ABSTRACT
Objective To construct the eukaryotic expression plasmids of Mycobacterium tuberculosis ESAT-6 antigen and to investigate its immunogenicity in mice. Methods The sequence encoding ESAT-6 was amplified by PCR from Mycobacterium tuberculosis H37Rv genomic DNA. Then, the amplified fragment was sub-cloned into pVAC expression vector. The eukaryotic expression plasmid pVAC-ESAT-6 was transfected into Vero E6 cells with liposome. The mRNA expression of pVAC-esat-6 in Vero E6 cells was detected by RT-PCR and its expression product was analyzed by Western-blot. The plasmid pVAC-esat-6 was introduced into the mice by gene gun injection. The specific antibody to ESAT-6 and the level of IFN-? in supernatant of spleno-lymphocyte cultures were detected by ELISA methods. Results The recombinant plasmid pVAC-esat-6 was successfully constructed, and the expression of ESAT-6 was also detected in vitro. After vaccinated three times, the mice produced specific antibody and the level of IFN-? in supernatant of spleno-lymphocyte cultures in group of pVAC-esat-6 was significantly higher than group of vector alone (P
ABSTRACT
Objective To construct a prokaryotic expression system containing the dense granule protein 4(GRA4) of Toxoplasma gondii,purify the expressed protein and detect its immunogenicity.Methods The specific fragment of GRA4 gene was amplified by PCR.After subcloning the prokaryotic expression recombinant pET,GRA4,the expressed product was purified with His?BindTM affinity chromatography and analyzed by Western blot.BALB/c mice were immunized with the GRA4 recombinant protein,and the antibody IgG titer was detected by ELISA.Results The pET,GRA4 prokaryotic expression system was obtained.The MW of the expressed protein was Mr 40 000 and formed in inclusion body.After purification,the recombinant protein could be specifically recognized by the T.gondii infected rabbit serum.Mice immunized with the purified recombinant protein elicited high titer of IgG antibody.Conclusion The pET,GRA4 recombinant protein was successfully expressed and purified,which shows the immunogenicity.