结核分枝杆菌优势蛋白PPE37重组载体的构建及原核表达

目的构建结核分枝杆菌ppe37基因的重组原核表达质粒,并将其转化到E.coli BL21中诱导表达。方法以结核分枝杆菌国际标准株H37Rv基因组DNA为模板,PCR法扩增ppe37基因,将其克隆至pEasyE2克隆载体中,构建pEasyE2-ppe37重组质粒。经双酶切后将ppe37基因片段亚克隆到pET30a载体中,构建重组原核表达质粒pET30a-ppe37,并转化到大肠杆菌BL21中,经IPTG诱导表达。利用SDS-PAGE及WesternBlot对表达产物进行鉴定。结果 PCR法扩增出约1600bp的条带。重组质粒pEasyE2-ppe37经PCR及双酶切鉴定构建正确,测序结果与GenBank中登录的PPE37蛋白基因序列一致。重组原核表达质粒pET30a-ppe37经PCR及双酶切鉴定构建正确。SDS-PAGE鉴定表达的组氨酸标签重组蛋白相对分子质量约为50KDa,Western blot验证重组蛋白可与抗组氨酸单抗发生特异性反应。结论成功构建结核分枝杆菌ppe37基因重组原核表达质粒pET30a-ppe37,并成功诱导表达,为PPE37蛋白作为结核病特异性诊断抗原的开发奠定了基础。

The Construction and Prokaryotic Expression of Mycobacterium Tuberculosis Dominant Protein PPE37 Recombinant Vector

Objective To construct a prokaryotic expression vector for the Mycobacterium tuberculosis ppe37 gene and to express the gene in E.coli BL21.Methods Mycobacterium tuberculosis ppe37 gene was amplified by PCR from Mycobacterium tuberculosis genome.The PCR product was cloned into the pEasyE2 vector.Ppe37 gene fragment after double enzyme digestion was cloned into pET30a.pET30a-ppe37 was transformed into E.coli BL21 through IPTG induction to express the target protein and the protein was analyzed by SDS PAGE and Western blot.Results PCR amplified bands of about 1600bp.pET30a-ppe37 was identified right by restriction endonuclease digestion analysis.DNA sequencing proved the sequence according to the GenBank.SDS-PAGE showed that the recombinant protein with relative molecular mass was 50 kD.SDS PAGE and Western blot analysis showed that a protein with molecular weight of5000 was expressed in E.coli BL21.Conclusion The extracellular fragment of ppe37 is successfully cloned into pET30a and expressed in E.coli BL21,which lays a foundation for the further functional research of PPE37.