携带ePNP基因的减毒伤寒沙门菌的构建及目的基因在胰腺癌细胞中的表达

Construction of attenuated Salmonella typhimurium containing PNP gene and expression in pancreatic cancer cells

目的构建含有pEGFP-C1-PNP质粒的减毒鼠伤寒沙门菌SL3261菌株,检测其在胰腺癌细胞BxPc-3中的表达。方法 PCR扩增得到PNP基因,构建真核表达载体pEGFP-C1-PNP,并进行酶切、PCR和测序鉴定。利用电转化法将重组质粒转入减毒鼠伤寒沙门菌SL3261,进行形态学和表面抗原稳定性检测。含质粒SL3261菌株与BxPc-3细胞体外共培养,利用荧光显微镜观察和RT-PCR检测绿色荧光蛋白的表达和PNP基因的转录。结果目的基因正确连接pEGFP-C1中,并成功转入减毒鼠伤寒沙门菌,感染细胞亦检测到目的基因的表达。结论成功构建了能携带ePNP基因真核表达质粒的SL3261菌株,且该基因能在胰腺癌细胞中正确表达。

Objective To develop attenuated Salmonella typhimurium SL3261 harboring recombinant expressing vector of pEGFP-C1-PNP and to detect the expression in pancreatic cancer cells. Methods PNP gene was amplified by PCR cloned into a recombinant eukaryotic expressing plasmid pEGFP- C I-ePNP and then identified by enzyme digesting,PCR,and sequencing analysis, pEGFP-C1-PNP vector was electrotransfered to attenuated Salmonella typhimurium SL3261. Then the stability of the morphology and suface antigen of attenuated Salmonella typhimunium containing plasmid were detected. Meanwhile the reconstructed SL3261 was cocultured with BxPc-3 ceils to detect their invasion. Fluorescent microscope observasion and RT-PCR was applied to observe GFP and PNP mRNA in the host cells. Results It was proved that PNP gene segments were inserted into pEGFP-C1 correctly and transfered into attenuated Salmonella typhimunium successfully. Conclusion Attenuated Salmonella typhimunium SL3261 containing reeombined eukaryotic expressing plasmid pEGFP-C1-PNP was successfully established and correctly expressed in pancreatic cancer cells.