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Cloning and Expression of Soluble Recombinant HIV-1 CRF35 Protease-HP Thioredoxin Fusion Protein.


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 ?g/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.

SUBMITTER: Azarnezhad A 

PROVIDER: S-EPMC5124254 | biostudies-literature | 2016 Oct-Dec

REPOSITORIES: biostudies-literature

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Cloning and Expression of Soluble Recombinant HIV-1 CRF35 Protease-HP Thioredoxin Fusion Protein.

Azarnezhad Asaad A   Sharifi Zohreh Z   Seyedabadi Rahmatollah R   Hosseini Arshad A   Johari Behrooz B   Sobhani Fard Mahsa M  

Avicenna journal of medical biotechnology 20161001 4


<h4>Background</h4>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 <i>E. coli</i> and investigation of its immunoreactivity.<h4>Methods</h4>Protease coding region was isolated from  ...[more]

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