TPT1真核表达载体的构建及在肝癌细胞系SMMC-7721中的表达

目的：构建TPT1真核表达载体，并转染肝癌细胞系SMMC-7721，为进一步研究奠定基础。方法：通过PCR的方法在TPT1 ORF（open reading frame，开放读码框）两侧添加EcoRI和BamHI的识别序列，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，VeeScreen和BLAST分析测序结果，lipofeetAMINE转染肝癌细胞系SMMC-7721，荧光显微镜及RT-PCR方法检测表达效率。结果：成功扩增了带有特异性核酸内切酶识别位点的TPT1 ORF片段，双酶切后连入pEGFP-N3，测序结果证明，rPT1正确插入pEGFP-N3中，转导SMMC-7721后，在荧光显微镜下，可见细胞发出明亮的绿色荧光，转染效率在30％左右。RT-PCR分析显示，pEGFP-N3TPT1转导的细胞，TPT1 mRNA水平较对照高0．78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SMMC-7721细胞内高效表达。

Construction of TPT1 eukaryotic report expression vector and its expression in Hepatocarcinoma cell line SMMC-7721

Objective:To construct the eukaryotic report expression vector of TPT1 and transfect it into Hepatocarcinoma cell line SMMC-7721 for further study.Methods:The specific recognize sites of EcoRI and BamHI were introduced respectively into the two ends of TPT1 ORF (open reading frame)through PCR, this PCR product and vector pEGFP-N3 were 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 analyse the sequence, ultrapure plasmids of pEGFP-N3TPT1 and pEGFP-N3 were extracted by QIAGEN plasmids nimi kit, the lipofectAMINE is selected to transfect the plasmids into the Hepatocarcinoma cell line SMMC-7721 the transfection efficiency were checked under the fluorescent microscope and RT-PCR analysis.Results:TPT1 ORF fragment with EcoRI and BamHI recognize sites was obtained and ligated into the pEGFP-N3 vector, the result of sequencing confirmed that TPT1 ORF is correctly inserted into pEGFP-N3 vector.When the Hepatocarcinoma cell line SMMC-7721 were transfected with pEGFP-N3TPT1, the bright green florescence was observed, The transfection efficiency is about 30%.RT-PCR showed in pEGFP-N3TPT1 group,TPT1 mRNA level is 1.78 times of that control.Conclusion:The TPT1 eukaryotic report expression vector pEGFP-N3TPT1 was constituted,this vector could express efficiently in hepatocarcinoma cell line SMMC-7721.