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

摘要

[目的]克隆细粒棘球绦虫(Eg)表膜糖抗原的热休克蛋白70 (HSP70)基因,构建原核表达载体,鉴定EgHSP70蛋白的免疫学特性.[方法]以Eg表膜糖抗原高免鼠血清为探针,从Eg成虫cDNA文库中筛选得到阳性克隆EgHSP70.构建EgHSP70基因的原核表达载体pET28a-EgHSP70,诱导表达重组蛋白,纯化,免疫鼠,ELISA、Western-blot和IFA法分别检测血清抗体效价、特异性及抗原在虫体组织中的位置.[结果]从Eg成虫cDNA文库中筛选获得特异性EgHSP70基因,酶切和测序结果均表明该基因已克隆到pET28a.EgHSP70融合蛋白在A600=0.6,0.4 mmol IPTG,37℃表达5h为最佳表达.经His亲和层析法纯化获得了HSP70融合蛋白相对分子质量约为33 kDa.免疫小鼠可产生高滴度(最高1∶640000)特异性抗体.Westernblot分析结果表明,制备的抗血清与EgHSP70融合蛋白、原头蚴粗提抗原、Eg虫体蛋白和Eg虫体表面蛋白均能特异性结合,其他带科绦虫有此蛋白表达IFA检测结果证实EgHSP70在细粒棘球绦虫成虫体表膜及体表内有表达.[结论]实验得到的EgHSP70融合蛋白具有较好的免疫原性和抗原性,可用于研制包虫病疫苗及相关诊断试剂为包虫病的防控提供技术支持.%[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.