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目的构建前列腺癌特异性启动子PSAe(AREc)-PSMAe-TARPp[P(A)PTp],并在腺病毒水平评价其启动目的基因表达的效率和特异性。方法巢式PCR扩增基因组DNA获得PSAe的AREc区基因、PSMAe基因和TARPp基因,并依次连接获得嵌合启动子P(A)PTp。采用Adeasy系统构建并制备P(A)PTp启动增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)表达的重组腺病毒Ad-pSh-P(A)PTp-EGFP。不同感染复数(multi-plicity of infection,MOI)的Ad-pSh-CMV-EGFP感染各细胞系后48 h,流式细胞术确定最佳MOI;并以最佳MOI的Ad-pSh-P(A)PTp-EGFP感染各细胞系,检测EGFP的表达效率。结果 PCR成功扩增并连接获得嵌合启动子基因P(A)PTp。采用Adeasy系统,构建并制备了重组腺病毒Ad-pSh-P(A)PTp-EGFP。Ad-pSh-CMV-EGFP感染后,流式检测结果显示,前列腺癌细胞PC3和PC3M细胞的最佳MOI为100 MOI;而前列腺癌细胞DU145以及肝癌细胞SMMC-7721和HepG2为250 MOI。最佳MOI的Ad-pSh-P(A)PTp-EGFP感染后,EGFP在各细胞系中的表达效率[P(A)PTvsCMV]为:PC3(25.56%vs77.76%)、DU145(50.50%vs65.26%)、PC3M(30.81%vs89.70%)、HepG2(6.26%vs63.76%)、7721(2.27%vs67.84%)。结论成功构建前列腺癌特异性嵌合启动子P(A)PTp,并证实能在前列腺癌细胞中特异并有效地启动EGFP的表达。
Objective To construct PSAe (AREc) -PSMAe-TARPp [P (A) PTp], which is a prostatic cancer-specific promoter, and evaluate the efficiency and specificity of its initiation of gene expression at the adenovirus level. Methods Genomic DNA was amplified by nested PCR to obtain AREc region gene, PSMAe gene and TARPp gene of PSAe, and then the chimeric promoter P (A) PTp was ligated in turn. The recombinant adenovirus Ad-pSh-P (A) PTp-EGFP expressed by P (A) PTp promoter and enhanced green fluorescent protein (EGFP) was constructed and prepared by Adeasy system. The optimal MOI was determined by flow cytometry 48 h after infection with different concentrations of Ad-pSh-CMV-EGFP with multi-plicity of infection (MOI). Ad-pSh-P ) PTp-EGFP infected cells, detection of EGFP expression efficiency. Results PCR was successfully amplified and ligated to obtain the chimeric promoter gene P (A) PTp. The recombinant adenovirus Ad-pSh-P (A) PTp-EGFP was constructed and prepared by Adeasy system. After Ad-pSh-CMV-EGFP infection, flow cytometry showed that the optimal MOI of prostate cancer cells PC3 and PC3M cells was 100 MOI, while prostate cancer cell DU145 and hepatocellular carcinoma cells SMMC-7721 and HepG2 were 250 MOI. The expression efficiency of EGFP in each cell line after the optimal MOI of Ad-pSh-P (A) PTp-EGFP was [PC (25.56% vs77.76%), DU145 vs65.26%), PC3M (30.81% vs89.70%), HepG2 (6.26% vs63.76%), and 7721 (2.27% vs67.84%). Conclusion The prostate-specific chimeric promoter P (A) PTp was successfully constructed and confirmed to specifically and effectively activate EGFP expression in prostate cancer cells.