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目的构建FLK1265-2 493与C3d3融合基因真核表达质粒,并在体外进行表达和鉴定。方法采用PCR技术扩增FLK1265-2 493片段,将该片段插入到pMDT-18载体中后,亚克隆至真核表达载体pSG.SS.C3d3.YL,构建FLK1265-2 493与C3d3融合基因表达质粒。经限制性酶切鉴定和DNA序列测定结果证实后,将重组质粒pSG.SS.FLK1265-2 493.C3d3.YL转染Hela细胞,检测其体外表达。结果免疫印迹和免疫细胞化学分析结果分别表明转染细胞上清液和胞浆内均有目的分子的存在。结论构建的DNA疫苗可在真核细胞内正确表达,这为进一步的动物实验打下了基础。
Objective To construct eukaryotic expression plasmid of FLK1265-2 493 and C3d3 fusion gene and to express and identify in vitro. Methods The FLK1265-2493 fragment was amplified by PCR. The fragment was inserted into pMDT-18 vector and subcloned into eukaryotic expression vector pSG.SS.C3d3.YL to construct FLK1265-2493 fusion gene expression plasmid with C3d3 . After confirmed by restriction enzyme digestion and DNA sequence determination, the recombinant plasmid pSG.SS.FLK1265-2 493.C3d3.YL was transfected into Hela cells to detect the expression in vitro. Results The results of immunoblotting and immunocytochemistry showed that the target molecules were present in both supernatants and cytoplasm of transfected cells. Conclusion The constructed DNA vaccine can be correctly expressed in eukaryotic cells, which lays the foundation for further animal experiments.