TIPE2基因过表达慢病毒载体的构建及转染人脐静脉血管内皮细胞

Construction of TIPE2 over-expressing lentiviral vector and transfection of human umbilical vascular endothelial cells

目的构建肿瘤坏死因子α诱导蛋白8样分子2（TIPE2）基因过表达慢病毒载体，包装慢病毒并体外感染人脐静脉血管内皮细胞（HUVEC）。方法设计TIPE2基因扩增引物，PCR扩增获得人源TIPE2目的基因片段并进行纯化，构建重组质粒GV287-TIPE2经琼脂糖凝胶电泳鉴定并进行测序鉴定。重组质粒GV287-TIPE2转染293T细胞构建GV287-TIPE2慢病毒载体，收获并浓缩病毒颗粒，实时定量PCR法检测病毒滴度。GV287-TIPE2过表达慢病毒体外转染HUVEC，荧光显微镜下鉴定转染效率，免疫印迹法检测转染细胞的TIPE2蛋白表达。结果PCR及测序鉴定证实重组质粒GV287-TIPE2中TIPE2基因目的片断插入位置和序列正确。包装的GV287-TIPE2慢病毒载体其病毒滴度为2．0×10^8TU／ml。GV287-TIPE2过表达慢病毒转染293T细胞后在荧光显微镜下可见绿色强荧光；收获病毒转染HUVEC后，在荧光显微镜下可见绿色荧光，且荧光强度不随培养时间延长而衰退。免疫印迹显示，GV287-TIPE2过表达慢病毒转染HUVEC后其TIPE2蛋白表达明显升高（P〈0．001）。结论成功构建GV287-TIPE2慢病毒载体，包装得到高浓度病毒液，转染HUVEC能稳定过表达TIPE2蛋白。

Objective To construct tumor necrosis factor-ct induced protein-8-like molecule 2 （TIPE2） over-expressing lentiviral vector, packaging the lentivirus and transfecting human umbilical vascular endothelial cells （HUVECs） in vitro. Methods The TIPE2 gene amplification primers were designed. The human TIPE2 gene fragment obtained by PCR amplification was purified. The recombinant plasmid GV287-TIPE2 was constructed and identified by agarose gel electrophoresis and sequencing. The recombinant plasmid GV287-TIPE2 was transfected into 293T cells to construct GV287-TIPE2 lentiviral vector, and the virus particles were harvested and concentrated. The virus titer was determined by real-time quantitative PCR. HUVECs was transfected with GV287-TIPE2 over-expressing lentivirus in vitro. The transfection efficiency was assessed by fluorescent microscope, and the protein expression of TIPE2 in transfected cells was examined by Western blotting. Results The PCR and sequencing analysis identified that the target insertion site and sequence of TIPE2 fragment in the recombinant plasmid GV287-TIPE2 were correct. The virus titer of the packaged GV287-TIPE2 lentiviral vector was 2.0 x l0^8 TU/ml. After the transfection of 293T ceils with GV287-TIPE2 overexpression lentivirus, a strong green fluorescence was observed under fluorescence microscope. After the transfected HUVECs was obtained, the green fluorescence was observed under fluorescence microscope, and the fluorescence intensity did not decline with the incubation time. After transfection with GV287-TIPE2 over-expressing lentivirus, the Western blotting showed that the protein expression of TIPE2 significantly increased in HUVECs （P〈0.001）. Conclusion The GV287-TIPE2 lentiviral vector is successfully constructed, and the highly concentrated virus solution is obtained after packaging. Transfection of HUVECs can stably overexpress TIPE2 protein, which lays the experimental foundation for further studies on the function and mechanism of TIPE2.