A型产气荚膜梭菌α毒素基因的克隆及高效表达  

Cloning and expression of alpha toxin gene from Clostridium perfringens type A in E.coli

作　　者：王景林[1] 王利春[2] 吴东林[3] 康琳[1] 姜永强[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,北京100071 [2]内蒙古大学生物工程中心,呼和浩特010020 [3]解放军军需大学军事兽医研究所,长春130062

出　　处：《军事医学科学院院刊》2004年第1期28-30,共3页Bulletin of the Academy of Military Medical Sciences

摘　　要：目的 :实现产气荚膜梭菌α毒素 (Clostridiumperfringensalphatoxin ,CPa)基因的克隆与表达。方法 :用PCR技术从A型产气荚膜梭菌中扩增出CPa基因 ,克隆到原核表达载体pQE30中 ,构建了重组质粒pQE30_CPa ,转化E .coliM15获得了CPa的高效表达。结果 :序列测定和双酶切表明 ,CPa基因正确插入载体。序列分析发现CPa基因与GenBank中的 4种CPa基因相似率为 76 .7%～ 99.9%。工程菌裂解液经SDS_PAGE分析显示在相对分子质量 4 30 0 0处有一条表达很强的蛋白区带 ,与CPa相对分子质量相符 ,约占菌体总蛋白的 6 2 .88% ,主要以包涵体形式存在。免疫印迹结果表明 ,表达的重组CPa蛋白同抗CPa血清有特异性结合。结论 :为进一步研究CPa重组疫苗和制备CPa的特异性抗体奠定了基础。Objective:To clone and express the gene of α-toxin from Clostridium perfringens(CPa).Methods:CPa gene was amplified from C.perfringens type A strain by PCR,then cloned into the expression vector pQE30 and expressed in E.coli M15.Results:CPa gene was correctly amplified and inserted into the vectors as confirmed by sequencing and digestion with restriction enzymes. The sequences analysis showed that cloned CPa gene was homologous between 76.7% and 99.9% to other four reported CPa genes. A strong expression band with moleclar weight of 43?000 was detected by SDS-PAGE, and the target protein accounted for 62.88% of total cell protein and was mainly in inclusion form. Western blot analysis revealed that antiserum against CPa had a specific affinity for the expressed protein. Conclusion:The α-toxin from C.perfringens is expressed in E.coli and it could be obtained in large quantity, which lays the foundation for the further study on recombinant vaccine and preparation of anti-CPa monoclonal antibody.

关 键 词：A型产气荚膜梭茵 Α毒素 基因表达 序列分析 

分 类 号：R346[医药卫生—基础医学]