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目的:构建野生型PTEN基因真核表达载体pcDNA3.0PTEN,并进行鉴定。方法:参照野生型PTEN基因mRNA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。从正常人外周血淋巴细胞中提取mRNA作为模板合成PTENcDNA第一链,并扩增目的基因全表达序列片段,经双酶切后定向克隆至pcDNA3.0真核表达载体中,蓝白斑试验筛选阳性重组质粒。分别经双酶切、特异PCR及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为1.2kb;测序法进一步证实该基因为野生型PTEN编码基因,经NCBIBLAST分析与GenBank中PTEN基因序列同源性为99.92%,编码的氨基酸同源性为100%。结论:成功构建了PTEN真核表达载体pcDNA3.0PTEN,为研究PTEN在肿瘤发生发展中的作用奠定了基础。
Objective: To construct the eukaryotic expression vector pcDNA3.0PTEN of wild type PTEN gene and to identify it. Methods: With reference to the full-length sequence of wild-type PTEN gene, a corresponding primer was designed at both ends of the cDNA and introduced into each restriction site. The mRNA of PTEN cDNA was extracted from normal peripheral blood lymphocytes as a template to amplify the first strand of PTEN cDNA. The full-length sequence of the target gene was amplified and double-digested to be cloned into pcDNA3.0 eukaryotic expression vector. Recombinant plasmids. The recombinant plasmids were identified by double digestion, specific PCR and sequencing. Results: The results of double digestion and PCR showed that the cloned gene fragment was about 1.2kb. The sequence of wild-type PTEN gene was further confirmed by sequencing. The nucleotide sequence of PTEN gene in GenBank was 99.92% Amino acid homology is 100%. Conclusion: The PTEN eukaryotic expression vector pcDNA3.0PTEN was successfully constructed, which laid the foundation for the study on the role of PTEN in tumorigenesis.