牛分枝杆菌mpb51-mpb70融合基因的原核表达与抗原活性初步鉴定

目的 通过获得牛分枝杆菌保护性抗原MPB51、MPB70的融合蛋白MPB51-MPB70,以提高单一蛋白的抗原性。方法 采用重叠拼接PCR技术将牛分枝杆菌mpb51与mpb70基因融合,获得融合基因mpb51-mpb70,克隆至pMD19中,构建重组质粒pMD-51-70。酶切、纯化后连接至pET28a(+)中,构建重组表达质粒pET-51-70。通过SDS-PAGE和Western blotting分析、鉴定融合蛋白的表达及抗原性。以间接ELISA比较MPB51、MPB70、MPB51-MPB70的抗原性。结果 表达、纯化了45 ku的融合蛋白MPB51-MPB70,并与牛结核血清发生特异性反应,且比单一蛋白反应性强。结论 成功地获得了具有良好抗原性的牛分枝杆菌融合蛋白MPB51-MPB70。

Prokaryotic expression of the Mycobacterium bovis fusion gene mpb51-mpb70 and preliminary identification of antigenic activity

To improve antigenicity of the simple protein, Mycobacterium bovis MPB51 and MPB70 were fused using (Gly4Ser)3 to obtain a fusion protein MPB51-MPB70. The mpb51 and mpb70 genes were fused by splicing overlapping extension(SOE) polymerase chain reaction(PCR) to acquire the fusion gene mpb51-mpb70 which was then cloned into pMD19 vector to construct recombinant plasmid pMD-51-70. After enzyme digestion by BamH I and EcoR I, the purified fusion gene was subcloned into the expression vector pET28a(+). The expression of mpb51-mpb70 was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and the antigenic activity was determined by Western-blotting. Indirect-ELISA was used to compare the antigenicity among MPB51, MPB70 and MPB51-MPB70. A 1359bp fusion gene was amplified and the recombinant plasmid pMD-51-70 was constructed successfully. The expressed fusion protein, approximately 45 ku, exhibited a reaction activity with bovine tuberculosis serum. Besides, the reaction activity was better than that of solo protein. These results indicated that the MPB51-MPB70 fusion protein was obtained successfully.