新疆农业科学2013，Vol.50Issue(5)：949-958,10.DOI:10.6048/j.issn.1001-4330.2013.05.025
细粒棘球绦虫表膜糖抗原HSP70基因的克隆、表达及免疫特性分析
Cloning, Expressing and Analyzing of HSP70 from Tegument Surface Glycoantigen of Echinococcus granulosus
摘要
Abstract
[Objective] To clone heat shock protein 70 gene (HSP70) from tegument surface glycoantigen of Echinococcus granulosus (Eg) for the construction of a prokaryotie expression vector.The immune characteristics of EgHSP70 were identified.[Methods] Eg adult cDNA library was screened by sera from glycoantigen immunized in mice,and positive clones EgHSP70 were obtained.EgHSP70 gene was amplified by PCR and cloned into prokaryotic expression vector pET28a,EgHSP7O protein were induced,expressed and purified.Then,the Balb/c mice were immunized with purified EgHSP70 protein via hypodermic for anti-serum,which was collected to measure immunoglobulin (IgG) and serum titer by ELISA,to determine specificity by Western-blot.To localize site of the antigen composition in the Eg adult worm by IFA.[Results] Full-length of EgHSP70 gene was obtained from Eg adult cDNA.restriction endonuclease analysis and DNA sequencing showed that EgHSP70 was cloned into the plasmid pET28a.Based on the optimization experiments,it was concluded that the best soluble expression conditions for the EgHSP70 protein are using 0.4 mmol/L IPTG when bacterial cells grew to OD600 =0.6 and induced for 5 h at 37℃.THE SDS-PAGE showed encoding a protein mass of 35 Ku.ELISA showed that the titers of the anti-serum were above 1:640,000,and Western blot's analytical results showed that the anti-serum could specifically be bound with EgHSP70 protein and native proteins.IFA proved that the EgHSP70 was located in the surface membrane of Eg adult worm.[Conclusion] The EgHSP70 fusion protein has good immunogenicity and reactivity.The protein is immunogenic and can be a vaccine candidate against Echinococcus infection and diagnosis material.关键词
细粒棘球绦虫成虫cDNA文库/细粒棘球绦虫表膜糖抗原/热休克蛋白HSP70/EgHSP70融合蛋白/免疫学特性Key words
Echinococcus granulosus adult cDNA/ Echinococcus granulosus tegument surface glycoantigen/ heat shock protein 70/ EgHSP70 fusion protein/ the immune characteristics分类
农业科技引用本文复制引用
古努尔·吐尔逊,米晓云,赵莉,张旭,张壮志,石保新,吐尔洪·依米提,艾力麦麦提·麦麦提力,马合木提·塔力普..细粒棘球绦虫表膜糖抗原HSP70基因的克隆、表达及免疫特性分析[J].新疆农业科学,2013,50(5):949-958,10.基金项目
新疆少数民族科技人才特殊培养计划科研项目(201023108) （201023108）
