亚洲带绦虫烯醇化酶基因的原核表达、鉴定与组织定位

Prokaryotic Expression,Identification and Histolocalization of the Taenia asiatica Enolase Gene

目的表达亚洲带绦虫(Taenia asiatica,Ta)成虫烯醇化酶(enolase,ENO)基因,并对其进行组织定位和免疫反应性分析。方法通过大规模测序从亚洲带绦虫cDNA文库中确定ENO基因,PCR扩增目的片段,将其克隆至表达质粒pET-30a(+),在大肠埃希菌BL-21/DE3中用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)观察重组蛋白的表达情况,用镍离子金属螯合剂亲和层析柱纯化重组蛋白,蛋白质印迹(Western blotting)分析该重组蛋白的免疫反应性。将重组蛋白免疫SD大鼠制备免疫血清,ELISA检测抗体水平,间接免疫荧光检测确定ENO在亚洲带绦虫成虫组织中的定位。结果PCR、双酶切和DNA测序结果均表明,重组质粒pET-30a(+)-TaENO构建成功。SDS-PAGE结果显示,重组蛋白TaENO的相对分子质量(Mr)为47000。经亲和层析获高纯度的蛋白,蛋白浓度为0.37mg/ml。重组蛋白TaENO能被SD大鼠抗血清、感染亚洲带绦虫的猪血清和患者血清识别。间接免疫荧光检测结果显示,TaENO主要定位于成虫的表膜。结论纯化后的亚洲带绦虫成虫烯醇化酶重组蛋白具有较强的免疫反应性,烯醇化酶主要定位于成虫表膜。

Objective To express enolase gene of Taenia asiatica,investigate the immunoreactivity of the recombinant TaENO pretein,and immuno-histolocalize the presence of the recombinant TaENO in adults of T.asiatica.Methods The gene encoding enolase of T.asiatica（TaENO） was cloned by high throughput sequencing from the cDNA library of adult T.asiatica.The coding region of TaENO was amplified by PCR,and cloned into a prokaryotic expression vector pET-30 a（＋ ）.The recombinant plasmid was transformed into E. coli BL-21/1）E3 and followed by expression of the protein induced by IPTG. The protein was purified by Ni-IDA affinity chromatography, and tested by SDS-PAGE. Its immunoreactivity was examined by Western blotting. The mice were immunized subcutaneously with purified TaENO formulated in Freund＇s adjuvant. Serum samples were collected and analyzed for specific antibodies by ELISA. The localization of TaENO in adult worms was demonstrated by immunofluorescent technique. Results The recombinant expression plasmid was identified by PCR, double endonuclease digestion and sequencing. The recombinant TaENO was about Mr 47 000 with a concentration of 0.37 mg/ml. It was recognized by antisera of SD rats immunized with TaENO, sera of taeniasis patients and sera of infected swine. The immunofluorescence assay revealed that TaENO immune serum located in the tegument of T. asiatica aduh. Conclusion The TaENO gene has been expressed with immunoreactivity, and the recombinant TaENO is immunolocalized in the tegument of T. asiatica adult.