PRL-3真核绿色荧光蛋白表达载体的构建及其在结肠癌SW480细胞中的表达

Construction of pEGFPN1-PRL-3 vector and its expression in SW480 cells

目的:构建蛋白酪氨酸磷酸酶-3(PRL-3)绿色荧光蛋白真核表达载体pEGFP-N1-PRL-3,为PRL-3基因功能研究奠定基础.方法:设计人PRL-3特异性引物,提取人大肠癌细胞系SW620细胞总RNA,应用RT-PCR方法获取人PRL-3全长cDNA,分别克隆至T载体及真核绿色荧光蛋白表达载体pEGFP-N1,应用PCR、酶切及DNA测序进行鉴定,确认后转染大肠癌细胞SW480,应用G418进行筛选获取PRL-3稳定表达细胞克隆,应用荧光定量PCR检测PRL-3基因表达.结果:获得522 bp的PRL-3基因编码序列并成功构建了真核绿色荧光蛋白表达载体pEGFP.N1-PRL-3,该重组载体能够在SW480细胞中稳定表达.结论:成功构建真核绿色荧光蛋白表达载体pEGFP-N1-PRL-3和建立PRL-3稳定表达SW480细胞,为进一步深入研究PRL-3基因在大肠癌发生发展中的作用奠定基础.

AIM：To construct the eukaryotic fluorescent expression vector of PRL-3 pEGFP-N1-PRL-3 and establish the PRL-3 gene over-expression cell model of human colon carcinoma. METHODS：PRL-3 full length cDNA was amplified by RT-PCR with total RNA extracted from human colon carcinoma SW480 cells as template, and cloned into pGEM-T easy vector or eukaryotic expression vector pEGFP-N1.Recombinant plasmid of pEGFP-N1-PRL-3 was identified by restriction endonuclease analysis and DNA sequencing,pEGFP-N1-PRL-3 plasmid was transfected into SW480 cells.PRL-3 over-expression in SW480 cells was screened by G418 selection. PRL-3 expression was determined by real-time reverse transcription polymerase chain reaction （PCR）analysis. RESULTS：The recombinant pEGFP-N1-PRL-3 plasmid with entirely coding elements of human PRL-3 gene was constructed and identified by restriction endonuclease analysis and DNA sequencing.The PRL-3 expression in SW480 cells was 2.78-fold that of control group. CONCLUSION：Establishment of PRL-3 gene over-expression cell model of human colon carcinoma provides a fundamental tool to study the role of PRL-3 gene in metastasis of human colorectal carcinoma.