弓形虫微线体分泌蛋白TgMIC10基因的克隆和序列分析

目的从弓形虫RH株cDNA中扩增出微线体分泌蛋白TgMIC10编码基因并进行克隆,为利用其表达的重组蛋白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,通过与线性克隆载体pGEM-T连接,经进行酶切和PCR鉴定及DNA测序证实。结果PCR法扩增出了一个大小约597bp左右的DNA片断,将重组质粒pGEM-T-TgMIC10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,对插入片断的测序结果表明,TgMIC10具有一个长度为597bp的完整开放阅读框(ORF),编码198个氨基酸,理论分子量23kDa,与GenBank收录(编号为AF293654)的弓形虫Tg-MIC10基因具有高度的同源性(99.5%)。结论本实验成功地克隆了TgMIC10编码基因,为进一步研究提供了条件。

Clonng and Sequencing of TgMIC10 Gene of Toxoplasma Gondii

Objective To set up a way of quickly and accurately detecting toxoplasmosis by use of recombinant protein, the micronal seceted protein TgMIC10 was amplified from Toxoplasma gondii (RH strain) and coloned into pGEM-T vector. Methods The specific primers were designed and synthesized. The DNA fragment encoding T. gondii TgMIC10 was amplified by RT-PCR from RNA of RH strain tachyzoites. The PCR products were cloned into pGEM-T vector and recombinants were screened by PCR? enzyme digestion and sequencing. Results The size of pGEM-T-TgMIC10 digested with EcoR I and Xba I was identical to the length of the PCR generated products. DNA sequencing indicated that the TgMIC10 gene has an open reading frame of 597bp, which encodes 198 amino acids with an approximate molecular weight of 23kDa and has high homology with the TgMIC10 gene submitted to GenBank (AF293654). Conclusion The findings suggested that TgMIC10 gene had been successfully cloned. It provided the basis for further study on TgMIC10 expression and its immunological diagnosis.