结核分枝杆菌Rv1886c蛋白的表达、纯化及其多克隆抗体的制备

Expression and purification of Rv1886c protein of Mycobacterium tuberculosis and preparation of its polyclonal antibody

目的表达、纯化结核分枝杆菌(Mycobacterium tuberculosis,M.tb)Rv1886c重组蛋白(Ag85B),并免疫小鼠制备多克隆抗体。方法采用PCR技术从M.tb(H37Rv)基因组中扩增Rv1886c基因,克隆至pET-28a质粒中,构建重组表达质粒pET-28a-Rv1886c,转化E.coli BL21(DE3),IPTG诱导表达Ag85B蛋白,并对表达条件进行优化。采用Ni-NTA His亲和层析柱纯化目的蛋白;将纯化的蛋白经皮下注射BALB/c小鼠,制备多克隆抗体,ELISA法检测抗体滴度,Western blot法检测抗体特异性。结果重组表达质粒pET-28a-Rv1886c经双酶切和测序鉴定证明构建正确;重组蛋白的最适表达条件为0.5 mmol/L IPTG 37℃诱导表达4 h,获得了相对分子质量约30000的重组蛋白;纯化的重组蛋白纯度达90%以上,蛋白浓度约为1.5 mg/mL;免疫BALB/c小鼠后获得的抗Ag85B血清具有良好的抗原结合特性,且抗体滴度达1︰51200。结论成功获得了高纯度的Rv1886c蛋白Ag85B,并制备了小鼠抗Ag85B多克隆抗体,为进一步研究TB的血清学早期诊断奠定了基础。

Objective To express and purify recombinant protein of Mycobacterium tuberculosis(M.tb)Rv1886 c protein(Ag85 B)and prepare polyclonal anti-Ag85 B antibody by immunizing mice.Methods Rv1886 c gene was amplified from M.tb H37 Rv genome with PCR,and inserted into pET-28 a plasmid,which was then transformed into E.coli BL21(DE3).Ag85 B expression was induced with IPTG and expressed protein was purified through Ni-NTA His affinity chromatography.Purified Ag85 B was subsequently injected into BALB/c mice to prepare polyclonal antibody.The titer and specificity of the antibody were detected with ELISA and Western blotting,respectively.Results The recombinant plasmid pET-28 a-Rv1886 c was successfully constructed and verified through both double restriction enzyme digestion and sequencing.For optimal expression,transformed bacteria were treated with 0.5 mmol/L IPTG,and cultured at 37℃for4 h.Soluble Ag85 B protein with relative molecular weight of 30000 was then obtained,with a purity of 90%and concentration of 1.5 mg/mL.The anti-Ag85 B serum obtained from immunized BALB/c mice had good antigen binding properties,and the antibody titer was 1∶51200.Conclusion Highly purified Rv1886 c protein Ag85 B was successfully obtained,and the polyclonal antibody against Ag85 B was prepared,which laid a foundation for further study of early serodiagnosis of TB.