[Expression, purification and renaturation of Pol P51 antigen of HIV-1 strain CN54 and its application in antibody detection].
To obtain the pure and soluble P51 antigen of HIV-1 strain CN54, we transformed the Escherichia. coli strain BL21 codonplus-RIL with recombinant plasmid pTHioHisA51 which carries a gene encoding the Polymerase (Pol) P51 antigen of HIV-1 CN54 formerly, and induced protein expression by IPTG. We purified the recombinant protein with Chelating Sepharose FF-Ni and DEAE-Sepharose FF column chromatography, then renatured the recombinant protein by dialyzation. Purified protein was identified by Western blotting. We labeled and coated antigen P51 in a dual-antigen sandwich system, and tested it with serum samples from HIV-infected individuals. The results showed that P51 was expressed as inclusion body, and represented about 50% of total cellular protein. After purification and renaturation, the purity of P51 was up to 95%. Western blotting and sandwich ELISA demonstrated that recombinant P51 had good anti-HIV antibody specificity and sensitivity. The results suggested that recombinant HIV-1 P51 can be prepared as diagnostic reagent, and provides valuable support for HIV-1 detection and vaccine research.
Hou
Jue
J
College of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, China.
Sun
Jing
J
Xu
Zhiyong
Z
Fan
Wenling
W
Zhang
Yixuan
Y
Liu
Yong
Y
Hao
Yanling
Y
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015483:HIV Antibodies / Q000097:blood* / Q000276:immunology; D015658:HIV Infections / Q000276:immunology* / Q000821:virology; D054303:HIV Reverse Transcriptase / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D015497:HIV-1 / Q000145:classification / Q000276:immunology*; D006801:Humans; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity
D015483:HIV Antibodies; D011994:Recombinant Proteins; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase