隐孢子虫截短热休克蛋白70(HSP70)基因的原核表达及其抗原性分析  

Prokaryotic expression of truncated HSP70 of Cryptosporidium and antigenicity analysis of the recombinant protein

作　　者：彭昊[1,2,3] 唐林生[3] 李军[1,2] 陶立[1,2] 陈泽祥[1,2] 潘艳[1,2] 谢永平[1,2] 许力干[1,2] 杨威[1,2] 黄维义[3] 

机构地区：[1]广西兽医研究所,广西南宁530001 [2]广西畜禽疫苗新技术重点实验室,广西南宁530001 [3]广西大学动物科技学院,广西南宁530005

出　　处：《畜牧与兽医》2014年第12期5-9,共5页Animal Husbandry & Veterinary Medicine

基　　金：公益性行业(农业)专项(201103008);广西基本科研业务费(桂科专项12-2);广西水产畜牧兽医局科技项目(桂渔牧科1304525;桂渔牧财[2011]52号)

摘　　要：根据Gen Bank上的安氏隐孢子虫HSP70基因主要抗原片段区序列设计特异性引物,用RT-PCR方法扩增出目的片段,构建克隆载体,双酶切后连接到p ET-32a表达载体,成功构建了表达质粒p ET-32a-HSP70。将鉴定正确的重组质粒转化到表达菌中,IPTG诱导表达,表达产物经过SDS-PAGE和Western blot鉴定和分析,证实获得HSP70融合蛋白。以鼠抗安氏隐孢子虫卵囊高免血清为一抗进行Western blot检测,试验结果证实重组HSP70蛋白具有良好的免疫反应性,为建立隐孢子虫病免疫学诊断方法奠定了基础。Specific primers were designed based on the truncated gene encoding the antigenic domain of Cryptosporidium andersoni HSP70 from GenBank.The target fragment was amplified by RT-PCR and the cloning vector was constructed.Double enzyme digestion was performed,and then the product was subcloned into the prokaryotic expression vector pET-32a.The recombinant plasmid pET-32a-HSP70 were successfully constructed and transformed into Escherichia coli BL21 （DE3）.With the induction of IPTG,the recombinant protein was expressed.The analyses of SDS-PAGE and Western blot showed that HSP70 fusion protein was successfully expressed,and the recombinant protein showed good reactinogenicity using the positive serum against C.andersoni.The study provides a potential candidate antigen for development of immunodiagnostic method of cryptosporidiosis.

关 键 词：隐孢子虫 热休克蛋白70 表达 免疫原性 

分 类 号：S852.723[农业科学—基础兽医学]