单纯疱疹病毒胸苷激酶与绿色荧光蛋白融合基因表达载体的构建

Construction of coexpression vector for HSV-1 thymidine kinase and green fluorescent protein

目的建立既有TK活性又表达绿色荧光的融合蛋白治疗系统用于实验研究,为建立荧光活体成像技术,更为科学地进行肿瘤自杀基因增效中药成分的研究打下基础。方法DNA重组技术构建HSV1-TK cDNA与EGFP基因融合的真核细胞表达载体pTKGFP,用酶切鉴定并进行序列测定;polyFect转染液对鼠黑色素瘤细胞B16进行转染,荧光显微镜观察基因表达情况,MTT法检测转化细胞B16对GCV的敏感性。结果重组质粒pTKGFP经限制性酶切鉴定和DNA序列测定分析显示,TK基因已经成功与GFP基因连接,连接处无移码突变产生;荧光显微镜下观察显示,TK-GFP融合基因在B16中获得表达。其在细胞中的定位与文献资料一致,TKGFP主要集中于胞核区而GFP弥散分布于胞质中。TKGFP组细胞对GCV的敏感性大于对照组细胞,差异有显著性。结论构建了pTKGFP融合蛋白基因表达载体,并成功在B16中进行表达,所表达的融合蛋白具有TK活性和GFP的荧光活性,可用于后续性的体外与体内实验。

[Objective] To construct a functional TKGFP fusion protein therapeutic system for monitoring therapeutic transgene expression by noninvasive imaging of HSV-1-tk marker gene expression and establishing basis for the antitumour studies of Chinese medicine cooperating with suicide gene therapeutic system. [Methods] The eukaryotic HSV1-tk eDNA and EGFP fusion gene expression vector pTKGFP was constructed by using DNA recombinant technique. The recombinant plasmid was identified by restriction endonuclease digestion and DNA sequencing, and then mixed with PolyFect Transfection reagent and transfected into the melanoma cell line B16. A standard fluorescence microscope was used for fluorescent detection. MTT assay was used to determine the ganciclovir （GCV） sensitivity of transfected B16. [Results] The results of restriction endonuclease digestion and DNA sequencing showed that tk gene had been connected with GFP gene and there had been no shift-mutation in the linkage site. The levels of EGFP-expression were assessed days after transfection and were visually of similar intensity in TKGFP and native GFP transfected cells. The intracellular expression pattern of the TKGFP was different from the native EGFP. TKGFP was mostly concentrated in the nucleus, whereas the native EGFP displayed a predominantly cytoplasmic distribution. GCV sensibility examination showed that the pTKGFP transfected cells were much more sensifive to GCV than the pEGFP-N1 transfected cells in a dose-dependent manner. [Conclusion] A TKGFP fusion protein expression vector has been obtained successfully, which can be used for the antitumour studies of Chinese medicine cooperating with suicide gene therapeutic system.