Cloning and expression of soluble recombinant HIV-1 CRF35 protease-HP thioredoxin fusion protein

Azarnezhad, A. and Sharifi, Z. and Seyedabadi, R. and Hosseini, A. and Johari, B. and Fard, M.S. (2016) Cloning and expression of soluble recombinant HIV-1 CRF35 protease-HP thioredoxin fusion protein. Avicenna Journal of Medical Biotechnology, 8 (4). pp. 175-181.

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Abstract

Background: As a drug target and an antigenic agent, HIV-1 protease (HIV-1 PR) is at the center of attention for designing anti-AIDS inhibitors and diagnostic tests. In previous studies, the production of the recombinant protease has been faced with several difficulties; therefore, the aims of this study were the easy production, purification of the soluble form of protease in E. coli and investigation of its immunoreactivity. Methods: Protease coding region was isolated from the serum of an infected individual, amplified by RT-PCR and cloned into PTZ57R using TA-cloning. Protease coding frame was isolated by PCR and cloned in pET102/D. TOPO expression vector and cloned protease was expressed in Escherichia coli (E. coli) BL21. Produced recombinant protein was purified by affinity Ni-NTA column and protein concentration was checked by BCA protein assay kit. Subsequently, immunoreactivity of recombinant protease (rPR) was assayed by Western blotting and ELISA. Results: Cloning of the HIV protease by TOPO cloning system in pET102/D.TOPO was confirmed with PCR and sequencing. The concentration range of purified recombinant protein was 85 to 100 µg/ml. Immunogenicity of rPR was confirmed by Western blotting and ELISA. Conclusion: Soluble production of recombinant HIV-1 protease (HIV-1 rPR) was performed successfully. This recombinant protein disclosed 86 specificity and 90 sensitivity in immunoassay tests. © 2016, Avicenna Journal of Medical Biotechnology. All rights reserved.

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