胃癌相关基因GCRG213正反义真核表达载体的构建及鉴定

Construction and identification of eukaryotic expression vector of gastric cancer associated gene GCRG213

目的:构建胃癌相关基因GCRG213正、反义真核表达载体．方法:从pGEM-T质粒上扩增出的胃癌相关基因GCRG213的DNA片段,两端分别引入限制性内切酶KpnI,BamHI和EcoRI, BamHI识别位点．按正向、反向克隆入真核表达载体pcDNA3．1(+)．测序正确的重组子pcDNA3．1-a(含GCRG213正向克隆), pcDNA3．1-b(含GCRG213反向克隆)和空载体经脂质体转染人胃癌细胞系MKN45细胞, G418筛选获得稳定转染的细胞株,采用半定量RT-PCR及Wlestern blot比较转染不同质粒的 MKN45细胞中GCRG213在mRNA和蛋白质水平上的表达差异．结果:经测序证实,GCRG213正向克隆和反向克隆正确插入真核表达载体 pcDNA3．1(+),组成重组子pcDNA3．1-a(含正向克隆),pcDNA3．1-b(含反向克隆)．重组子 pcDNA3．1-a,pcDNA3．1-b和空载体经脂质体转染人胃癌细胞系MKN45细胞,G418筛选获得稳定转染的细胞株．与对应的空载体比较,RT-PCR结果显示转染pcDNA3．1-a的 MKN45细胞中其mRNA的表达上调35．4％,而转染pcDNA3．1-b的MKN45细胞中其mRNA 的表达下调32．1％;Western blot结果显示转染 pcDNA3．1-a的MKN45细胞中其蛋白的表达上调49．4％,而转染pcDNA3．1-b的MKN45细胞中其蛋白的表达下调50．3％．结论:成功构建胃癌相关基因GCRG213正、反义真核表达载体．

AIM： To construct and identify the gastric cancer associated gene GCRG213 eukaryotic expression vector. METHODS： The sense and anti-sense fragment of GCRG213 were obtained by polymerase chain reaction （PCR）, which were GCRG213-a and GCRG213-b respectively. They were cloned into eukaryotic expression vector pcDNA3.1（＋） after introduced the sites of restrictive endonuclease enzyme KpnI, BamHI and EcoRI, BamHI. The recombinant plasmid pcDNA3.1-a, pcDNA3.1-b and the vector pcDNA3.1 were transfected separately into MKN45 cells conducted by lipofectamine^TM 2000. G418 selection was used to obtain the stably transfected cells. The expression of GCRG213 was detected at both mRNA and protein level with semi-quantitative reverse transcription PCR （RT-PCR） and Western blot, respectively. RESULTS： After sequencing, sense GCRG213 and anti-sense GCRG213 were confirmed to be successfully cloned into eukaryotic expression vector pcDNA3.1, which were named pcDNA3.1-a and pcDNA3.1-b correspondingly. The recombinant plasmid pcDNA3.1-a, pcDNA3.1-b, and the vector pcDNA3.1 were transfected successfully into MKN45 cells by lipofectamineTM 2000. After G418 selection, the stably transfected cells were obtained. Transfection with pcDNA3.1-a significantly increased the expression of GCRG213 in MKN45 cells both at mRNA （35.4%） and protein （49.4%） level, while transfection of pcDNA3.1-b significantly decreased the expression of GCRG213 both at mRNA （32.1%） and protein （50.3%） level. CONCLUSION： The eukaryotic expression vector of gastric cancer associated gene GCRG213 is successfully constructed and identified.