亚洲带绦虫六钩蚴Ta8基因的克隆表达及免疫原性分析  

Cloning , expression and immunogenicity analysis of Ta 8 gene of Taenia asiatica oncospheres

作　　者：王宇[1,2] 程金芝[1] 黄江[1] 戴佳琳[1] 陈小睿 廖兴江[1] 

机构地区：[1]贵阳医学院寄生虫学教研室,贵阳550004 [2]贵州省疾病预防控制中心,贵阳550004

出　　处：《中国人兽共患病学报》2012年第11期1102-1106,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(No.30760227);贵州省科技攻关项目[黔科合NY字(2008)3060]联合资助~~

摘　　要：目的原核克隆表达亚洲带绦虫六钩蚴8kDa基因(Ta8),探索其表达蛋白在检测亚洲带绦虫感染中的应用价值。方法用RT-PCR方法从亚洲带绦虫六钩蚴cDNA中获取Ta8基因,克隆到原核表达质粒pET-28a(+)中,在大肠埃希菌BL21/DE3中用IPTG诱导表达,表达产物通过SDS-PAGE进行鉴定,用镍离子金属螯合剂亲和层析柱进行纯化,纯化的重组蛋白用蛋白印迹进行免疫学分析。结果 PCR、双酶切及DNA测序结果均表明pET-28a(+)-Ta8重组质粒构建成功。SDS-PAGE结果表明目的基因在大肠埃希菌BL21/DE3中获得高效表达,亲和层析获得了高纯度蛋白。重组蛋白可被其免疫的SD大鼠血清识别但不能够被正常大鼠血清识别,表明其具有免疫原性。结论成功地构建了亚洲带绦虫六钩蚴8kDa基因重组质粒,在大肠埃希菌BL21/DE3中获得了成功表达;证明了8kDa基因表达产物具有免疫原性;还发现该绦虫成虫的头节、成节、孕节中也存在8kDa基因。The novel gene named oncosphere 8kDa gene of Taenia asiatica(Ta8)was cloned and expressed in order to evaluate the value of the recombinant protein in the diagnosis.The full-length cDNA of Ta8gene was cloned fromTaenia asiatica by using RT-PCR and cloned into the prokaryotic expression vector pET-28a(+),and then expressed in E.coli BL21 with IPTG induction.The recombinant protein was detected by SDS-PAGE and purified by Ni-IDA affinity chromatography.Its immunogenicity was examined by Western blot.PCR,double enzyme digestion and DNA sequencing confirmed that the recombinant expression plasmid was successfully constructed.The expression products were obtained and purified by Ni-IDA affinity chromatography.Western blot analysis of Ta8recombinant protein testified that it could be recognized by immunized SD rat serum while could not be recognized by normal SD rat,therefore indicated its immunogenicity.A novel gene coding Ta8of Taenia asiatica was cloned,expressed,purified and identified successfully,which proofing that Ta8protein had immunogenicity.Additionally,the Ta8gene was also detected in scolex and mature and gravid proglottids of adult Taenia asiatica.

关 键 词：亚洲带绦虫 六钩蚴 基因克隆 原核表达 免疫原性 

分 类 号：R383[医药卫生—医学寄生虫学]