牛分枝杆菌cfp10-esat6-cfp7融合基因的原核表达及抗原反应性分析

Prokaryotic expression of the Mycobacterium Bovis fusion gene cfp10-esat6-cfp7 and antigen reactivity analysis

为了增强单个蛋白在牛结核病诊断中的敏感性和抗感染中的免疫原性,试验采用重叠延伸PCR将牛分枝杆菌cfp10基因与esat6基因融合,得到cfp10-esat6融合基因,再行重叠延伸PCR将cfp10-esat6融合基因与cfp7基因融合,得到融合基因cfp10-esat6-cfp7,并克隆至T-Vector pMD19中,构建重组质粒pMD-cfp10-esat6-cfp7。对pMD-cfp10-esat6-cfp7和pET-28a(+)进行BamHⅠ和EcoRⅠ双酶切,将纯化的cfp10-esat6-cfp7连接到pET-28a(+)中,获得表达质粒pET-cfp10-esat6-cfp7,通过IPTG诱导,SDS-PAGE分析表达质粒在E.coli BL21(DE3)中的表达情况。对表达的融合蛋白进行镍柱纯化,通过Western-blot和间接ELISA分析其抗原反应性。结果表明:获得了融合基因cfp10-esat6-cfp7,表达了约36.3 ku的融合蛋白CFP10-ESAT6-CFP7。融合蛋白CFP10-ESAT6-CFP7与10份牛结核阳性血清反应的OD492值均在0.6以上,而融合蛋白CFP10-ESAT6与阳性血清反应的OD492值在0.1~0.3之间;融合蛋白CFP10-ESAT6-CFP7与10份牛结核阴性血清的OD492值也明显高于CFP10-ESAT6,其与牛结核阳性血清呈现良好的反应性。说明CFP10-ESAT6-CFP7融合蛋白具有作为牛结核病诊断抗原用于新型疫苗研究的潜在价值。

To enhance the diagnostic sensitivity and anti-infectious immunogenicity of bovine tuberculosis, Mycobacterium bovis CFP 10, ESAT 6 and CFP 7 were fused by a(Gly4Ser)3 linker. In this study, cfp10 was first connected with esat6 to obtain the fusion gene cfp10-esat6 by overlapping extension(SOE) polymerase chain reaction(PCR) method, and then the fusion gene cfp10-esat6 was fused with the cfp 7 gene by SOE PCR to obtain the fusion gene cfp10-esat6-cfp7, which was cloned into T-Vector pMD19 vector to construct the recombinant plasmid pMD-cfp10-esat6-cfp7. The pMD-cfp10-esat6-cfp7 and pET-28 a(+) were digested by BamHⅠ and EcoRⅠ double enzymes. The purified cfp10-esat6-cfp7 was subcloned into vector pET-28 a(+) to obtain the expression plasmid pET-cfp10-esat6-cfp7. Plasmid containing pET-cfp10-esat6-cfp7 was transformed into competent Escherichia coli BL21(DE3), then induced by isopropyl-β-D-thiogalactopy-ranoside(IPTG) and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE). The expressed fusion protein was purified by nickel column and its antigenic reactivity was analyzed by Western-blot and indirect enzyme linked immunosorbent assay(ELISA). The results showed that the cfp10-esat6-cfp7 fusion gene was obtained and an approximate 36.3 ku fusion protein CFP10-ESAT6-CFP7 was successfully expressed. The OD492 values of fusion protein CFP10-ESAT6-CFP7 and 10 M. bovis positive serum reactions were all above 0.6, while the OD492 values of fusion protein CFP10-ESAT6 and positive serum reactions were between 0.1 and 0.3. The results of fusion protein CFP10-ESAT6-CFP7 with 10 M. bovis negative serum were also significantly higher than that of CFP10-ESAT6, which showed a good reaction with M. bovis positive serum.The results demonstrated that CFP10-ESAT6-CFP7 fusion protein had potential value as a diagnostic antigen and a novel vaccine for bovine tuberculosis.