日本血吸虫编码抱雌沟蛋白保守区cDNA片段的克隆表达及其免疫保护功能  

Clone Expression and Protective Immunity Evaluating of the Conservative Region with cDNA Encoding Gynecophoral Canal Protein of Schistosoma japonicum Chinese Strain

作　　者：朱建国[1] 林矫矫[1] 冯新港[1] 陈永军[1] 吴祥甫[2] 周元聪[2] 蔡幼民[1] 

机构地区：[1]中国农业科学院上海家畜寄生虫病研究所农业部动物寄生虫学重点开放实验室,上海200232 [2]中国科学院上海生物化学研究所,上海200031

出　　处：《中国预防兽医学报》2001年第5期348-353,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家高技术"86 3"基金 (N .10 2_0 7_0 4_0 3)资助项目。

摘　　要：本研究根据曼氏血吸虫抱雌沟蛋白 (Gynecophoralcanalprotein)基因SmGCP保安区片段设计一对引物 ,以日本血吸虫中国大陆株成虫mRNA为模板 ,用TR_PCR法扩增出一大小为 86 8bp的基因片段。测序后分析序列推断该片段与SmGCP相应片段碱基同源性 86 .6 % ,说明所克隆的基因为编码日本血吸虫中国大陆株抱雌沟蛋白基因 ,推断的氨基酸序列同源性为84.1%。将其克隆到表达载体pET2 8(a)中 ,在大肠杆菌BL2 1中获得了较高量的融合表达 ,表达产物分子量约 2 9kD。利用日本血吸虫成虫粗抗原免疫大白兔所得抗血清对该表达产物进行Westernblot检测 ,在预测位置出现了明显的识别条带 ,显示了该表达产物良好的抗原性。将该表达产物用佐剂处理免疫昆明系小鼠 ,攻击血吸虫尾蚴 ,7周后剖杀小鼠冲虫 ,进行成虫和虫卵计数 ,其减虫率为 35 .2 % ,减卵率为 37.8% ,与对照组比较差异显著 ,初步证明血吸虫抱雌沟蛋白具有一定的免疫保护功能。A 868 bp cDNA fragment was amplified by RT_PCR from adult Schistosoma japonicum (Chinese strain)mRNA with primer designed according to published SmGCP encoding Gynecophoral canal protein.Sequence analysis indicated that this fragment, named SjGCP1,with86.6%homology to SmGCP,was a part of gene encoding Gynecophoral canal protein of Schistosoma japonicum(Chinese strain).This fragment was cloned into the expression vector pET28a(+)and subsequently expressed in Escerichia coli.SDS_PAGE analysis revealed that the molecular weight of this expressed product was 29kD.Western Blot showed that the recombinant protein can be recognized by the rabbit serum immunized with Sj worm antigen,indicating that this expressed product had good antigenicity.In order to observe the protective immunity induced by Vaccination in animals with the recombinant protein of SjGCP1,Kuming mice were vaccinated 3 times with rSjGCP1 and challenged with 40 cercariae of Sj.The mice were perfused 7 weeks post_challenge infection.The reduction percentage of worm and tissue egg were count in both vaccinated and control group of mice,getting the 35.2% worm reduction rate and 37.8% egg reduction rate and indicating the significantly protection in animal of rSjGCP1.

关 键 词：日本血吸虫 抱雌沟蛋白 保守区编码基因 基因克隆 表达 免疫保护功能 

分 类 号：S852.735[农业科学—基础兽医学]