弓形虫表面抗原SAG1基因的克隆及在大肠杆菌中的表达

Molecular cloning and expresssion of T.gondii surface antigen SAG1 gene in E.coli

目的 体外扩增弓形虫 RH株膜表面抗原 SAG1编码基因 ,构建原核表达质粒 ,并表达 SAG1编码基因序列。方法 收集、纯化 RH株速殖子 ,提取 RNA,据已知的 SAG1基因序列 ,在设计合成的 1对引物中引入 Eco RI和 Hind 酶切位点。应用 RT-PCR技术 ,扩增 SAG1基因片段 ,插入原核表达质粒 p ET2 8a中 ,转化大肠杆菌 BL2 1 感受态细胞 ,重组子经 Eco RI和 Hind 双酶切、PCR鉴定 ,转化宿主菌 BL2 1 ,并以 IPTG诱导表达。结果 从弓形虫 RH株 RNA中扩增出 10 1 1 bp的 SAG1基因片段 ,构建重组质粒 p ET2 8a/ SAG1 ,酶切和 PCR鉴定产物大小均与预期值相符 ;IPTG诱导 ,SDS-PAGE显示表达产物的大小约 3 4.8k D。结论 成功地从弓形虫 RH株 RNA中获取 SAG1基因 ,构建了 p ET2 8a/ SAG1重组质粒并获得了高效表达 。

Objective To clone and express surface antigen SAG1 gene from Toxoplasma gondii RH strain.Methods T.gondii RH strain tachyzoites,which maintained by mouse passage,were harvested from ascites of mice and total RNA was prepared. A pair of primers were designed and synthesized based on the sequence of SAG1 cDNA. A specific fragment of SAG1 gene was obtained by RT-PCR amplification. The RT-PCR products were ligated to pGEM-T. The EcoRI/HindⅢ restricted fragments,confirmed by PCR and EcoRI/HindⅢ digestion,were cloned into expression vector pET28a and the recombinants were transformed into E.coli BL 21.Fusion expression was induced by beta-D-thiogalactosidase(IPTG) and identified by western blot with anti-Toxoplasma sera.Results The molecular size of SAG1 was 1 011 bp,which was identical to the previous report cloned from the parasites of intestinal epithelial stage in cat. High expression was obtained in pET28a/SAG1/E.coli BL 21when confirmed by western blot.Conclusions The recombinant construction of SAG1 is generated and expression is induced.The recombinant P30 has a specific immunoactivity and potentiality in immunological diagnosis.