title: 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 inE. coliand 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 inEscherichia 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 100μg/ml. Immunogenicity of rPR was confirmed by Western blotting and ELISA.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Cellular and Molecular Research Center, Kurdistan University of Medical Sciences, Sanandaj, Iran; Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Asaad
authors.initials: A
authors.lastname: Azarnezhad
authors.affiliation: Blood Transfusion Research Center, Institute for Research and Education in Transfusion Medicine, Tehran, Iran.
authors.forename: Zohreh
authors.initials: Z
authors.lastname: Sharifi
authors.affiliation: Department of Molecular Medicine and Genetics, Faculty of Medicine, Hamedan University of Medical Sciences, Hamedan, Iran.
authors.forename: Rahmatollah
authors.initials: R
authors.lastname: Seyedabadi
authors.affiliation: Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran.
authors.forename: Arshad
authors.initials: A
authors.lastname: Hosseini
authors.affiliation: Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran.
authors.forename: Behrooz
authors.initials: B
authors.lastname: Johari
authors.affiliation: Department of Immunology, Faculty of Medicine, Hamedan University of Medical Sciences, Hamedan, Iran; Student Research Center, Hamedan University of Medical Sciences, Hamedan, Iran.
authors.forename: Mahsa
authors.initials: M
authors.lastname: Sobhani Fard
keywords: Human immunodeficiency virus; Molecular cloning; Protease; Recombinant proteins