弓形虫依钙蛋白激酶Calpain—like基因的克隆及表达

目的克隆弓形虫RH株Calpain—like基因片段，构建原核表达载体，诱导表达Calpain-like基因重组蛋白。方法收集、纯化弓形虫RH株速殖子，提取总RNA，在设计合成的引物中引入SalI和EcoRI酶切位点。应用FIT—PCR扩增弓形虫RH株Calpain—like基因片段，插入pGEM—T载体，提取重组质粒，双酶切获得目的基因，亚克隆到原核表达质粒pET32a，重组子经双酶切、PCR和DNA序列分析鉴定，转化大肠杆菌BL21并以IPTG诱导表达。结果从弓形虫RH株速殖子eDNA中扩增出316bp的Calpain-like基因片段；含pET32a／Calpain—like的重组质粒在宿主菌经诱导后，获得与预期分子量相符的表达产物。结论成功地克隆和表达弓形虫RH株Calpain—like基因，弓形虫Calpain—like基因的克隆表达为进一步筛选弓形虫疫苗候选分子和治疗药物的靶位提供了研究基础。

Cloning and Expression of Calpain-like Gene in Toxoplasma gondii

Objective To clone, identify and express calpain-like gene from Toxoplasma gondii RH strain. Method RH strain tachyzoites, by laboratory mouse passage, were harvested from ascites of mice, and used to prepare RNA. RT-PCR was used to amplify the calpain-like cDNA. The PCR products were ligated to pGEM-T. The recombinants were confirmed by Sal I / EcoR I digestion, PCR, and DNA sequencing, and were subcloned into expression vector pET32a. The recombinants were transformed into E.coli BL 21 （DE3） for the expression of recombinant protein, which was analyzed by SDS-PAGE. Result 316 bp calpain-like cDNA of T.gondii was cloned, which is highly homologous to the sequence of other parasites previously reported such as schistosome. The recombinant calpain-like protein of T.gondii was expressed in E.coli. Conclusion This work paved the way for the further study of vaccine against T.gondii.