Alstine J, van Reis R. An exclusion mechanism in ion exchange chromatography. Biotechnol Bioeng. 2006;95(5):775-87.
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19. Lendero N, Vidic J, Brne P, Podgornik A, Strancar A. Simple method for determining the amount of ion-exchange groups on chromatographic supports. J Chromatogr A. 2005;1065(1):29-38.
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20. Urmann M, Graalfs H, Joehnck M, Jacob LR, Frech C. Cation-exchange chromatography of monoclonal antibodies: Characterisation of a novel stationary phase designed for production-scale purification. MAbs. 2010;2(4).
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21. Levison PR, Jones RMH, Toome DW, Badger SE, Streater M, Pathirana ND. Influence of flow-rate on the chromatographic performance of agarose-and cellulose-based anion-exchange media. J Chromatogr A. 1996;734(1):137-43.
21
22. Martin C, Coyne J, Carta G. Properties and performance of novel high-resolution/high-permeability ion-exchange media for protein chromatography. J Chromatogr A. 2005;1069(1):43-52.
22
23. Sheth B. Characterisation of chromatography adsorbents for antibody bioprocessing: UCL (University College London); 2009. Pages.
23
24. Roy R. A primer on the Taguchi method. Editor, editor^editors.: Van Nostrand Reinhold; 1990.
24
25. Yuen CT, Storring PL, Tiplady RJ, Izquierdo M, Wait R, Gee CK, et al. Relationships between the N-glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures. Br J Haematol. 2003;121(3):511-26.
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26. Simonian MH, Smith JA. Spectrophotometric and colorimetric determination of protein concentration. Curr Protoc Mol Biol. 2006;Chapter 10:Unit 10 1A.
26
27. Mihelic I, Podgornik A, Koloini T. Temperature influence on the dynamic binding capacity of a monolithic ion-exchange column. J Chromatogr A. 2003;987(1-2):159-68.
27
28. Shukla AMRE, Shishir G. Process Scale Bioseparations for the Biopharmaceutical Industry. Editor, editor^editors.: Taylor & Francis Group; 2007.
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29. Hunter AK, Carta G. Protein adsorption on novel acrylamido-based polymeric ion-exchangers. III. Salt concentration effects and elution behavior. J Chromatogr A. 2001;930(1-2):79-93.
29
30. Ljunglof A, Lacki KM, Mueller J, Harinarayan C, van Reis R, Fahrner R, et al. Ion exchange chromatography of antibody fragments. Biotechnol Bioeng. 2007;96(3):515-24.
30
31. Endo I, Nagamune T, Katoh S, Yonemoto T. Bioseparation and bioengineering: progress in Biotechnology. Elsevier Scie BV. 2000.
31
ORIGINAL_ARTICLE
Construction of a Minigenome Rescue System for Measles Virus, AIK-c Strain
Background:In the recent decade, the reverse genetics method has been broadly used for rescue of negative-stranded RNA viruses from cDNA or viral minigenomes. This technique has been applied to study different steps in virus replication and virus-host interactions. Reverse genetics could also be implemented for design of new vaccines. The T7 RNA polymerase activity as well as virus (nucleocapsid protein) N, (phosphoprotein) P and (Large) L proteins are necessary to rescue the virus or viral minigenome. Measles virus is a negative-stranded non-segmented RNA virus. There are useful vaccine strains to prevent measles disease. Objectives:Here, we describe the construction of a new helper cell line for rescue of measles virus minigenome. The helper cell line stably expresses T7 RNA polymerase as well as measles virus N and P proteins by tricistronic mRNA. Materials and Methods:For rescue of measles virus minigenome a stable helper cell line by using tricistronic expression vector was developed which expressed T7 RNA polymerase as well as measles virus N and P proteins. To construct the tricistronic expression vector, T7 RNA polymerase gene was cloned after cytomegalovirus (CMV) promoter and measles virus N and P proteins were under control of IRES (internal ribosome entry site) sequences. Results:Our results indicated that measles virus minigenome could be rescued in this constructed helper cell line. Conclusions:Through this system, the measles virus minigenome was rescued. Further studies are necessary to improve the rescue efficiency. This may be possible by replacing the CMV promoter with the T7 promoter.
