大肠埃希菌嘌呤核苷磷酸化酶基因载体的构建及对胰腺癌细胞的作用  

Construction of Vector Expressing Escherichia Coli Purine Nucleoside Phosphorylase Gene and Study of Its Effect on Pancrease Cancer Cells

作　　者：李文林[1] 李克强[2] 饶泽昌[3] 石小玉[4] 赵林[3] 

机构地区：[1]南昌大学医学院病理生理学教研室,南昌市330006 [2]宁波市第二医院普外科,浙江省宁波市315000 [3]江西省医学科学研究所医学生物高技术重点实验室,南昌市330006 [4]南昌大学医学院组织胚胎学教研室,南昌市330006

出　　处：《医学分子生物学杂志》2011年第4期313-317,共5页Journal of Medical Molecular Biology

基　　金：江西省自然科学基金（No.2007GZY1000）,江西省卫生厅科技课题（No.20072017）,浙江省医药卫生科学研究基金（No.2006）

摘　　要：目的构建大肠埃希菌（Escherichia coli）嘌呤核苷磷酸化酶（purine nucleoside phosphorylase，PNP）基因表达载体，研究其生物活性，为肿瘤的基因治疗奠定基础。方法PCR扩增大肠埃希菌K12的PNP基因，T4连接酶将PNP连接人pMSCV逆转录病毒载体，构建重组逆转录病毒载体pMSCV／PNP。pM—SCV／PNP转化感受态大肠埃希菌XLI-Blue，提取pMSCV／PNP，酶切、PCR和测序鉴定。病毒包装细胞293产生重组逆转录病毒pMSCV／PNP，流式细胞仪测病毒滴度。pMSCV／PNP转染胰腺癌细胞BXPC-3，倒置荧光显微镜观察，FACS分离转染阳性细胞（GFP阳性）。RT—PCR检测PNPmRNA在胰腺癌细胞BXPC-3细胞中的表达，MTT法检测PNP基因的生物活性。结果PCR扩增出大肠埃希菌PNP基因（738bp），酶切和PCR的电泳条带显示pMSCV／PNP，测序结果正常。293包装细胞产生高滴度（3．6×10^7U／m1）重组逆转录病毒pMSCV／PNP。RT—PCR实验结果表明，pMSCV／PNP转染的胰腺癌细胞BXPC-3表达PNPmRNA。前药6-甲基嘌呤-2’-脱氧核苷（MePdR）作用72h浓度达1．00mg／L，BXPC-3／PNP细胞存活率为10．09％，随着MePdR浓度加大，BXPC-3／PNP细胞存活率继续下降直至为0。结论构建了pMSCV／PNP载体，获得了表达大肠埃希菌PNP基因的BXPC-3细胞克隆，PNP／MePdR自杀基因系统对胰腺癌细胞BXPC-3有较强的抑杀作用。Objective To construct recombinant retroviral vector pMSCV/PNP containing pufine nucleoside phosphorylase （PNP） , determine the biological activity of PNP in pancrease cancer cells, with the aim to explore PNP in gene therapy. Methods PNP was amplified from Escherichia coli K12 by PCR and cloned into retroviral vector pMSCV. The recombinant vector pMSCV/PNP was analyzed with restriction endonucleases, PCR and sequencing. The retroviruses containing PNP were produced in 293 package cells, with viral titer was tested by FACS analysis. Pancreas cancer BXPC- 3 cells were infected with PNP containing retroviruses. The infected cells （ GFP positive） were ob- served using fluorescence microscope and sorted by FACS. mRNA expression of PNP in BXPC-3/ PNP cells was detected by RT-PCR. PNP activity was identified by MTr assay. Results PNP gene with 738bp was cloned into retroviral vector pMSCV, as verified by PCR and sequencing. Packaging cell line 293 produced high titer of pMSCV/PNP retroviruses （3.6 107 U/ml） . pMSCV/PNP infec- ted BXPC-3 pancreas cancer cells expressed high level of PNP mRNA and responded to the cytotoxie and antiproliferative effects of MePdR （6-Methylpurine-2- Deoxyriboside ） in a concentration de- pendent manner, displaying 10% survival at 1.00 mg/L MePdR and up to 100% cell death with the increase of MePdR. Conclusion PNP sensitive to MePdR treatment.

关 键 词：大肠埃希菌嘌呤核苷磷酸化酶 自杀基因 载体 胰腺癌 

分 类 号：Q782[生物学—分子生物学]