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:: Volume 20, Issue 5 (Bimonthly 2016) ::
Feyz 2016, 20(5): 420-426 Back to browse issues page
Reverse staining: Effective method for purification of HIV-1 Nef protein in prokaryotic expression system
Behnaz Sadat Jafarzade, Seyyed Mehdi Sadat, Ramin Yaghobi, Azam Bolhassani *
Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, I. R. Iran. , azam.bolhassani@yahoo.com
Abstract:   (2327 Views)

Background: Nef protein is one of the HIV regulatory proteins. This protein has various conserved epitopes inducing the efficient immune responses in HIV-1 infected individuals. Thus, Nef protein has been proposed as a suitable candidate for vaccine design. In current study, our goal was the cloning and expression of Nef protein in prokaryotic expression system and its purification using the reverse staining method. 

Materials and Methods: The coding sequence of Nef protein was amplified from pUC19-nef vector by PCR. Then, nef gene was inserted into the pGEX6p2 expression vector. This construct was transformed into the E.coli BL21 E.coli strain and subsequently protein expression was induced by IPTG anti-repressor. The protein expression was confirmed by SDS-PAGE and Western blot using anti-Nef antibody. Protein purification was performed by reverse staining method.

Results: The PCR and digestion analysis showed a clear band of 648 bp in agarose gel indicating the correct cloning of HIV-nef in pGEX6p2 expression vector. In addition, the detection of a clear 50 kDa band   in Western blotting using Anti-Nef antibody suggests the Nef protein expression induced by IPTG. Finally, the purified protein was obtained by reverse staining method.

Conclusion: The recombinant Nef protein expressed in E.coli was purified by reverse staining method. The Nef protein has the potential of antigenicity for vaccine designing against HIV infections.

Keywords: HIV virus, Nef, Cloning, Expression and purification, Reverse staining, E. coli
Full-Text [PDF 624 kb]   (612 Downloads)    
Type of Study: Research | Subject: medicine, paraclinic
Received: 2016/11/13 | Accepted: 2016/11/13 | Published: 2016/11/13
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Jafarzade B S, Sadat S M, Yaghobi R, Bolhassani A. Reverse staining: Effective method for purification of HIV-1 Nef protein in prokaryotic expression system. Feyz. 2016; 20 (5) :420-426
URL: http://feyz.kaums.ac.ir/article-1-3173-en.html

Volume 20, Issue 5 (Bimonthly 2016) Back to browse issues page
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