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目的 :提取并鉴定已构建的EB病毒表达载体pDR2 TK ,利用脂质体介导的基因转染技术将 pDR2 TK转染人前列腺癌细胞并对单纯疱疹胸苷激酶 (HSV TK)表达状况进行检测。方法 :采用DNA大量制备及纯化系统提取pDR2 TK ,酶切和DNA测序进行鉴定 ,采用阳离子脂质体法将 pDR2 TK导入激素非依赖性人前列腺癌细胞系PC 3m ,逆转录PCR(RT PCR)法和SABC免疫组化法检测TKmRNA和蛋白的表达。结果 :扩增提取的质粒经PstI和EcoRV酶切后各获得 4个及 2个片段 ,与原基因酶切图谱一致 ;所提取质粒PCR产物经DNA测序 ,与NCBI公布的HSV TK基因序列对照 ,证实所提取质粒含目的基因序列。脂质体法转染PC 3m细胞后 ,mRNA和蛋白均有HSV TK的表达 ,其蛋白表达率约为 2 2 %。结论 :pDR2 TK质粒含有目的基因HSV TK ,阳离子脂质体法可将 pDR2 TK导入人前列腺癌细胞并获得较高效率的表达
OBJECTIVE: To extract and identify the constructed EB virus expression vector pDR2 TK and to transfect pDR2 TK into human prostate cancer cells by liposome-mediated gene transfection and to detect the expression of herpes simplex thymidine kinase (HSV TK) . Methods: pDR2 TK was extracted by DNA preparation and purification system, and digested by restriction endonucleases and DNA sequencing. The pDR2 TK was introduced into PC3m cell line by cationic liposome method and reverse transcriptase PCR (RT PCR) Method and SABC immunohistochemical detection of TKmRNA and protein expression. Results: The amplified plasmids were digested with PstI and EcoRV, respectively, and got 4 and 2 fragments respectively, which were consistent with the original gene digestion map. The PCR products of the extracted plasmids were sequenced by DNA and compared with the HSV TK gene sequences published by NCBI, Confirmed that the extracted plasmid containing the gene sequence of interest. After transfection with PC 3m cells by liposome, the expression of HSV TK mRNA and protein were detected, and the protein expression rate was about 22%. CONCLUSION: The pDR2 TK plasmid contains the target gene HSV TK. The cationic liposome method can be used to introduce pDR2 TK into human prostate cancer cells with higher efficiency