CLONING, EXPRESSION AND PURIFICATION OF THE RECOMBINANT HIV-1 TAT-NEF FUSION PROTEIN IN PROKARYOTIC EXPRESSION SYSTEM

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KADKHODAYAN SOMAYEH; IRANI SHIVA; SADAT SEYED MEHDI; FOTOUHI FATEMEH; BOLHASSANI AZAM

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Journal: Journal of Arak University Medical Sciences Year:2016 | Volume:19 | Issue:4 (109) Page(s): 60-68

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Title

CLONING, EXPRESSION AND PURIFICATION OF THE RECOMBINANT HIV-1 TAT-NEF FUSION PROTEIN IN PROKARYOTIC EXPRESSION SYSTEM

Author(s)

Keywords

HIV VIRUSQ1

NEFQ1

TATQ1

RECOMBINANT PROTEINQ2

E. COLIQ1

VACCINEQ1

Abstract

Background: NEF is one of the HIV-1 critical proteins, because it is essential for viral replication and AIDS disease progression and induction of immune response against it can partially inhibit viral infection. Moreover, a domain of the HIV-1 Trans-Activator of Transcription (Tat, 48-60 aa) could act as a cell penetrating peptide (CPP). In current study, cloning and expression of TAT- NEF fusion protein was performed in E. COLI for the first time. The protein expression was confirmed by western blot analysis and was purified using reverse staining method.Materials and Methods: In this experimental study, primarily, cloning of TAT-Nef fusion gene was done in pGEX6p2 expression vector. Then, the expression of TAT- NEF recombinat protein in E.coli BL21 (DE3) strain was performed by using IPTG inducer. The protein expression was confirmed by SDS- PAGE and western blotting using anti-Nef monoclonal antibody. Then, the recombinant fusion protein was purified from gel using reverse staining method.Results: The results of PCR analysis and enzyme digestion showed a clear band of ~ 726 bp in agarose gel indicating the correct TAT-Nef fusion cloning in pGEX6p2 prokaryotic expression vector. In addition, a 54 kDa band of TAT-Nef on SDS-PAGE revealed TAT-Nef protein expression that western blot analysis using anti-Nef monoclonal antibody confirmed it.Conclusion: The purified TAT-Nef recombinant fusion protein will be used as an antigen for protein VACCINE design against HIV infection.

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References

CONSTRUCTION OF HUMAN LRIG1-TAT FUSIONS AND TAT-MEDIATED LRIG1 PROTEIN DELIVERY

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APA: Copy

KADKHODAYAN, SOMAYEH, IRANI, SHIVA, SADAT, SEYED MEHDI, FOTOUHI, FATEMEH, & BOLHASSANI, AZAM. (2016). CLONING, EXPRESSION AND PURIFICATION OF THE RECOMBINANT HIV-1 TAT-NEF FUSION PROTEIN IN PROKARYOTIC EXPRESSION SYSTEM. ARAK MEDICAL UNIVERSITY JOURNAL (AMUJ), 19(4 (109)), 60-68. SID. https://sid.ir/paper/69898/en

Vancouver: Copy

KADKHODAYAN SOMAYEH, IRANI SHIVA, SADAT SEYED MEHDI, FOTOUHI FATEMEH, BOLHASSANI AZAM. CLONING, EXPRESSION AND PURIFICATION OF THE RECOMBINANT HIV-1 TAT-NEF FUSION PROTEIN IN PROKARYOTIC EXPRESSION SYSTEM. ARAK MEDICAL UNIVERSITY JOURNAL (AMUJ)[Internet]. 2016;19(4 (109)):60-68. Available from: https://sid.ir/paper/69898/en

IEEE: Copy

SOMAYEH KADKHODAYAN, SHIVA IRANI, SEYED MEHDI SADAT, FATEMEH FOTOUHI, and AZAM BOLHASSANI, “CLONING, EXPRESSION AND PURIFICATION OF THE RECOMBINANT HIV-1 TAT-NEF FUSION PROTEIN IN PROKARYOTIC EXPRESSION SYSTEM,” ARAK MEDICAL UNIVERSITY JOURNAL (AMUJ), vol. 19, no. 4 (109), pp. 60–68, 2016, [Online]. Available: https://sid.ir/paper/69898/en

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