刚地弓形虫蛋白质二硫键异构酶基因的克隆及原核表达

Gene-cloning,prokaryotic expression and immunoreactivity analysis of protein disulfide isomerase in Toxoplasma gondii

目的克隆刚地弓形虫蛋白质二硫键异构酶(TgPDI)基因,原核表达、纯化重组TgPDI蛋白,并分析其免疫反应性。方法制备弓形虫RH株速殖子总RNA,根据TgPDI基因全长编码序列(登录号为AJ306291.2)的开放阅读框设计合成引物,并进行逆转录-聚合酶链式反应(RT-PCR),扩增产物经双酶切后连接入pET-30a(+)载体,重组质粒pET-30a(+)-TgPDI转化大肠埃希菌(E.coli)DH5α,阳性菌落经PCR、双酶切并测序鉴定。将重组质粒pET-30a(+)-TgPDI转化至大肠埃希菌Rosetta(DE3),并加入异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)结合考马斯亮蓝染色检测表达产物;分别以抗His抗体和兔抗弓形虫血清为一抗,蛋白质印迹(Western blotting)鉴定重组蛋白及其免疫反应性;重组蛋白His-TgPDI通过Ni2+琼脂糖凝胶亲和层析柱纯化,纯化蛋白经SDSPAGE分离,考马斯亮蓝染色结合凝胶成像系统分析其纯度。结果 RT-PCR扩增产物约为1 427 bp。菌落PCR、双酶切及测序结果显示,重组质粒pET-30a(+)-TgPDI构建成功。SDS-PAGE结果显示,经IPTG诱导获得约57 kDa的可溶性重组蛋白。Western blotting结果表明,诱导表达的重组蛋白能被兔抗弓形虫血清识别。亲和纯化后获得纯度大于95%的重组TgPDI蛋白质。结论克隆得到弓形虫PDI基因,原核表达并纯化出该基因编码的蛋白,且该重组蛋白具有免疫反应性。

Objective To clone, prokaryotic express and purify the protein disulfide isomerase （PDI） gene of RH strain of Toxoplosma gondii,analyze the immunoreactivity of recombinant protein. Methods Total RNA was extracted from tachyzoites of RH strain of T. gondii. Open reading frame of target gene was amplified with a pair of specific primers designed according to the coding sequence of TgPDI gene （GenBank accession No：AJ306291.2） by RT-PCR. The RT-PCR product was digested with restrict enzymes and ligated into a pET-30a（＋） vector. The recombinant pET-30a（＋）-TgPDI plasmid was transferred into E. coli DH5c~ and the positive clones were selected via the colony-PCR and confirmed through the double restrict enzymes digestion and DNA sequencing. The pET-30a（＋）-TgPDI construct was transformed into E. coli Rosetta （DE3） and induced to express by IPTG treatment. The recombinant TgPDI was purified through Ni2＋-NTA gel column and the expression products were analyzed via SDS-PAGE followed by coomassie blue staining.Western blotting assay with His primary antibody and rabbit anti-T.gondii serum was used to confirm the expression of rTgPDI and its immunoreactivity was analyzed. Results The product of RT-PCR was 1 427 bp. The recombinant plasmid pET-30a （＋）-TgPDI was confirmed successfully by colony-PCR,double restrict enzymes digestion and DNA sequencing. The rTgPDI proteins were successfully expressed in E. coli and the molecular weight was approximately 57 kDa. Western blotting analysis indicated that the rTgPDI band was able to react with the rabbit anti-T.gondii serum polyclonal antibody. The isolated protein was water soluble and showed 90% purity based on SDS-PAGE and gel imaging analysis. Conclusion The TgPDI gene was cloned and the recombinant TgPDI protein was produced in E. coli with specific immunoreactivity. Key words： Toxoplasma gondii; Protein disulfide isomerase; Clone; Prokaryotic expression; Immunoreactivity