TPT1表达载体的构建及在肝细胞系L-02中的表达

目的构建肝细胞癌高表达基因TPT1真核表达载体,并转导肝细胞系L-02,为进一步研究其在肝细胞癌发生、发展过程中的作用奠定基础。方法通过PCR方法在TPT1 ORF两侧添加EcoRⅠ和BamHⅠ的识别序列而将其定向插入真核报告表达载体pEGFP-N3中,构成pEGFP-N3TPT1重构体,卡那霉素筛选阳性克隆,碱裂解法抽提质粒,Sanger法测序,VecScreen和BLAST分析测序结果,lipofectAM INE转导肝细胞系L-02,荧光显微镜及RT-PCR方法检测表达情况。结果将TPT1成功插入pEGFP-N3中,转导L-02后,在荧光显微镜下,可见明亮的绿色荧光。pEGFP-N3TPT1转导的细胞,TPT1 mRNA水平较对照高1.59倍(P<0.05)。结论成功构建了TPT1的真核报告表达载体pEGFP-N3TPT1,pEGFP-N3TPT1可在L-02细胞内高效表达。

Construction of TPT1 eukaryotic report expression vector and its expression in liver cell line L-02

Objective To construct the eukaryotic report expression vector of TPT1 and transduct it into liver cell line L-02 for further study.Methods The specific recognize sites of EcoRⅠ and BamHⅠ were introduced respectively into the two ends of TPT1 ORF through PCR,this PCR product and vector pEGFP-N3 were doubled digested with EcoRⅠ and BamHⅠ restriction enzyme respectively and then ligated together,kanamycin was used to screen the positive reconstituted vector,plasmids were extracted by miniprep and checked through sequencing.VecScreen and BLAST were used to analyze the sequence,ultrapure plasmids of pEGFP-N3TPT1 and pEGFP-N3 were extracted by QIAGEN plasmids nimi kit,the lipofectAMINE was selected to transduct the plasmids into the L-02 cell and transduction was repeated three times in 24 h.The transduction efficiency was checked under the fluorescent microscope and RT-PCR analysis.Results TPT1 ORF was correctly inserted into pEGFP-N3 vector.When the L-02 cells were transducted with pEGFP-N3TPT1,the bright green florescence was observed.In pEGFP-N3TPT1 group,TPT1 mRNA level was 2.59 times of that in pEGFP-N3 control group.Conclusion The TPT1 eukaryotic report expression vector pEGFP-N3TPT1 was constituted,this vector could express efficiently in liver cell line L-02.