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:: Volume 17, Issue 2 (summer 2019) ::
jmj 2019, 17(2): 46-53 Back to browse issues page
Cloning, Optimization of Expression Condition and Purification of the Recombinant Nef- MPER-V3 Protein of HIV-1 in Prokaryotic Expression System
AHMAD FAGHIH Mr, SEYED MEHDI SADAT Dr , AZAM BOLHASSANI Dr, SHIVA IRANI Dr
Abstract:   (202 Views)
Background: Nef regulatory protein as well as structural proteins MPER and V3 of HIV-1 is important targets in vaccine studies. Therefore, in the current study, the Nef-MPER-V3 fusion protein was cloned and expressed in prokaryotic expression system that will be considered as a candidate for future vaccine studies.
Materials and Methods: The Nef-MPER-V3 sequence was synthesized and inserted into pET-28a(+) vector using NotI/HindIII enzymes. Then, the recombinant construct was expressed in BL21 (DE3) strain of E.coli. Several factors including time course, IPTG concentration and optical density (OD) were evaluated for optimization of protein expression. The protein expression was purified on a Ni-NTA agarose column and analyzed by 12% SDS-PAGE. Finally, the fusion protein was identified and confirmed by western blotting.
Results: The target gene was cloned successfully into the recombinant vector and after selection of the best condition for the protein expression such as; 2 mM of IPTG, OD= 0.7 at 600nm and 5 hours after induction, purification of the recombinant protein by affinity chromatography in denaturing condition by urea yielded about 300 µg/ mL. The purified fusion protein migrated as a clear band of around 35 kDa in SDS-PAGE and was detectable using anti-Nef antibody in western blotting.
Conclusion: Our results showed that the Nef-MPER-V3 protein was successfully expressed in prokaryotic system and purified protein may provide the antigen for future experiments for immunogenicity evaluation in mice are currently undertaken.
 
Keywords: HIV-1, Nef-MPER-V3, Prokaryotic expression system, Affinity chromatography.
Full-Text [PDF 680 kb]   (35 Downloads)    
Type of Study: Research | Subject: VirocShenaci
Received: 2019/09/7 | Accepted: 2019/10/22 | Published: 2019/10/22
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FAGHIH A, SADAT S M, BOLHASSANI A, IRANI S. Cloning, Optimization of Expression Condition and Purification of the Recombinant Nef- MPER-V3 Protein of HIV-1 in Prokaryotic Expression System. jmj. 2019; 17 (2) :46-53
URL: http://jmj.jums.ac.ir/article-1-1390-en.html


Volume 17, Issue 2 (summer 2019) Back to browse issues page
فصلنامه دانشگاه علوم پزشکی جهرم Journal of Jahrom University of Medical Sciences
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