脓肿分枝杆菌非溶血性磷脂酶C的原核表达及活性鉴定

Prokaryotic expression and activity of non-hemolytic phospholipase C of Mycobacterium abscess

目的原核表达脓肿分枝杆菌非溶血性磷脂酶C,并检测其生物学活性。方法以脓肿分枝杆菌标准株(ATCC19977)基因组DNA为模板,PCR扩增非溶血性磷脂酶C基因,克隆至pQE-30载体,构建重组原核表达质粒pQE-30-MAB_0555,转化大肠杆菌DH5α,IPTG诱导表达。表达的重组蛋白经Ni2+-NTA层析纯化后,采用杯碟法检测其生物学活性。结果重组原核表达质粒pQE-30-MAB_0555经PCR、双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约为53 000,纯化后蛋白浓度为0.40 mg/ml,具有溶解蛋黄中卵磷脂的酶活性,无溶血活性。结论成功地在大肠杆菌DH5α中表达了非溶血性磷脂酶C,为进一步研究其生物学功能及其在脓肿分枝杆菌致病机制中的作用提供了材料,也为研制脓肿分枝杆菌临床治疗药物及寻找新的药物作用靶点提供了思路。

Objective To express the non-hemolytic phospholipase C（PLC） of Mycobacterium abscess in prokaryotic cells and determine its biological activity.Methods Non-hemolytic PLC gene was amplified by PCR using the genomic DNA of standard M.abscess strain（ATCC19977） as a template,and cloned into vector pQE-30.The constructed recombinant plasmid pQE-30-MAB0555 was transformed to E.coli DH5α for expression under induction of IPTG.The expressed recombinant protein was purified by Ni2＋-NAT chromatography and determined for biological activity by cylinder plate method.Results PCR,restriction analysis and sequencing proved that the recombinant plasmid pQE-30-MAB0555 was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 53 000,reached a concentration of 0.40 mg / ml after purification,and showed activity of dissolution of lecithin in yolk while showed no hemolytic activity.Conclusion Non-hemolytic PLC was successfully expressed in E.coli DH5α,which provided a material for further study on its biological function and its role in pathogenic mechanism of M.abscess,and a route for development of novel drugs for clinical therapy of M.abscess infection.