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目的构建含结核分枝杆菌(M.tb)rv2352c基因原核表达载体,经转化E.coli以表达Rv2352c融合蛋白,并研究其抗原性。方法用PCR扩增M.tb rv2352c基因,克隆入pET30a(+)质粒,构建pET30a(+):rv2352c重组质粒,阳性克隆测序验证正确后转化入表达宿主大肠杆菌BL21(DE3),经IPTG诱导Rv2352c蛋白表达。经Ni+-NTA层析柱纯化融合蛋白,通过SDS-PAGE和结核患者血清Western blot进行鉴定。将纯化的重组蛋白分别免疫家兔,制备抗Rv2352c抗血清,抗血清的效价测定采用酶联免疫吸附试验法(ELISA),取兔抗血清与纯化蛋白Rv2352c通过Western blot方法,检测抗体特异性。结果经酶切鉴定和测序分析证实rv2352c原核表达质粒构建正确,SDS-PAGE和Western blot结果显示,在45 kD处呈现单一蛋白条带。用重组蛋白Rv2352c免疫接种后可诱导出高滴度的特异性抗体。纯化蛋白通过Western blot鉴定证实为目的蛋白,有较强的免疫原性。结论成功构建原核表达重组质粒pET30a(+):rv2352c,制备和纯化的Rv2352c融合蛋白具有较好的纯度和生物学功能,为进一步研究结核病的潜在分子标志物奠定基础。
Objective To construct prokaryotic expression vector containing M. tuberculosis rv2352c gene and transform it into E.coli to express Rv2352c fusion protein and study its antigenicity. Methods The gene of M.tb rv2352c was amplified by PCR and cloned into pET30a (+) plasmid. The recombinant plasmid pET30a (+): rv2352c was constructed. The positive clones were verified by sequencing and transformed into E. coli BL21 (DE3). IPTG induced Rv2352c Protein. The fusion protein was purified by Ni + -NTA column and identified by SDS-PAGE and Western blot. Anti-Rv2352c antiserum was prepared by immunizing rabbits with purified recombinant protein. The titer of antiserum was determined by enzyme-linked immunosorbent assay (ELISA), rabbit antiserum and purified protein Rv2352c by Western blot, and the antibody specificity . Results The recombinant plasmid rv2352c was confirmed by restriction enzyme digestion and sequencing analysis. SDS-PAGE and Western blot showed that a single protein band appeared at 45 kD. Immunization with the recombinant protein Rv2352c induced high titers of specific antibodies. The purified protein was verified by Western blot for the purpose of protein, strong immunogenicity. Conclusion The prokaryotic recombinant plasmid pET30a (+): rv2352c was successfully constructed. The purified and purified Rv2352c fusion protein has good purity and biological function, which lays the foundation for further research on potential molecular markers of tuberculosis.