摘要
利用反转录聚合酶链式反应(RT-PCR),从猪囊尾蚴虫体中扩增出小热休克蛋白(small Heat shock protein,sHSP)基因,克隆至原核表达载体pGEX-4T-1,在大肠杆菌BL21中以GST融合蛋白的形式表达。SDS-PAGE分析证明,表达产物为64Ku的融合蛋白,经凝胶扫描分析,表达量约占菌体可溶性蛋白的30%。免疫学分析(Westernblot,ELISA)结果表明,sHSP能与猪囊尾蚴血清反应。将表达产物点电泳后切胶回收免疫小鼠,进行Westernblot和ELISA检测,其抗血清能与猪囊虫粗抗原及纯化的sHSP重组融合蛋白产物发生免疫学反应。该研究为丰富新的诊断试剂和免疫用重组抗原具有重要意义,同时,预测糖基化、磷酸化作用在sHSP实现生物学功能方面也具有广泛的意义。
A gene encoding small heat-shock protein （sHSP） from Cysticereus cellulosae was amplified by reverse transcript polymerase chain reaction（RT-PCR） technique. The PCR product was cloned into the expressing plasmid vector pGEX-4T-1,it was expressed in E.coli as a fusion protein with glutathione S-transferase protein,and was indentified by SDS-PAGE,and a specific protein band pf 64 000 was found,amounting to 30 % of the total proteins of the induced recombinant bacteria at the presence of IPTG. Western blot and ELISA showed good immunoreactiviW of the expressed product. The specific band of expression was excised from the gel and used to immunize mice. The antisera were collected from the immunized mice and examined by ELISA and Western blot. The results showed that immunization of mice generated antibodies and the antibodies specifically reacted with recombinant sHSP. It was concluded from these results that the expressed protein of sHSP gene as a filsion protein has antigenicity. From the results of predication sHSP of Cysticercus cellulosae may be a glycol-protein. In the processing of sHSP,the phosphorylated lnodification may be important.These results suggest that its function may be complicated,related with the regulation of gene expression and maintance of the celhllar cytoskeleton and so on.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2006年第2期167-171,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国家重大基础研究发展规划(973)项目
关键词
猪囊尾蚴
小热休克蛋白
免疫原性
Cysticercus celhllosae
small heat shock protein
immunogenicity