https://www.ijbiotech.com/article_7255_b0ceb5c928325205673ed9a1e8a655f1.pdf
2014-04-01
56
62
10.5812/ijb.18002
Measles Virus
Reverse Genetics
Minigenome
Mostafa
Ghaderi
1
Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, I.R. IRAN
AUTHOR
Farzaneh
Sabahi
2
Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, I.R. IRAN
LEAD_AUTHOR
Majid
Sadeghizadeh
sadeghma@modares.ac.ir
3
Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, I.R. IRAN
AUTHOR
Zahra
Khanlari
4
Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, I.R. IRAN
AUTHOR
Azam
Jamaati
5
Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, I.R. IRAN
AUTHOR
Seyed Dawood
Mousavi Nasab
6
Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, I.R. IRAN
AUTHOR
Nasrin
Majidi Garenaz
7
Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, I.R. IRAN
AUTHOR
1. Robert AL, Griffith DP. Fields Virology. 5th ed.; 2007.
1
2. Kingston RL, Baase WA, Gay LS. Characterization of nucleocapsid binding by the measles virus and mumps virus phosphoproteins. J Virol. 2004;78(16):8630-40.
2
3. Parks CL, Lerch RA, Walpita P, Wang HP, Sidhu MS, Udem SA. Analysis of the noncoding regions of measles virus strains in the Edmonston vaccine lineage. J Virol. 2001;75(2):921-33.
3
4. Kohl A, Hart TJ, Noonan C, Royall E, Roberts LO, Elliott RM. A bunyamwera virus minireplicon system in mosquito cells. J Virol. 2004;78(11):5679-85.
4
5. Radecke F, Spielhofer P, Schneider H, Kaelin K, Huber M, Dotsch C, et al. Rescue of measles viruses from cloned DNA. EMBO J. 1995;14(23):5773-84.
5
6. Combredet C, Labrousse V, Mollet L, Lorin C, Delebecque F, Hurtrel B, et al. A molecularly cloned Schwarz strain of measles virus vaccine induces strong immune responses in macaques and transgenic mice. J Virol. 2003;77(21):11546-54.
6
7. Takeda M, Takeuchi K, Miyajima N, Kobune F, Ami Y, Nagata N, et al. Recovery of pathogenic measles virus from cloned cDNA. J Virol. 2000;74(14):6643-7.
7
8. Herfst S, de Graaf M, Schickli JH, Tang RS, Kaur J, Yang CF, et al. Recovery of human metapneumovirus genetic lineages a and B from cloned cDNA. J Virol. 2004;78(15):8264-70.
8
9. de Wit E, Spronken MI, Vervaet G, Rimmelzwaan GF, Osterhaus AD, Fouchier RA. A reverse-genetics system for Influenza A virus using T7 RNA polymerase. J Gen Virol. 2007;88(Pt 4):1281-7.
9
10. Kumada A, Komase K, Nakayama T. Recombinant measles AIK-C strain expressing current wild-type hemagglutinin protein. Vaccine. 2004;22(3-4):309-16.
10
11. Yuri K, Masaaki S, Makoto S, Seiji O, Chieko K, Kyoko TK. Evaluation of a recombinant measles virus as the expression vector of hepatitis C virus envelope proteins. World J vaccines. 2011;2011.
11
12. Mok H, Cheng X, Xu Q, Zengel JR, Parhy B, Zhao J, et al. Evaluation of Measles Vaccine Virus as a Vector to Deliver Respiratory Syncytial Virus Fusion Protein or Epstein-Barr Virus Glycoprotein gp350. Open Virol J. 2012;6:12-22.
12
13. Reyes-del Valle J, de la Fuente C, Turner MA, Springfeld C, Apte-Sengupta S, Frenzke ME, et al. Broadly neutralizing immune responses against hepatitis C virus induced by vectored measles viruses and a recombinant envelope protein booster. J Virol. 2012;86(21):11558-66.
13
14. Liniger M, Zuniga A, Tamin A, Azzouz-Morin TN, Knuchel M, Marty RR, et al. Induction of neutralising antibodies and cellular immune responses against SARS coronavirus by recombinant measles viruses. Vaccine. 2008;26(17):2164-74.
14
15. Brandler S, Marianneau P, Loth P, Lacote S, Combredet C, Frenkiel MP, et al. Measles vaccine expressing the secreted form of West Nile virus envelope glycoprotein induces protective immunity in squirrel monkeys, a new model of West Nile virus infection. J Infect Dis. 2012;206(2):212-9.
15
16. Brandler S, Ruffie C, Najburg V, Frenkiel MP, Bedouelle H, Despres P, et al. Pediatric measles vaccine expressing a dengue tetravalent antigen elicits neutralizing antibodies against all four dengue viruses. Vaccine. 2010;28(41):6730-9.
16
17. Sleeman K, Bankamp B, Hummel KB, Lo MK, Bellini WJ, Rota PA. The C, V and W proteins of Nipah virus inhibit minigenome replication. J Gen Virol. 2008;89(Pt 5):1300-8.
