转录因子AP-4基因真核表达载体构建及表达分析  

Construction and Expression Analysis of Eukaryotic Expression Vector of Transcription Factor AP-4

作　　者：吴华彰[1] 公丕海 吴涛[1] 赵云利 汤必奎[1] 陈文静 刘牧林[3] 樊红[2] Wu Huazhang;Gong Pihai;Wu Tao;Zhao Yunli;Tang Bikui;Chen Wenjing;Liu Mulin;Fan Hong(School of Life Science, Bengbu Medical College, Bengbu, 233030;Medical School of Southeast University, The Key Laboratory of Developmental Genes and Human Diseases, Ministry of Education, Nanjing, 210009;Department of Gastrointestinal Surgery, The First Affiliated Hospital of Bengbu Medical College, Bengbu, 233030)

机构地区：[1]蚌埠医学院生物科学系,蚌埠233030 [2]东南大学医学院,发育与疾病相关基因教育部重点实验室,南京210009 [3]蚌埠医学院第一附属医院胃肠外科,蚌埠233000

出　　处：《基因组学与应用生物学》2019年第5期2270-2275,共6页Genomics and Applied Biology

基　　金：国家自然科学基金(21707002);安徽省高校自然科学研究项目(KJ2017A213; KJ2017A219; KJ2015B041by);大学生创新创业项目(201710367028; 201610367002)共同资助

摘　　要：本研究利用生物信息学分析AP-4与胃癌患者临床病理信息的相关性,根据GenBank中人AP-4基因cDNA序列设计并合成特异性引物,以胃癌细胞总RNA逆转录的cDNA为模板,利用高保真酶扩增AP-4基因CDS (Coding DNA sequence)序列并构建入pcDNA3.1+载体,并通过限制性内切酶酶切分析和测序法进行进一步验证;脂质体法将AP-4重组表达载体及对照pcDNA3.1+载体转染胃癌细胞,qRT-PCR(Quantitative real time polymerase chain reaction)和Western blotting检测分别检测AP-4在m RNA和蛋白水平的表达。生物信息学分析发现,AP-4的表达与胃癌分期及预后显著相关;酶切及测序分析表明,转录因子AP-4真核表达载体构建成功,并能够在胃癌细胞中实现转录和蛋白水平的高效表达。此研究为深入研究转录因子AP-4在胃癌等肿瘤发生发展中的作用及分子机制奠定了基础。In this research, a pair of specific primers were designed and synthesized based on the coding DNA Sequence of human AP-4 gene in GenBank, the AP-4 CDS sequence was amplified by PCR and inserted into pcDNA3.1+vector, and AP-4 expression vector was further verified by restriction analysis and DNA sequencing after it was constructed. The recombinant plasmid AP-4 and control pcDNA3.1 +vector were transfected into gastric cancer cells by lipofectamine 2000, and AP-4 mRNA and protein expression in gastric cancer cells were detected by q RT-PCR and western blot, respectively. The research results showed that high levels of AP-4 expression are significantly associated with the degree of malignancy and prognosis of gastric cancer patients by bioinformatics analysis. Restriction enzyme digestion and sequencing analysis revealed that AP-4 eukaryotic expression vector was successfully constructed and showed significantly higher m RNA and protein expression levels of AP-4 in gastric cancer cells. These results indicated that the constructed vector works well that can use for further study of the role of transcription factor AP-4 in the tumorigenesis of gastric cancer.

关 键 词：转录因子 AP-4 载体构建 真核表达 

分 类 号：Q78[生物学—分子生物学] R735.2[医药卫生—肿瘤]