猪带绦虫CDC37基因的克隆、表达与组织定位研究  

Prokaryotic Expression and Histolocalization of the Taenia solium CDC37 Gene

作　　者：黄江[1,2] 李波[1] 戴佳琳[1] 张爱华[2] 

机构地区：[1]贵阳医学院寄生虫学教研室,贵阳550004 [2]贵阳医学院公共卫生学院毒理学教研室,贵阳550004

出　　处：《中国寄生虫学与寄生虫病杂志》2013年第1期23-26,共4页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.30760227;81260256);贵州省科技攻关项目[黔科合NY字(2009-3074)];贵州省优秀科技教育人才省长资金项目[黔省专合字(2009)82号]~~

摘　　要：目的原核表达猪带绦虫(Taeniasolium)细胞分裂周期蛋白37(cell division cycle 37,TsCDC37),并对其进行组织定位和免疫原性研究。方法通过Blastx分析从猪带绦虫成虫cDNA质粒文库中筛选出TsCDC37基因,PCR扩增CDC37基因,以pET-28a(+)为载体构建目的基因原核表达体系,在大肠埃希菌(E.coli)BL-21/DE3中诱导表达,纯化的重组蛋白免疫SD大鼠制备免疫血清,Western blotting分析重组蛋白的免疫原性,免疫组织化学方法观察TsCDC37在猪带绦虫成虫中的组织定位。结果 PCR、双酶切和DNA测序结果均表明重组质粒pET-28aTsCDC37构建成功。目的基因在BL-21/DE3菌株中得到稳定表达,重组蛋白的相对分子质量(M_r)为52 000。纯化的重组蛋白可被其免疫的SD大鼠血清和猪带绦虫病、亚洲带绦虫病或牛带绦虫病患者血清识别。免疫组化结果显示,TsCDC37分布于猪带绦虫成虫表膜和子宫中的虫卵。结论纯化后的猪带绦虫细胞周期分裂蛋白TsCDC37具有较强的免疫原性,主要定位于猪带绦虫成虫的表膜和子宫中的虫卵。Objective To express Taenia solium gene encoding cell division cycle 37 protein （TsCDC37） and investigate its antigenicity and localization in adults of Taenia solium. Methods The complete coding sequence of TsCDC37 was amplified by PCR based on the recombinant plasmid clone from the cDNA library of adult Taenia solium. The PCR product was cloned into a prokaryotic expression vector pET-28a （＋）. The recombinant expression plasmid was identified by PCR, double endonuclease digestion and sequencing. The recombinant plasmid was transformed into E. coli BL21/DE3 and followed by expression of the protein induced by IPTG. The mice were immunized subcutaneously with purified recombinant TsCDC37 formulated in Freund＇s adjuvant. The antigenicity of the recombinant protein was examined by Western blotting. The localization of TsCDC37 in adult worms was demonstrated by immunofluorescent technique. Results The recombinant expression vector was constructed successfully. The recombinant protein was about M, 52 000, it was then purified and specifically recognized by immunosera of SD rats and sera from patients infected with Taenia solium, Taenia saginata or Taenia asiatica. The immunofiuorescence assay revealed that TsCDC37 located at the tegument of T. solium adult and the eggs. Conclusion TsCDC37 gene has been expressed with immnnoreactivity. The recombinant protein is mainly expressed in tegument and egg, and is a common antigen of the three human taenia cestodes.

关 键 词：猪带绦虫 细胞分裂周期蛋白37 抗原性 组织定位 

分 类 号：R383.32[医药卫生—医学寄生虫学]