马尔尼菲青霉菌溶血磷脂酶基因的克隆、表达及纯化  

Molecular cloning, expression and purification of the gene encoding lysophospholipase from Penicillium marneffi

作　　者：李凌华[1] 胡凤玉[1] 陈万山[1] 宋伟南[1] 蔡卫平[1] 唐小平[1] 

机构地区：[1]广州市第八人民医院,广州510060

出　　处：《中华实验和临床感染病杂志（电子版）》2012年第2期18-21,共4页Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition)

基　　金：广州市卫生局重点科研项目(2009-Zdi-016);广东省自然基金(10151006002000000)

摘　　要：目的研究马尔尼菲青霉菌(PM)溶血磷脂酶(LysoPLs)基因的克隆、表达和纯化,为研究其致病机制奠定基础。方法采用生物信息学方法,从PM酵母相全长cDNA文库中识别出PMLysoPLs基因的同源序列及其全长编码区。通过PCR方法扩增PMLysoPLs基因的编码区序列,构建原核表达载体pET30a(+)-PMLysoPLs,经DNA序列测定鉴定其序列,在大肠埃希菌BL-21/DE3中用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,重组产物采用His-镍蛋白纯化柱进行纯化。结果 PMLysoPLs基因长度为991bp,其全长编码序列长度为732bp,编码243个氨基酸,其编码蛋白理论分子量为26.8kDa。重组表达载体经序列测定及酶切鉴定与理论推测结果相符。经IPTG诱导,该基因在大肠埃希菌BL-21/DE3中得到高效的可溶性上清表达,纯化后的重组蛋白分子量为25～35kDa,其单一蛋白纯度达95%以上。结论本研究成功构建了PMLysoPLs基因的pET30a(+)原核重组质粒,获得的可溶性重组蛋白表达效率高,可用于进一步研究PM溶血磷脂酶的功能。Objective To clone, express and purify the gene encoding lysophospholipase of penicillium marneffei （PMLysoPLs） for establishing a solid foundation and to investigate its functions in pathogenesis. Methods The homologous sequence and full-length coding region of PMLysoPLs gene were identified from the full-length cDNA library of P. marneffei in yeast phase by bioinformatics tools. Then, the coding region sequence of PMLysoPLs gene was amplified to construct the prokaryotic expression plasmid of pET 30a （＋）-PMLysoPLs. The PCR products were verified by sequencing and expressed in E.coli BL-21/DE3 induced by isopropy-β-D-thiogalactoside （IPTG）. The recombinant products were purified by Nickel-affinity chromatography column. Results The gene was 991 bp and its full-length gene encoding lysophospholipase was presumed to be 732 bp encoding 243 amino acids with theoretical molecular weight being 26.8 kDa. The recombinant expression vector was proved to be concordant with the theoretical presumption by sequencing and endonuclease cleavage. Induced by IPTG, this gene was highly and efficiently expressed in lysate supernatants in E.coli BL-21/DE3. The purity of the purified recombinant protein was over 95%. Conclusions The prokaryotic recombinant plasmid pET 30a （＋）-PMLysoPLs was successfully established. Moreover, the dissoluble recombinant protein was highly expressed and appropriate for further study on the function of PMLysoPLs.

关 键 词：马尔尼菲青霉菌 溶血磷脂酶 克隆 原核表达 

分 类 号：R440[医药卫生—诊断学]