17
18. Brown DD, Collins FM, Duprex WP, Baron MD, Barrett T, Rima BK. 'Rescue' of mini-genomic constructs and viruses by combinations of morbillivirus N, P and L proteins. J Gen Virol. 2005;86(Pt 4):1077-81.
18
19. Flick K, Hooper JW, Schmaljohn CS, Pettersson RF, Feldmann H, Flick R. Rescue of Hantaan virus minigenomes. Virology. 2003;306(2):219-24.
19
20. Halpin K, Bankamp B, Harcourt BH, Bellini WJ, Rota PA. Nipah virus conforms to the rule of six in a minigenome replication assay. J Gen Virol. 2004;85(Pt 3):701-7.
20
21. Rennick LJ, Duprex WP, Rima BK. Measles virus minigenomes encoding two autofluorescent proteins reveal cell-to-cell variation in reporter expression dependent on viral sequences between the transcription units. J Gen Virol. 2007;88(Pt 10):2710-8.
21
22. Jiang Y, Liu H, Liu P, Kong X. Plasmids driven minigenome rescue system for Newcastle disease virus V4 strain. Mol Biol Rep. 2009;36(7):1909-14.
22
23. Ho SC, Bardor M, Feng H, Mariati, Tong YW, Song Z, et al. IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. J Biotechnol. 2012;157(1):130-9.
23
24. Zhu J, Musco ML, Grace MJ. Three-color flow cytometry analysis of tricistronic expression of eBFP, eGFP, and eYFP using EMCV-IRES linkages. Cytometry. 1999;37(1):51-9.
24
25. Teschendorf C, Warrington KH, Jr., Siemann DW, Muzyczka N. Comparison of the EF-1 alpha and the CMV promoter for engineering stable tumor cell lines using recombinant adeno-associated virus. Anticancer Res. 2002;22(6A):3325-30.
25
26. Choi KH, Basma H, Singh J, Cheng PW. Activation of CMV promoter-controlled glycosyltransferase and beta -galactosidase glycogenes by butyrate, tricostatin A, and 5-aza-2'-deoxycytidine. Glycoconj J. 2005;22(1-2):63-9.
26
ORIGINAL_ARTICLE
In Vivo Toxicity Assessment of Bovine Serum Albumin and Dimercaptosuccinic Acid Coated Fe3O4 Nanoparticles
Background: Recently, applications of nanoparticles in many fields of medicine have been developed, due to their specific physical and chemical properties. Therefore assessment of their toxicity specially in the in vivo condition is necessary. Objectives: The aim of this study is to evaluation the effect of Fe3O4 nanoparticles coating by biocompatible compounds on their toxicity and also comparison by noncoated nanoparticles. Materials and Methods: Wetted chemical method was used in order to synthesize Fe3O4 nanoparticles. The synthesized nanoparticles were coated by BSA (Bovine Serum Albumin) and DMSA (Dimercaptosuccinic Acid) and the coating interactions were investigated by FTIR. Magnetic and structure properties of Fe3O4 and coated Fe3O4 nanoparticles were evaluated by AGFM (Alternating Gradient Force Magnetometer), TEM (Transmission Electron Microscope) and XRD (X Ray Diffraction). Toxicity assessment of Fe3O4 and coated Fe3O4 nanoparticles were studied in mice by intra peritoneally injections during a month. Liver enzymes (SGPT, SGOT, ALP, and LDH) were measured 7, 15 and 30 days post injection. Result: The synthesized nanoparticles are single phase and have the spinel structure which their size distribution in the net from is around 5 to 11 nm and in the coated form is 17 to 25 nm. Some liver enzymes were changed due to the injection of both uncoated and coated nanoparticles to mice (especially in groups who received concentrations more than 100 mg per kg of mice weight). The liver enzymes changes were more considerable in the groups received DMSA or DMSA coated in comparison with the groups received BSA or BSA coated. Chemical toxicity studies showed that there is not any irreversible effect in concentrations less than 200 mg/kg for all control and treated groups. Conclusions: The results indicate that, liver enzymes were changed during 7 and 15 days post injection measurements especially in high doses (200 mg/kg). The results of 30 days post injection measurements were changed less in comparison with the control and this is indicates that there is not any irreversible effect in liver. Moreover, DMSA coated nanoparticles were more toxic in comparison with BSA coated nanoparticles.
