RESUMO
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.
Assuntos
Humanos , Escherichia coli , Genética , Metabolismo , Anticorpos Anti-HIV , Sangue , Alergia e Imunologia , Infecções por HIV , Alergia e Imunologia , Virologia , Transcriptase Reversa do HIV , Genética , Alergia e Imunologia , HIV-1 , Classificação , Alergia e Imunologia , Renaturação Proteica , Proteínas Recombinantes , Genética , Alergia e Imunologia , Sensibilidade e EspecificidadeRESUMO
Objective To construct the recombinant plasmid of human MMP 1 clone and study its antigenecity of MMP 1 fusion protein. Methods The total RNA was extracted from human liver and used as a template for reverse transcription. After PCR amplification, a 1 432 bp fragment was obtained and cloned into T vector. After digested with restriction enzyme, the target fragment was subcloned into plasmid pMAL c2x. The recombinant plasmid was transferred into JM109 which can express a fusion protein. We analyze the protein with SDS PAGE and Western blot. Results We obtained human MMP 1 gene and its recombinant plasmid clone. The expressed protein can be recognized by MMP 1 polyclonal antibody in Western blot.Conclusions We obtained the MMP 1 protein with antigenicity. The fusion protein can be used to prepare polyclonal antibody against MMP 1.