弓形虫环核苷酸依赖性蛋白激酶真核表达载体构建及在Vero细胞内的定位分析

Construction of eukaryotic expression vector of Toxoplasma gondii nucleotide-dependent protein kinase and its localization analysis in Vero cells

为研究弓形虫环核苷酸依赖性蛋白激酶（PKAR）基N在Vero细胞内的定位情况，本研究构建弓形虫TgPKAR—RFP—C2真核表达质粒，并将其真核转染至Vero细胞内。试验中利用PKAR基N序列设计引物，以反转录获得的cDNA模板进行PCR扩增，成功获得目的片段，并构建克隆质粒pMD-PKAR。经BglⅡ和HindⅢ双酶切后构建真核表达质粒PKAR-RFP-C2，利用脂质体转染法将其导至Vero细胞内，利用Leica激光共聚焦显微镜观察PKAR基因在Vero细胞内的表达情况及其定位。结果显示，成功获得了PKAR基因，与GenBank公布的基因序列相似性为100％，并成功构建真核表达质粒及在Vero细胞内表达。Western blot试验成功检测到目的条带；激光共聚焦显微镜观察发现PKAR基因主要在Vero细胞的细胞质中呈点状表达。结果为进一步研究PKAR基因生物学功能及核酸疫苗研制奠定基础。

In the study,eukaryotic expression plasmid of cyclic nucleotide dependent protein kinase regulatory subnit（PKAR）of Toxoplasma gondii was constructed to explore the expression and location in Vero cell in vitro. The primers were designed according to the gene sequence fr6m Gen- Bank. The gene was amplified with the cDNA template by PCR amplification. Subsequently, the pMD-PKAR plasmid was constructed and digested by Bgl Ⅱ and Hind Ⅲ double enzyme for constructing the eukaryotic expression vector PKAR-RFP C2. The expression plasmid was transfected into Vero cell by liposome 2000. The expression and location were observed by Leiea laser scanning confocal microscope. The results showed that PKAR target fragment was successful obtained and 100% the homology with corresponding gene sequence in GenBank. PKAR and RFP were also found by Western blot analysis. The location of PKAR showed dotted distribution in the eytoplasma of Vero cell,which provided a key reference for the further studies on the biological function and vaccine development of Toxoplasma gondii.