mTIGIT-mLumin跨膜融合蛋白慢病毒载体和真核表达载体的构建及体外的稳定表达

Construction of lentiviral and eukaryotic expression vectors of m TIGIT-m Lumin transmembrane fusion protein and stable expression in target cells

目的:构建以mLumin荧光蛋白为指示系统的小鼠T细胞免疫球蛋白和结构域蛋白(mouse T cell Ig and ITIM domin,mTIGIT)胞外区真核表达载体,并表达mTIGIT-mLumin融合蛋白。方法:分别设计mTIGIT和mLumin的特异性引物,通过PCR技术扩增两个目的基因序列,经酶切、连接,克隆至plenti-puro和pcDNA3.1载体。将构建的plenti-puro-mTIGIT-mLumin慢病毒载体进行病毒包装并感染HEK-293T细胞,而pcDNA3.1-mTIGIT-mLumin真核表达质粒转染非洲绿猴肾成纤维Cos7细胞,利用两种表达载体的药物抗性进行筛选感染或转染的细胞,检测融合蛋白的表达情况。结果:通过PCR顺利扩增获得mTIGIT和mLumin,经酶切、连接插入至载体,基因测序结果证实克隆的mTIGIT和mLumin片段序列正确,并成功正确插入到plenti-puro载体和pcDNA3.1载体中;荧光显微镜和共聚焦显微镜观察显示,转染的HEK-293T细胞和Cos7细胞经过筛选能够稳定表达mTIGIH-mLumin融合蛋白。结论:成功构建plenti-puro-mTIGIT-mLumin慢病毒载体和pcDNA3.1-mTIGIT-mLumin真核表达载体,且能够在目的细胞中稳定表达mTIGIT-mLumin融合蛋白。

Objective： To construct the lentiviral and eukaryotic expression systems of mouse T cell Ig and ITIM domin（TIGIT） cell transmembrane protein fused with mLumin fluorescent protein as an indicator system and to obtain mTIGIT-mLumin fusion protein for the development of a monoclonal antibody product. Methods ： The gene fragments encoding mLumin or mTIGIT which containing extracellular and transmembrane regions,were amplified by PCR with special primers from existed plasmid or mouse genomic DNA, and inserted into plenti-puro and pcDNA3. 1 vectors respectively. The recombinant vector or lentivirus packaging in HEK-293 T cells with plasmid pMD2.0 G were transfected into African green monkey kidney fibroblast Cos7 cells and human renal epithelial 293 T cell respectively. Stable cell lines were selected with G418（geneticin） and purimycin respectively; fluorescence microscope and confocal microscope were used to detect the mLumin expression. Results ： mTIGIT and mLumin were successfully amplified by PCR, and digested, inserted into and ligated with the vector subsequently. The sequence of the cloned mTIGIT and mLumin fragments was proved to be correct and inserted into the plenti-puro and pcDNA3. 1 vectors successfully. Fluorescence and confocal microscopy observation showed that transfected HEK-293 T cells and Cos7 cells could stably express mTIGIT-mLumin fusion protein after screening with G418 or purimycin respectively. Conclusion： The expression vectors of mTIGIT-mLumin were construted correctly and the fusion protein were expressed stably in cells.