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Title

OPTIMIZATION OF MULTI-EPITOPIC HIV-1 RECOMBINANT PROTEIN EXPRESSION IN PROKARYOTE SYSTEM AND CONJUGATION TO MOUSE DEC-205 MONOCLONAL ANTIBODY: IMPLICATION FOR IN-VIVO TARGETED DELIVERY OF DENDRITIC CELLS

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 Start Page 145 | End Page 152

Abstract

 Objective(s): Multi-epitopic protein vaccines and direction of vaccine delivery to dendritic cells (DCs) are promising approaches for enhancing immune responses against mutable pathogens. Escherichia coli is current host for expression of RECOMBINANT PROTEINs, and it is important to optimize expression condition. The aim of this study was the optimization of multi-epitopic HIV-1 tat/pol/gag/env RECOMBINANT PROTEIN (HIVtop4) expression by E. coli and conjugation of purified protein to anti DEC-205 monoclonal antibody as candidate vaccine.Materials and Methods: In this study, expression was induced in BL21 (DE3) E. coli cells by optimization of induction condition, post induction incubation time, temperature and culture medium formula. Some culture mediums were used for cell culture, and isopropyl-beta-Dthiogalactopyranoside was used for induction of expression. Protein was purified by Ni-NTA column chromatography and confirmed against anti-His antibody in western-blotting. To exploit DCs properties for immunization purposes, RECOMBINANT PROTEIN chemically coupled to aDEC-205 monoclonal antibody and confirmed against anti-His antibody in western-blotting.Results: The optimum condition for expression was 1 mM IPTG during 4 hr cultures in 2XYT medium, and final protein produced in soluble form. Conjugation of purified protein to aDEC-205 antibody resulted in smears of protein: antibodies conjugate in different molecular weights.Conclusion: The best cultivation condition for production of HIVtop4 protein is induction by 1 mM IPTG during 4 hr in 2XYT medium. The final concentration of purified protein was 500 mg/ml.

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