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目的对耐甲氧西林金黄色葡萄球菌(MRSA)临床分离株携带的ldh-1基因进行克隆、表达,为L-乳酸脱氢酶(L-LDH)功能研究奠定基础。方法根据金黄色葡萄球菌Newman菌株的基因序列,设计一对特异性引物,以MRSA临床分离株(菌株号:1758)DNA为模板PCR扩增ldh-1,纯化DNA进行BamH I、Xhol I双酶切鉴定及测序鉴定,并与做相应酶切的pET-28a(+)连接,转化大肠杆菌BL21,对质粒进行双酶切鉴定及基因序列分析,用IPTG诱导融合蛋白的表达,His标签单克隆抗体进行免疫印迹验证融合蛋白的表达。诱导表达后的重组菌经超声破碎离心后,使用Ni2+-NTA亲和层析柱纯化目的蛋白。结果成功构建重组表达质粒pET-28a-ldh1,基因测序结果显示,扩增的ldh1基因全长956 bp,与金黄色葡萄球菌Mu50株ldh1基因的序列同源性为100%。IPTG诱导表达后,聚丙烯酰胺凝胶电泳在39×103Mr处见目的条带,Western blot验证了重组蛋白L-LDH的表达。该蛋白呈可溶性表达,纯化后获得0.5 g/L的重组蛋白。结论在MRSA(菌株号:1758)临床分离株中成功克隆及表达了ldh-1基因,获得了纯化的重组蛋白,为进一步进行L-LDH的功能研究奠定了基础。
Objective To clone and express ldh-1 gene of methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates and lay a foundation for the study of L-lactate dehydrogenase (L-LDH) function. Methods According to the gene sequence of Staphylococcus aureus Newman strain, a pair of specific primers was designed. PCR was used to amplify ldh-1 with DNA of MRSA clinical isolates (strain No. 1758) as template. The purified DNA was subjected to BamH I, Xhol I double enzyme The recombinant plasmid pET-28a (+) was ligated with pET-28a (+). The recombinant plasmid was transformed into E. coli BL21. The plasmid was identified by double enzyme digestion and gene sequence analysis. The fusion protein was induced by IPTG. Antibodies were immunoblotted to verify the expression of the fusion protein. Recombinant bacteria after induced expression were centrifuged and centrifuged, and the target protein was purified by Ni2 + -NTA affinity chromatography. Results The recombinant plasmid pET-28a-ldh1 was successfully constructed. The sequencing results showed that the amplified ldh1 gene was 956 bp in length and 100% identical to the ldh1 gene of Staphylococcus aureus Mu50 strain. IPTG induced expression, polyacrylamide gel electrophoresis at 39 × 103Mr see the purpose of the band, Western blot confirmed the recombinant protein L-LDH expression. The protein was expressed in soluble form and purified to obtain 0.5 g / L recombinant protein. Conclusions The ldh-1 gene was successfully cloned and expressed in MRSA (strain no. 1758) and the purified recombinant protein was obtained, which laid the foundation for the further study on the function of L-LDH.