Optimization of multi-epitopic HIV-۱ recombinant protein expression in prokaryote system and conjugation to mouse DEC-۲۰۵ monoclonal antibody: implication for in-vivo targeted delivery of dendritic cells

Publish Year: 1394
نوع سند: مقاله ژورنالی
زبان: English
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JR_IJBMS-18-2_006

تاریخ نمایه سازی: 4 آبان 1400

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-۱ tat/pol/gag/env recombinant protein (HIVtop۴) expression by E. coli and conjugation of purified protein to anti DEC-۲۰۵ monoclonal antibody as candidate vaccine. Materials and Methods: In this study, expression was induced in BL۲۱ (DE۳) 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-D-thiogalactopyranoside 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 αDEC-۲۰۵ monoclonal antibody and confirmed against anti-His antibody in western-blotting. Results: The optimum condition for expression was ۱ mM IPTG during ۴ hr cultures in ۲XYT medium, and final protein produced in soluble form. Conjugation of purified protein to αDEC-۲۰۵ antibody resulted in smears of protein: antibodies conjugate in different molecular weights. [AGA۱] . Conclusion: The best cultivation condition for production of HIVtop۴ protein is induction by ۱ mM IPTG during ۴ hr in ۲XYT medium. [AGA۲] The final concentration of purified protein was ۵۰۰ µg/ml.

Authors

Roghayeh Rahimi

Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran , Iran

Massoumeh Ebtekar

Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran , Iran

Seyed Mohammad Moazzeni

Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran , Iran

Ali Mostafaie

Biotechnology Research Center, Kermanshah University, Kermanshah, Iran

Mehdi Mahdavi

Department of Virology, Pasteur Institute of Iran, Tehran, Iran

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