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目的原核表达并纯化牛型结核分枝杆菌MPT64蛋白。方法以牛型结核分枝杆菌Vallee株全基因组DNA为模板,PCR扩增MPT64基因,克隆至pET-28a(+)载体,构建重组原核表达质粒pET-MPT64,转化至E.coli BL21(DE3)中,IPTG诱导表达。表达产物经SDS-PAGE鉴定后,用Ni-NTA亲和层析纯化,Western blot鉴定纯化蛋白。结果获得的MPT64基因测序结果与GenBank中登录的MPT64基因核苷酸序列同源性为100%;重组表达质粒pET-MPT64经酶切鉴定表明构建正确;表达的重组MPT64蛋白相对分子质量约为26 000,主要以可溶性形式表达,表达量约占全菌总蛋白的11%;纯化的重组MPT64蛋白纯度达93%,可与抗结核牛血清发生特异性反应。结论 成功原核表达并纯化了牛型结核分枝杆菌MPT64蛋白,其可作为诊断抗原用于新型牛结核病的检测。
Objective To express and purify Mycobacterium bovis MPT64 protein in prokaryotic cells. Methods The full-length genomic DNA of Mycobacterium bovis Vallee strain was used as a template to amplify MPT64 gene by PCR and cloned into pET-28a (+) vector to construct recombinant prokaryotic expression vector pET-MPT64. The recombinant plasmid was transformed into E. coli BL21 (DE3) IPTG induced expression. The expressed product was identified by SDS-PAGE and purified by Ni-NTA affinity chromatography. The purified protein was identified by Western blot. Results The nucleotide sequence of the MPT64 gene was 100% identical to that of the MPT64 gene registered in GenBank. The recombinant plasmid pET-MPT64 was confirmed by restriction enzyme digestion and the recombinant protein MPT64 had a relative molecular mass of 26 000, mainly expressed in soluble form, the expression amount accounted for about 11% of the total bacterial total protein; Purified recombinant MPT64 protein purity of 93%, with anti-TB bovine serum specific reaction. Conclusion The prokaryotic expression and purification of Mycobacterium bovis MPT64 protein can be successfully used as a diagnostic antigen for the detection of novel bovine tuberculosis.