日本血吸虫(中国大陆株)Cathepsin L基因的克隆、表达及其免疫保护功能的研究  

Cloning and expression of cathepsin L gene from Schistosoma japonicum (Chinese mainland strain) and its protective efficiency

作　　者：张慧[1] 苑纯秀[1] 冯新港[1] 傅志强[1] 刘金明[1] 蔡幼民[1] 林矫矫[1] 

机构地区：[1]中国农业科学院上海兽医研究所农业部动物寄生虫学重点实验室上海动物生物技术研究中心,上海200232

出　　处：《中国人兽共患病学报》2007年第1期11-15,18,共6页Chinese Journal of Zoonoses

基　　金：国家"863"计划资助项目(No.2004AA2Z3510);科技部科研院所社会公益研究专项项目(No.2004DIB4J158);上海市重大科技公关项目(No.03DZ19231)

摘　　要：目的开展日本血吸虫组织蛋白酶L(SjCL)功能研究,为研发血吸虫病抗病疫苗提供基础。方法应用RT-PCR技术分离、克隆日本血吸虫中国大陆株组织蛋白酶L保守功能域编码基因,并在大肠杆菌系统中表达,应用免疫亲和层析法纯化组织蛋白酶L重组抗原,用该基因纯化重组表达产物进行小鼠免疫保护实验。结果获得了672 bp的血吸虫组织蛋白酶L保守功能域cDNA序列,成功构建了原核表达载体pET28a(+)-SjCL,并在大肠杆菌中进行了表达,经免疫亲和层析获得了高纯度的SjCL融合蛋白,以该纯化物免疫小鼠,结果实验组减虫率为36.04%,肝组织减卵率为34%,粪卵减少率达49%,与对照组进行方差分析,差异均显著。结论获得日本血吸虫组织蛋白酶L保守功能域cDNA克隆,其原核表达产物免疫小鼠对抗血吸虫感染具有一定的保护性。For further studies on the cathepsin L gene from Schistosoma japonicum in order to provide the basis for vaccine development against schistosomiasis, the coding gene in the conserved domain of cathepsin L gene was isolated and cloned into the high level expression vector pET28a（＋） by RT-PCR, thus obtaining a recombinant plasmid pET28a（＋）-SjCL. The recombinant fusion protein was expressed through induction with IPTG, and then analyzed by SDS-PAGE and Western blotting. Mice were immunized 4 times with 50μg of purified recombinant cathepsin L protein plus adjuvant or adjuvant alone, and then challenged with 46 cercariae after immunization. Adult worms as well as the eggs from liver tissues of mice were counted 42 days later. From the fact that the size of the amplified cathepsin L gene was in accordance with the expected one, it indicates that the recombinant plasmid pET28a（＋）-SjCL was successfully constructed. It was also demonstrated the recombinant cathepsin L protein was expressed in E. coli. This recombinant protein had a molecular weight of approximate 31 kDa and could be recognized specifically by rabbit antiserum against Schlstosoma adult worm antigen preparation（SWAP）, showing that the expressed product possessed good antigenicity. Compared with the control group, the rates of adult worm burdens, EPG in livers and excrement reduced to 36.04%, 34.0% and 39.0% respectively, when challenged with 40 carcariae. These results suggest that cathepsin L might be considered as a suitable candidate of vaccine development against schistosomiasis.

关 键 词：日本血吸虫 组织蛋白酶L 基因克隆表达 免疫 

分 类 号：R383.2[医药卫生—医学寄生虫学]