柱状黄杆菌胶原蛋白酶的克隆与表达  

Clone and expression of collagenase gene in Flavobacterium columnare

作　　者：张立强[1,2] 谢海侠[1] 李楠[1] 聂品[1] 

机构地区：[1]淡水生态与生物技术国家重点实验室,中国科学院水生生物研究所,湖北武汉430072 [2]中国科学院研究生院,北京100039

出　　处：《中国水产科学》2009年第5期786-790,共5页Journal of Fishery Sciences of China

基　　金：国家重点基础研究发展计划项目资助(2009CB118703)

摘　　要：利用PCR方法从柱状黄杆菌(Flavobacterium columnare)G4株中克隆了1个胶原蛋白酶基因(GenBank登录号EF501979)。同源性比对发现该基因与柱状黄杆菌的另外1种胶原酶基因(GenBank登录号EAZ95511.1)最为相近,有84%的一致性和93%的相似性。该属的另外2种细菌,约氏黄杆菌(Flavobacterium johnsoniae)和嗜冷黄杆菌(F.psychrophilum)都有与该胶原酶基因相似的基因,其相似性分别为92%和83%。该基因经KpnI和SalI酶切后连接到表达载体pET-32a上,转入大肠杆菌BL21(DE3)plysS内进行表达,经SDS-PAGE电泳后显示重组融合蛋白在44kD处有明显表达带,与预期分子量大小一致,且主要以不溶的包涵体形式存在,变性条件下利用His·Bind树脂成功纯化了融合蛋白,将其免疫家兔,获得兔抗柱状黄杆菌胶原蛋白酶抗体,Western blotting表明该胶原酶在柱状黄杆菌的胞内和胞外都存在。这些结果为进一步研究这种胶原蛋白酶的功能及柱状黄杆菌的致病机理奠定了基础。Collagenase gene of Flavobacterium columnare was cloned using genome walking PCR method. The sequence analysis revealed that it had 87% identify and 93% similarity with another collagenase（ gb｜EAZ95511.1｜） also in F. columnare. This gene has 92% and 83% similarity with its counterparts in F. johnsoniae and F. psychrophilum,respectively. In the present study,collagenase was ligated to prokaryotic expressing plasmid pET-32α. Then the recombinant plasmid pET-32α-coll was transformed into E. coli BL21（DE3） plysS under the induction of IPTG. SDS-PAGE demonstrated that a recombinant 45 kD protein was expressed as an insoluble protein. The protein was further purified by His Bind column. Polyclonal antibody was then developed against the recombinant protein, and western blotting analysis revealed that the collagenase was present in both broth supernatant and cell body indicating it is a secretory protein. [Journal of Fishery Sciences of China,2009,16 （5）： 786-790]

关 键 词：柱状黄杆菌 胶原蛋白酶 克隆 表达 

分 类 号：S94[农业科学—水产养殖]