人PTEN绿色荧光蛋白真核表达载体的构建及鉴定

目的 构建人PTEN绿色荧光蛋白真核表达载体并进行鉴定.方法 参照PTEN基因全序列,在cDNA两端各设计一条对应引物,并引入各自的酶切位点.从正常人胎盘组织中提取mRNA作为模板合成第一链,并扩增目的基因序列片段,经双酶切后定向克隆至绿色荧光蛋白真核表达载体pEGFP-C1中,筛选阳性重组质粒,分别经双酶切、测序法对重组质粒进行鉴定.结果 双酶切和特异PCR结果表明克隆的基因片段约1.2 kb;测序法进一步证实该基因为PTEN编码基因,经NCBIBLAST分析与Gene Bank中基因序列完全相同.结论 成功构建了人PTEN绿色荧光蛋白真核表达载体pEGFP-C1-PTEN,为研究在肿瘤发生发展中的作用奠定了基础.

Construction and identification of human PTEN and green fluorescent protein eukaryotic vector

Objective Aim:To construct and identify eukaryotic vector of human PTEN and green fluorescent protein.Methods a pair of primers matching the each end of PTEN cDNA and supplemented with appropriate endonuclease sites were designed according to the sequence of PTEN published in NCBI GeneBank.The PTEN mRNA was extracted from human placenta tissue and used as template to synthesize the first strand of PTEN cDNA.The PTEN gene with complete expression sequence was amplified,which was cloned into pEGFP-C1 vector after being double-digested by endonucleases.The recombinants were identified with double-endonuclease digestion,specific PCR sequencing.Results The results of double-endonuclease and specific PCR showed that a 1.2kb fragment had been cloned into pEGFP-C1 vector which was further identified by sequencing and NCBI BLAST analysis.Conclusion PTEN and green fluorescent protein eukaryotic vector-pEGFP-C1-PTEN has been successfully constructed which may provide the base for further study of PTEN in tumorigenesis mechanism.