弓形虫微线体分泌蛋白TgMIC10基因的克隆、表达及鉴定

Molecular cloning,expression and identification of the TgMIC10 gene from Toxoplasma gondii

目的克隆、表达及鉴定弓形虫(RH株)微线体分泌蛋白TgMIC10编码基因,为建立一种灵敏检测弓形虫感染的实验方法作准备。方法提取弓形虫速殖子总RNA,设计并合成引物,RT-PCR扩增TgMIC10编码基因,与克隆载体pGEM-T连接,经酶切和PCR鉴定及DNA测序证实后,亚克隆入表达载体pBK-CMV,IPTG诱导表达pBK-TgMIC10阳性重组子转化宿主菌E.coliBL21,Western blot鉴定。结果PCR法扩增出了一597bp左右的DNA片断,将重组质粒pGEM-TgMIC10、pBK-TgMIC10分别作EcoRI和XbaI双酶切,均能得到一个大小与PCR扩增产物一致的插入片断,表明Tg-MIC10基因的克隆和亚克隆均获成功,用IPTG诱导带有重组质粒pBK-TgMIC10的大肠杆菌,产物行SDS-PAGE,可得到一约21kDa融合蛋白,Western-blot法鉴定该蛋白能被弓形虫感染的兔血清所识别。

To set up a way to rapid and accurate detection of toxoplasmosis by use of recombinant protein, the micronnemal secreted protein TgMIC10 was amplified from Toxoplasma gondii RH strain, and subcloned to the expression vector pBK-CMV. At first, the specific primers were designed and the DNA fragment encoding T. gondii TgMIC10 was amplified by RT-PCR from RNA of the RH strain tachyzoites. The PCR products were then cloned into vector pGEM-T, and the recombinants were screened by PCR, enzyme digestion and sequencing. Then, TgMIC10 was subcloned into expression vector pBKCMV and the recombinant plasmid was transformed into E. coli BL21 with IPTG induction. The expression of the recombinam proteins was identified by Western blotting. It was found that the sizes of recombinant plasmid pGEM-T-gMIC 10 and pBK- CMV-TgMIC 10 digested with,EcoR1 and Xba1 were identical to the length of the PCR products generated. It is evident that the TgMIC10 gene is successfully cloned and subcloned into vectors. With the positive clones induced by IPTC, a fusion protein recognized by sera of rabbits infected with T. gondii tachyzoites can be obtained.