耐甲氧西林金黄色葡萄球菌ldh-1基因的序列分析及原核表达

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BamHI、Xhol I双酶切鉴定及测序鉴定，并与做相应酶切的pET一28a（＋）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1PTG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—ldhl，基因测序结果显示，扩增的ldhl基因全长956bp，与金黄色葡萄球菌Mu50株ldhl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，Westernblot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0．5g／L的重组蛋白。结论在MRSA（菌株号：1758）1~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。

Sequence analysis and prokaryotic expression of ldh-1 gene from MRSA

Objective To clone and prokaryotic express Staphylococcus aureus ldh-1 gene from methicillin-resistant Staphylococcus aureus （MRSA） clinical isolated, and make foundation for L-LDH biological function research. Methods A pair of specific primers was designed according to Staphylococcus aureus Newman strain in the GenBank. Using MRSA clinical isolated DNA as template, ldh-1 gene was PCR amplified by specific primers, and the PCR product was digested with BamH I and Xhol I,was and ligated with BamH I and XhoI cut pET-28a （＋）,and then transformed into E.coli BL21. Recombinant plasmid was identified by double enzyme digestion and sequence analysis.The sequence results were compared with gene sequence in the GenBank. And the expression of recombinant fusion protein was induced by IPTG, and identified by western blotting probed with HRP conjugated anti his-tag mouse monoclonal antibody. After induction, the bacteria cells were collected, sonicated and centrifugation. The fusion protein was purified by using Ni2~-NTA affinity column. Results Recombinant pET-28a-ldh-1 plasmid was constructed. Sequence analysis showed that ldh-1 contained an open reading frame of 956 bp. Sequence comparison analysis showed that DNA and amino acid sequences were identical to ldh-1 gene of Staphylococcus aureus MuS0 strain. After induced by IPTG, a recombinant protein which is about 39×10^3 Mr was indentified by SDS-PAGE and western blot. The protein was expressed in soluble form with a yield of 0.5 g/L culture after purification. Conclusion ldh-1 of MRSA was successfully cloned and prokaryotic expressed, which laid a good foundation for further study on L-LDH.