构建PDCD5基因真核表达载体及电转染BGC823细胞研究.docVIP

构建PDCD5基因真核表达载体及电转染BGC823细胞研究 　　[摘要] 目的 构建PDCD5基因真核表达载体，并电转染BGC823细胞，获得稳定转染细胞。 方法 设计合成PDCD5基因片段，将其插入经EcoRⅠ和XhoⅠ双酶切的pcDNA3.1+表达载体，菌落PCR和基因测序进行鉴定。重组质粒电转染BGC823细胞，G418筛选数周后PCR鉴定稳定转染细胞。 结果 重组质粒经菌落PCR测序分析表明，PDCD5基因序列准确插入预期位置，且序列正确。重组质粒成功电转染BGC823细胞，并整合入细胞基因组DNA。结论 成功构建了PDCD5基因真核表达载体，并整合进BGC823细胞基因组，为研究PDCD5基因的功能及胃癌的基因治疗提供实验基础。 　　[关键词] PDCD5基因；pcDNA3.1+表达载体；电转染；BGC823细胞系 　　[中图分类号] R394 [文献标识码] A [文章编号] 1673-9701（2017）06-0033-03 　　[Abstract] Objective To construct eukaryotic expression vector of PDCD5 gene and transfect BGC823 cells to obtain stable transfected cells. Methods The PDCD5 gene fragment was designed and inserted into pcDNA3.1+ expression vector which was digested with EcoR Ⅰ and Xho Ⅰ. Colony PCR and sequencing were used for identification. The recombinant plasmid was electrically transfected into BGC823 cells， and PCR was used for identifying the stably transfected cells after G418 screening for weeks. Results It was suggested by colony PCR and sequencing analysis that the recombinant plasmid PDCD5 gene sequenc was accurately inserted into the desired position and the sequence was correct.Recombinant plasmids were successfully transfected into BGC823 cells and integrated into cell genomic DNA. Conclusion The eukaryotic expression vector of PDCD5 gene is successfully constructed and integrated into BGC823 cell genome， which provided experimental basis for studying the function of PDCD5 gene and gene therapy of gastric cancer. 　　[Key words] PDCD5 gene； pcDNA3.1+ expression vector； Electrical transfection； BGC823 cell line 　　程序性细胞死亡因子5（programmed cell death 5，PDCD5）原名为TFAR19，由北京大学人类疾病基因中心从白血病细胞株TF-1细胞中克隆得到，具有促进多种细胞凋亡和抑制增殖的效应，其异常表达与多种肿瘤及自身免疫性疾病有相关性[1]。研究表明，PDCD5基因在胶质瘤细胞[2]、前列腺癌细胞[3]、结肠癌[4]和胃癌[5]等多种恶性肿瘤组织中较正常组织表达水平下降，并且重组人PDCD5蛋白可以增强软骨肉瘤细胞[6]和肺癌细胞[7]等肿瘤细胞对化疗药物的敏感性。胃癌是最常见的恶性肿瘤之一，其5年生存率低于20%，是全球第二个癌症死亡相关的原因，而我国每年因胃癌死亡病例占全球人口的47.8%[8]。本研究以PDCD5基因和BGC823胃癌细胞为研究对象，构建pcDNA3.1+-PDCD5重组真核表达载体，电转染BGC823细胞，并筛选稳定转染细胞株，为进一步研究PDCD5基因的功能及进行胃癌的基因治疗奠定基础。 　　1 材料与方法 　　1.1