摘要
目的构建人胰十二指肠同源盒(PDX-1)基因的真核表达载体,观察其在NIH3T3细胞中的表达。方法以人胰岛细胞瘤cDNA为模板,采用聚合酶链式反应(PCR)扩增PDX-1基因编码区的全部序列,克隆入真核细胞表达载体pcDNA3.1中,经限制性内切酶双酶切及DNA序列分析鉴定目的基因。序列正确的重组质粒用脂质体转染NIH3T3细胞,Western blot观察基因表达情况。结果PCR扩增的特异性片段长度为852 bp,以此构建的重组质粒pcDNA3.1-PDX-1,经BamHⅠ和EcoRⅠ双酶切后显示5.5 kb和850 bp左右的两条片段,测序结果与GenBank中的人PDX-1基因cDNA(GenBank序列号NM_000209)序列一致。证明PDX-1基因已成功克隆到了真核细胞表达载体pcDNA3.1中。Western blot证实pcDNA3.1-PDX-1转染NIH3T3细胞24 h后有PDX-1的表达。结论成功构建了pcDNA3.1-PDX-1重组真核表达载体,并在NIH3T3细胞中表达。
Objective To construct eukaryotic expression vector of human pancreatic duodenal homeobox I（PDX-1） gene, and to detect its expression in NIH3T3 cell lines. Methods The whole coding sequence of PDX-1 gene was amplified by polymerase chain reaction （PCR） from human pancreatic -cell tumors cDNA. The fragment was inserted into eukaryotic expression vector pcDNA3.1 plasmid. The recombinant plasmid was verified by double digestion and DNA sequencing. The expression of PDX-1 gene in NIH3T3 cells was assayed by Western blot. Results The length of specific fragment amplified by PCR was 852 bp, and the recombinant plasmid pcDNA3.1- PDX-1 showed two bands of 5.5 kb and 852 bp by digestion using respective restriction enzymes BamH I and EcoR I. The sequence of PDX-1 gene was approved or confirmed by blasting to GenBank. It was suggested that PDX-1 gene had been cloned into pcDNA3.1 vector correctly. Western blot showed that PDX-1 gene was expressed, which was detected 24 h after pcDNA3.1-PDX-1 plasmid was transfected into NIH3T3 cells. Conclusion The recombinant eukaryotic expression vector pcDNA3.1-PDX-1 was successfully constructed and expressed in NIH3T3 cell lines.
出处
《上海交通大学学报（医学版）》
CAS
CSCD
北大核心
2007年第1期85-87,共3页
Journal of Shanghai Jiao tong University:Medical Science
关键词
胰十二指肠同源盒
基因克隆
真核表达载体
转染
pancreatic duodenal homeobox 1
gene clone
eukaryotic expression vector
transfection
