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目的构建结核分枝杆菌PPE68基因的原核表达质粒,并在大肠杆菌中进行表达和纯化。方法以结核分枝杆菌H37Rv基因组DNA为模板,PCR法扩增PPE68基因,将其克隆至pET-32a(+)载体中,构建重组原核表达质粒pET-32a(+)-PPE68,转化至大肠杆菌BL21(DE3)中,IPTG诱导表达。表达产物经SDS-PAGE及Western blot鉴定后,进行纯化。结果重组表达质粒pET-32a(+)-PPE68经PCR及双酶切鉴定构建正确,测序结果与GenBank中登录的PPE68基因序列一致。表达的Trx-PPE68融合蛋白相对分子质量约为57000,表达量约占菌体总蛋白的41%,可与结核分枝杆菌免疫小鼠血清发生特异性反应。纯化的重组蛋白纯度约为93%。结论已成功构建了结核分枝杆菌PPE68基因重组原核表达质粒pET-32a(+)-PPE68,原核表达并纯化了重组蛋白,为PPE68作为结核病特异性诊断抗原的开发及重组BCG疫苗的研制奠定了基础。
Objective To construct prokaryotic expression plasmid of Mycobacterium tuberculosis PPE68 gene and express and purify it in Escherichia coli. Methods Mycobacterium tuberculosis H37Rv genomic DNA was used as a template to amplify PPE68 gene by PCR and cloned into pET-32a (+) vector to construct recombinant prokaryotic expression vector pET-32a (+) - PPE68. The recombinant plasmid was transformed into E.coli In BL21 (DE3), IPTG induces expression. The expressed product was identified by SDS-PAGE and Western blot, then purified. Results The recombinant plasmid pET-32a (+) - PPE68 was identified by PCR and double enzyme digestion. The sequencing result was consistent with the sequence of PPE68 gene in GenBank. The expressed Trx-PPE68 fusion protein has a relative molecular mass of about 57000 and an expression level of about 41% of the total bacterial protein, and can specifically react with the serum of the immunized mice of Mycobacterium tuberculosis. Purified recombinant protein purity of about 93%. Conclusion The recombinant prokaryotic expression plasmid pET-32a (+) - PPE68 of Mycobacterium tuberculosis PPE68 gene has been successfully constructed, and the prokaryotic expression and purification of the recombinant protein have been established. The development of PPE68 as a specific diagnostic antigen for tuberculosis and the development of recombinant BCG vaccine have been established basis.