弓形虫微线体蛋白MIC9的原核表达及免疫反应性分析

Prokaryotic expression and immunoreactivity analysis of MIC9 of Toxoplasma gongdii

为深入了解弓形虫微线体蛋白9(MIC9)基因功能,以弓形虫RH株总基因组为模板PCR扩增MIC9基因片段,并构建MIC9的原核表达载体,采用Western blot对其免疫反应性进行鉴定分析。结果显示:扩增出大小为903 bp的MIC9基因片段,与预期结果一致;成功构建了pMD-18T-MIC9克隆质粒和pGEX-4T-1-MIC9原核表达载体,经过双酶切及测序鉴定正确,且能够在大肠杆菌中成功表达,表达蛋白的分子量为59 kDa;经免疫反应性鉴定,MIC9重组蛋白能够被弓形虫全虫抗体特异性识别,证明重组蛋白MIC9具有一定的免疫反应性。研究结果为弓形虫MIC9基因免疫功能的后续研究提供了参考。

In order to understand the genetic function of MIC9 of Toxoplasma gongdii, we expressed the MIC9 gene in the RH strain of Toxoplasma gondii and constructed the prokaryotic expression vector of MIC9, and its immunogenicity was identified by Western blot. The result showed that a 903 bp of MIC9 fragment was amplified by PCR, the pMD18-T-MIC9 clone plasmid and the prokaryotic expression vector of pGEX-4 T-MIC9 were constructed and the expression of the gene was confirmed by sequencing identification and double digestion. The molecular weight of the expressed protein was about 59 kDa in the E.coli expression system. In the immunogenicity identification, the MIC9 recombinant protein could be specifically recognized by T. gondii whole antibodies, which suggested that the recombinant protein MIC9 had some immunogenicity. The present results laid a foundation for further research on the immune function of MIC9 of T. gondii.