抑癌基因PTEN真核表达载体的构建及其在人舌鳞癌SCC-4细胞系中的表达

［摘要］ 目的：构建抑癌基因PTEN真核表达载体pEGFP-PTEN，并观察其在人舌鳞癌scc-4细胞系中的表达，为舌鳞癌的基因治疗提供理论依据。方法：提取人HeLa细胞总RNA，采用RT-PCR法获取PTEN全基因，克隆至pMD18-T载体，经EcoRⅠ和XhoⅠ双酶切后与真核表达载体pEGFP-N1连接，构建pEGFP-PTEN重组质粒。双酶切及测序鉴定后，经脂质体介导转染至体外培养的人舌鳞癌SCC-4细胞系，荧光显微镜观察绿色荧光蛋白在细胞内的表达；Western blotting法检测细胞内PTEN蛋白的表达。结果：pEGFP-PTEN重组质粒双酶切，克隆的目的基因片段约为1 200 bp，测序结果与GenBank上PTEN序列完全一致；荧光显微镜下观察到pEGFP-PTEN在SCC-4细胞系中成功表达；Western blotting结果，转染组PTEN蛋白表达强度为1.07±0.15，与空转染组（0.62±0.11）和未转染组（0.57±0.08）比较差异有统计学意义（P<0.05)。结论：成功构建pEGFP-PTEN重组真核表达载体，该载体能在人舌鳞癌SCC-4细胞系中表达。

Construction of PTEN gene eukaryotic expressing vector and its expression in tongue squamous cell carcinoma SCC-4 cell line

Objective To construct the eukaryotic expressing vector of PTEN gene and to express the gene in SCC-4 cell line and to provide theoretical foundation for gene therapy of tongue squamous cell carcinoma.Methods Human total length of PTEN gene was obtained from HeLa cell line by RT-PCR and was cloned into pMD18-T to analyze the sequence.The correct PTEN was inserted into pEGFP-N1 vector.pEGFP-PTEN was identified by digestion with EcoRⅠ and XhoⅠ.Scc-4 cell line was transfeted by pEGFP-PTEN and the expression of PTEN was observed by fluorescence microscope and Western blotting.Results A 1 200 bp fragment had been cloned into pEGFP-N1 vector which was further identified by sequencing and NCBI BLAST analysis.pEGFP-PTEN was expressed successfully in SCC-4 cell line.Western blotting showed the expression of PTEN protein in pEGFP-PTEN group was 1.07±0.15,there were significant differences compared with pEGFP-N1 group(0.62±0.11) and blank control group(0.57±0.08)(P<0.05).Conclusion The eukaryotic recombinant vector pEGFP-PTEN is constructed successfully and could be overexpressed in SCC-4 cell line.