弓形虫信号转导蛋白14-3-3基因的克隆与表达

目的　体外扩增弓形虫RH株信号转导蛋白1433(Toxo1433)基因编码序列,构建原核表达质粒,并表达Toxo1433。　方法　收集、纯化弓形虫RH株速殖子,提取RNA,在设计合成的引物中引入EcoRI和XhoI酶切位点。应用RTPCR扩增Toxo1433基因片段,插入原核表达质粒pET28a中,重组子双酶切、PCR和测序鉴定,转化大肠杆菌BL21并以异丙基βD硫代半乳糖苷(IPTG)诱导表达。　结果　从弓形虫RH株RNA中扩增出803bp的Toxo1433基因片段,构建重组质粒pET28a/1433;IPTG诱导,SDSPAGE显示表达产物的大小约30.7kDa,Western印迹鉴定为Toxo1433。　结论　成功地从弓形虫RH株基因组DNA中获取了1433基因,构建了pET28a/Toxo1433重组质粒,并获得高效表达。

Cloning and Expression of the Signaling Protein 14-3-3 of Toxoplasma gondii

Objective To clone and express the cell signaling protein 14-3-3 gene from Toxoplasma gondii RH strain. Methods Toxoplasma RH strain tachyzoites, which maintained by mouse passage, were harvested from ascites of mice and genomic DNA was prepared. A pair of primers were designed and synthesized based on the sequence of Toxo 14-3-3 cDNA. A specific fragment of Toxo 14-3-3 gene was obtained by RT-PCR amplification from Toxoplasma genomic DNA. The PCR products were ligated to pGEM-T. The EcoRI / Xho I restricted fragments, confirmed by PCR and EcoRI / XhoI digestion, were cloned into expression vector pET28a and the recombinants were transformd into E.coli BL21. Fusion expression was induced by isopropyl-beta-D-thiogalactoside (IPTG) and confirmed by Western blotting with rabbit anti-Toxoplasma sera. Results The molecular size of Toxo 14-3-3 was 803 bp, which is highly homologous to the previous report cloned from the parasites of intestinal epithelial stage in cat. High expression was obtained in pET28a/ Toxo 14-3-3/E.coli BL21 when confirmed by Western blotting. Conclusion The recombinant construction of Toxo 14-3-3 was generated and expression was induced.