弓形虫信号转导蛋白14-3-3基因的克隆与表达

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

目的 体外扩增弓形虫RH株信号转导蛋白 14 3 3 (Toxo 14 3 3 )基因编码序列 ,构建原核表达质粒 ,并表达Toxo 14 3 3。 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RT PCR扩增Toxo 14 3 3基因片段 ,插入原核表达质粒 pET2 8a中 ,重组子双酶切、PCR和测序鉴定 ,转化大肠杆菌BL2 1并以异丙基 β D 硫代半乳糖苷 (IPTG)诱导表达。 结果 从弓形虫RH株RNA中扩增出 80 3bp的Toxo14 3 3基因片段 ,构建重组质粒 pET2 8a/14 3 3 ;IPTG诱导 ,SDS PAGE显示表达产物的大小约 3 0 .7kDa ,Western印迹鉴定为Toxo 14 3 3。 结论 成功地从弓形虫RH株基因组DNA中获取了 14 3 3基因 ,构建了 pET2 8a/Toxo 14 3 3重组质粒 ,并获得高效表达。

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.