鞭虫乳清酸蛋白TtWAP-3的原核表达、纯化及其活性研究  

Prokaryotic expression and purification of the TtWAP-3,a whey acidic protein fromTrichuris trichiura and analysis of its biological activities

作　　者：李少金 邵正 司徒永立 彭礼飞 邓莉 

机构地区：[1]广东医科大学寄生虫学教研室,广东湛江524023

出　　处：《中国病原生物学杂志》2016年第7期653-656,660,共5页Journal of Pathogen Biology

基　　金：广东省高等学校科技创新项目(No.2013KJCX0087);广东医学院科技创新团队项目(No.STIF201107)

摘　　要：目的克隆、原核表达人鞭虫(Trichuris trichiura)乳清酸蛋白TtWAP-3,分析其生物学活性。方法分别以鞭虫成虫前端及尾端cDNA为模板,通过PCR扩增TtWAP-3编码基因;将TtWAP-3编码基因克隆至原核表达载体pET32a-sumo,构建重组表达质粒转化至大肠埃希菌BL21(DE3),IPTG诱导表达目的蛋白并进行Ni-NTA亲和纯化和SUMO酶酶切,用发色底物法检测rTtWAP-3对蛋白酶的抑制活性。结果在成虫前端及尾端均成功克隆获得TtWAP-3编码基因序列,构建的原核表达重组质粒pET32a-sumo/TtWAP-3在大肠埃希菌表达rTtWAP-3,该蛋白对人中性粒细胞弹性蛋白酶(NE)和人蛋白酶3(PR3)有抑制活性,其抑制人NE(20nmol/L)、人PR3(200nmol/L)50%活性的浓度分别约为2.25μmol/L和5.34μmol/L。结论成功获得原核表达的重组TtWAP-3,该蛋白对人NE及人PR3均有抑制作用,为研究TtWAP-3对宿主的免疫调节作用奠定了基础。Objectives To express and purify whey acid protein 3fromTrichuris trichiura（TtWAP-3）,and to analyze its biological activities. Methods The sequence encoding mature TtWAP-3from the anterior region and posterior region of adult Trichuris trichiura cDNA was amplified with PCR and inserted into the vector pET32a-sumo to construct the recombinant plasmid pET32a-sumo/TtWAP-3.Expression of recombinant（r）TtWAP-3in E.coli BL21（DE3）was induced with IPTG.The recombinant was purified with Ni-NTA affinity chromatography and digested with SUMO protease.The biological activities of rTtWAP-3were analyzed using chromogenic assays. Results The TtWAP-3gene was amplified from the anterior region and posterior region of adult Trichuris trichiuracDNA,and rTtWAP-3was obtained in E.coli.The purified recombinant protein inhibited human neutrophil elastase（HNE）and human proteinase 3（HPR3）.rTtWAP-3inhibited HNE（20nmol/L）activity by 50%at a concentration of about 2.25μmol/L and it inhibited HPR3（200nmol/L）activity by 50%at a concentration of about 5.34μmol/L. Conclusions rTtWAP-3,which was successfully obtained in a prokaryotic expression system,exhibited proteinase inhibitory activity against HNE and HPR3.This study has laid the foundation for investigation of the modulation of host immune function by TtWAP-3.

关 键 词：鞭虫 乳清酸蛋白 原核表达 中性粒细胞弹性蛋白酶 蛋白酶3 

分 类 号：R383.14[医药卫生—医学寄生虫学]