https://www.ijbiotech.com/article_7258_3d1719ca71b8f9de4ae611b5a1155d20.pdf
2014-04-01
63
68
10.5812/ijb.16858
BSA
DMSA
Fe3O4
Liver enzyme
Nanoparticle
Parisa
Hajshafii
1
Falavarjan Branch, Islamic Azad University, Isfahan, I.R. IRAN
LEAD_AUTHOR
Soheil
Fatahian
2
Falavarjan Branch, Islamic Azad University, Isfahan, I.R. IRAN
AUTHOR
Kahin
Shahanipoor
3
Falavarjan Branch, Islamic Azad University, Isfahan, I.R. IRAN
AUTHOR
Jayakumar OD, Ganguly R, Tyagi AK, Chandrasekharan DK, Nair CKK. Water dispersible Fe3O4 nanoparticles carrying doxorubicin for cancer therapy. J Nanosci Nanotechnol. 2009;9(11):6344-8.
1
Mahmoudi M, Sant S, Wang B, Laurent S, Sen T. Superparamagnetic iron oxide nanoparticles (SPIONs): Development, surface modification and applications in chemotherapy. Adv Drug Deliv Rev. 2011;63(1-2):24-46.
2
Weissleder R, Elizondo G, Wittenburg J, Rabito CA, Bengele HH, Josephson L. Ultrasmall superparamagnetic iron oxide: characterization of a new class of contrast agents for MR imaging. J Radiology. 1990;175(2):489-93.
3
Bregya A, Kohlera A, Steitzb B, Petri-Finkb A, Bognid S, Alfieria A, et al. Electromagnetic tissue fusion using superparamagnetic iron oxide nanoparticles: first experience with rabbit aorta. Open Surg J. 2008;2:3-9.
4
Qi H, Yan B, Lu W, Li C, Yang Y. A Non-Alkoxide Sol-Gel Method for the Preparation of Magnetite (Fe3O4) Nanoparticles. Current Nanoscience. 2011;7(3):381-8.
5
Amiri GhR, Yousefi MH, Aboulhassani MR, Keshavarz MH, Shahbazi D, Fatahian S, et al. Radar absorption of Ni0.7Zn0.3Fe2O4nanoparticles. Digest J Nanomaterials Biostructures. 2010;5(3):1025-31.
6
Amiri GhR, Yousefi MH, Aboulhassani MR, Keshavarz MH, Manouchehri S, Fatahian S. Magnetic properties and microwave absorption in Ni–Zn and Mn–Zn ferrite nanoparticles synthesized by low-temperature solid-state reaction. J Magn Magn Mater. 2011;323(6):730-4.
7
Amiri GhR, Fatahian S, Jelvani AR, Mousarezaei R, Habibi M. magnetic properties of cofe2o4 and co0.5 zn0.5fe2o4 ferrite nanoparticles synthesized by microwave method. Optoelectron Adv Mat. 2011;5(11):1178-80.
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Salata OV. Applications of nanoparticles in biology and medicine. J Nanobiotech. 2004;2(1):3.
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Kavitha AL, Prabu HG, Babu SA, Suja SK. Magnetite nanoparticles chitosan composite containing carbon paste electrode for glucose biosensor application. J Nanosci Nanotechnol. 2003;13(1):98-104.
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14
Garcia MP, Parca RM, Chaves SB, Silva LP, Santos AD, Lacava ZGM, et al. Morphological analysis of mouse lungs after treatment with magnetite-based magnetic fluid stabilized with DMSA. J Magn Magn Mater. 2005;293(1):277-82.
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Kim JS, Yoon TJ, Yu KN, Kim BG, Park SJ, Kim HW, et al. Toxicity and tissue distribution of magnetic nanoparticles in mice. Toxicol Sci. 2006;89(1):338-47.
17
Fatahian S, Shahbazi D, Pouladian M, Yousefi MH, Amiri GhR, Noori A. Biodistribution and toxicity assessment of radiolabeled and DMSA coated ferrite nanoparticles in mice. J Radioanal Nucl Chem. 2012;293(3):915-23.
18
Keshavarz M, Ghasemi Z. Coating of iron oxide nanoparticles with human and bovine serum albumins: A thermodynamic approach. J Phys Theor Chem. 2011;8(2):85-95.
19
Fashen L, Wang H, Wang L, Wang J. Magnetic properties of ZnFe2O4 nanoparticles produced by a low-temperature solid-state reaction method. J Magn Magn Mater. 2007;309(2): 295-9.
20
Fatahian S, Shahbazi D, Pouladian M, Yousefi MH, Amiri GhR, Shahi Z, et al. Preparation and magnetic properties investigation of Fe3O4 nanoparticles 99mTc labeled and Fe3O4 nanoparticles DMSA coated. Dig J Nanomater Bios. 2011;6(3):1161-5.
21