猪链球菌二氢硫辛酰胺脱氢酶的原核表达及其粘附相关功能初探  

Prokaryotic Expression and Adhesion Detection of Putative Dihydrolipoamide Dehydrogenase of Streptococcus suis

作　　者：张静[1,2] 孙建和[1,2] 

机构地区：[1]上海交通大学农业与生物学院 [2]上海市兽医生物技术重点实验室,上海200240

出　　处：《上海交通大学学报（农业科学版）》2012年第2期34-40,共7页Journal of Shanghai Jiaotong University(Agricultural Science)

基　　金：国家自然科学基金(31172381);家畜疫病病原生物学国家重点实验室开放基金(SKLVEB2010KFKT004)

摘　　要：目的:克隆、表达猪链球菌2型菌株ZY05719的膜蛋白二氢硫辛酰胺脱氢酶基因(hm6),分析其重组蛋白的生物学活性。方法:基于GenBank中S.suis P1/7SSU1634蛋白的基因序列设计引物,克隆ZY05719株的二氢硫辛酰胺脱氢酶基因并进行序列分析;构建pET28a-hm6原核表达质粒,在大肠杆菌BL21中诱导表达,重组HM6蛋白即二氢硫辛酰胺脱氢酶用His亲和层析镍柱纯化后,通过SDS蛋白电泳鉴定分析并利用新西兰大白兔制备抗血清,研究其酶活性及其在介导猪链球菌粘附HEp-2细胞中的作用。结果:克隆了1 761bp的二氢硫辛酰胺脱氢酶基因,纯化的70kD重组融合蛋白具有良好的酶活性和免疫原性,且在介导猪链球菌粘附HEp-2细胞中具有作用。结论:成功克隆、表达了ZY05719株的二氢硫辛酰胺脱氢酶基因,初步揭示了其生物学活性,为深入研究二氢硫辛酰胺脱氢酶在猪链球菌致病机制中的作用奠定了基础。To clone and express the putative dihydrolipoamide dehydrogenase（hm6） gene of Streptococcus suis type 2 strain ZY05719 isolated from Ziyang,Sichuan province,for the detection of its function on bacterial adherence,the hm6 gene fragment was amplified by PCR from ZY05719 and cloned into prokaryotic expression plasmid pET-28a to form pET28a-hm6.The expression of recombinant protein was induced in E.coli BL21 and was purified by Ni2＋-nitrilotriacetic acid（Ni-NTA） affinity chromatography.The expressed and purified proteins were analyzed by SDS-PAGE and its antiserum was raised from immunized rabbits.The effect of purified recombinant protein and its antiserum on adhesion of SS2 to HEp-2 cell was assayed.The results showed that the length of the hm6 gene was 1 761 bp.The prokaryotic expressed product was a fusion protein,whose molecular weight was about 70 kD and could keep down adhesion of SS2 to HEp-2 cell.The hm6 gene of ZY05719 has been successfully cloned and highly expressed in E.coli BL21,the recombinant protein facilitates the further studies on the bacterial adhesion,and even the pathogenesis of Streptococcus suis.

关 键 词：猪链球菌 二氢硫辛酰胺脱氢酶 酶活性 粘附特性 

分 类 号：S852.61[农业科学—基础兽医学]