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Cloning and Expression of Soluble Recombinant HIV-1 CRF35 Protease-HP Thioredoxin Fusion Protein
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...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Avicenna Research Institute
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5124254/ https://www.ncbi.nlm.nih.gov/pubmed/27920885 |
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author | Azarnezhad, Asaad Sharifi, Zohreh Seyedabadi, Rahmatollah Hosseini, Arshad Johari, Behrooz Sobhani Fard, Mahsa |
author_facet | Azarnezhad, Asaad Sharifi, Zohreh Seyedabadi, Rahmatollah Hosseini, Arshad Johari, Behrooz Sobhani Fard, Mahsa |
author_sort | Azarnezhad, Asaad |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-5124254 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Avicenna Research Institute |
record_format | MEDLINE/PubMed |
spelling | pubmed-51242542016-12-05 Cloning and Expression of Soluble Recombinant HIV-1 CRF35 Protease-HP Thioredoxin Fusion Protein Azarnezhad, Asaad Sharifi, Zohreh Seyedabadi, Rahmatollah Hosseini, Arshad Johari, Behrooz Sobhani Fard, Mahsa Avicenna J Med Biotechnol Original Article 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. Avicenna Research Institute 2016 /pmc/articles/PMC5124254/ /pubmed/27920885 Text en Copyright© 2016 Avicenna Research Institute This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. |
spellingShingle | Original Article Azarnezhad, Asaad Sharifi, Zohreh Seyedabadi, Rahmatollah Hosseini, Arshad Johari, Behrooz Sobhani Fard, Mahsa Cloning and Expression of Soluble Recombinant HIV-1 CRF35 Protease-HP Thioredoxin Fusion Protein |
title | Cloning and Expression of Soluble Recombinant HIV-1 CRF35 Protease-HP Thioredoxin Fusion Protein |
title_full | Cloning and Expression of Soluble Recombinant HIV-1 CRF35 Protease-HP Thioredoxin Fusion Protein |
title_fullStr | Cloning and Expression of Soluble Recombinant HIV-1 CRF35 Protease-HP Thioredoxin Fusion Protein |
title_full_unstemmed | Cloning and Expression of Soluble Recombinant HIV-1 CRF35 Protease-HP Thioredoxin Fusion Protein |
title_short | Cloning and Expression of Soluble Recombinant HIV-1 CRF35 Protease-HP Thioredoxin Fusion Protein |
title_sort | cloning and expression of soluble recombinant hiv-1 crf35 protease-hp thioredoxin fusion protein |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5124254/ https://www.ncbi.nlm.nih.gov/pubmed/27920885 |
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