野生型抑癌基因PTEN真核表达载体的构建及鉴定

Construction and identification of eukaryotic vector incorporated with wild-type PTEN

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。

Aim： To construct and identify the eukaryotic vector incorporated with wild-type PTEN （pcDNA 3. 0PTEN）. Methods： A pair of primers matching the each end of PTEN cDNA and supplemented with appropriate endonuclease sites were designed according to the sequence published in NCBI GenBank, The PTEN mRNA was extracted from peripheral blood lymphocytes, and used as the template to synthesize the first strand of PTEN cDNA. The PTEN gene with complete expression sequence was amplified, which was cloned into pcDNA 3.0 vector after being double-dlgested by endo- nucleases. The recombirlants were identified with double-endonuclease digestion, specific PCR and sequencing. Results： The results of double-endonuclease digestion and specific PCR showed that a 1.2 kb fragment had been cloned into pcDNA 3.0 vector, which was further identified by sequencing and NCBI BLAST analysis. Conclusion： pcDNA 3.0-PTEN has been successfully constructed which may provide the base for further study of PTEN in tumorigenesis mechanism.