HIGH-LEVEL EXPRESSION OF RECOMBINANT TRICHOSANTHIN IN ESCHERICHIA COLI AND EVALUATION OF ITS IMMUNOGENICITY / 解剖学报

ABSTRACT

Objective The TCS gene without N-and C-terminal sequences was cloned into pET-29b in order to get the recombinant trichosanthin(rTCS) at the relatively high level in E.coli,and the rTCS immunogenicity was compared with natural TCS(nTCS). Methods TCS gene without the N-and C-terminal coding sequences was amplified by RT-PCR,and then inserted into pET-29b.The soluble rTCS was induced with IPTG and purified through metal chelate affinity chromatography,the purity and content were showed by the thin-layer scan analysis and bradford assay.After the rabbits were immunized with purified rTCS and nTCS,respectively,the titer and specificity of polyclonal antibody were detected by indirect ELISA assay and Western blotting. Results 1.The vector pET-29b-TCS was constructed successfully after it was confirmed with the same restriction enzymes.2.The rTCS attained the maximum in the presence of 1 mmol/L IPTG for 4?h at 30℃.3.The soluble rTCS was expressed at relatively high level(36% of total protein in E.coli) and the yield of rTCS with a purity of 95% was 1.2?g in 1 L bacterium.4.The polyclonal antisera titer of nTCS was significantly higher than that of rTCS(1∶40?960 and 1∶5?120 respectively).5.The specific reaction between rTCS and anti-nTCS antibody was detected by Western blotting.Conclusion The high level expressing vector pET-29b-TCS,carrying TCS gene withoug the N-and C-terminal coding sequences,was constructed successfully.The immunogenicity of rTCS was remarkably lower than that of nTCS,which indicates that glycosylation of nTCS has a relationship with the antigenicity.