pEGFP-C2-hTim-3真核表达载体的构建和hTim-3质粒稳定转染16HBE细胞株的建立

Construction of a eukaryotic expression vector,pEGFP-C2-hTim-3,and establishment of a 16HBE cell line stably transfected with a plasmid containing human Tim-3

目的筛选表达Tim-3的人呼吸道相关细胞株,为进一步阐明Tim-3可能与人呼吸道疾病相关奠定基础;构建pEGFP-C2-hTim-3真核表达载体,以期用于hTim-3的功能研究。将测序正确的重组质粒pEGFP-C2-hTim-3转染入人支气管上皮细胞株16HBE中,筛选出阳性细胞克隆,为后续实验提供理论基础。方法用含10%胎牛血清的高糖DMEM培养基于37℃5%CO2饱和湿度培养箱中体外培养人呼吸道相关细胞株16HBE、A549和GLC-82,调整细胞到最佳状态,对数生长期时收集细胞,抽提总RNA,逆转录为cDNA。参照人GenBank中Tim-3基因的全长序列,使用Primer5.0设计并合成其引物,采用PCR检测以上3种细胞Tim-3的mRNA。将健康人外周血Tim-3基因全长cDNA片段克隆入真核表达载体pEGFP-C2,经菌落PCR、双酶切及测序鉴定,构建包含目的基因Tim-3的重组质粒pEGFPC2-hTim-3。利用脂质体介导pEGFP-C2-hTim-3质粒和pEGFP-C2质粒分别转染16HBE细胞,荧光显微镜下观察16HBE细胞的瞬时转染效率,并采用含G418的培养基筛选以获得整合pEGFP-C2-hTim-3和pEGFP-C2的阳性细胞克隆,用荧光显微镜观察pEGFP-C2-hTim-3和pEGFP-C2在16HBE细胞上的定位表达。结果 1)PCR显示人呼吸道相关细胞株16HBE、A549和GLC-82均表达Tim-3 mRNA;2)构建了具有筛选特征和能够稳定表达的pEGFP-C2-hTim-3质粒载体,测序结果与预期设计完全一致;3)瞬时转染后经荧光显微镜检查可见表达pEGFP-C2-hTim-3绿色荧光蛋白的16HBE细胞不足8%,表达pEGFP-C2绿色荧光蛋白的16HBE细胞不足15%。采用G418筛选后获得表达野生型hTim-3和外源性pEGFP-C2-hTim-3的16HBE细胞及转染pEGFP-C2空质粒的16HBE细胞,经荧光显微镜观察前者定位在胞膜上,后者定位于胞质中。结论人呼吸道相关细胞株16HBE、A549和GLC-82均表达Tim-3mRNA,Tim-3可能与人类呼吸道疾病的发生和发展相关。成功构建了真核表达载体pEGFP-C2-hTim-3并获得了高表达pEGFP-C2-hTim-3的16HBE阳性克隆细胞,可供后续研究使用。

Objectives To screen cell lines derived from the human respiratory tract for expression of Tim-3in order to lay the foundation for elucidation of the potential association between Tim-3and respiratory tract diseases;to construct a eukaryotic expression vector,pEGFP-C2-hTim-3,for further study of the function of human Tim-3（hTim-3）;and to transfect pEGFP-C2-hTim-3and pEGFP-C2 into 16HBE cells and screen for positive clones in order to facilitate further study. Methods The human bronchial epithelial cell lines 16 HBE,A549,and GLC-82 were cultured in DMEM with a high concentration of glucose supplemented with 10% fetal bovine serum.Cell lines were maintained at 37 ℃ in a 5%CO2atmosphere with saturated humidity.Peripheral blood was collected from healthy adults and treated with EDTA-K2 as an anticoagulant.Monocytes were separated using Ficoll lymphocyte separation medium.RNA was extracted from the aforementioned cell lines using TRIzol and then reverse-transcribed into cDNA.PCR was performed to detect Tim-3mRNA in the 3cell lines.Monocytes were harvested from the peripheral blood of healthy adults,and the full-length cDNA of the hTim-3gene was amplified.The full-length cDNA of the hTim-3gene was cloned into the plasmid pEGFP-C2.The resulting plasmid was verified using PCR,restriction enzyme analysis,and DNA sequencing.The eukaryotic expression vector pEGFP-C2-hTim-3 was successfully constructed.The recombinant plasmid pEGFP-C2-hTim-3and the plasmid pEGFP-C2 were transfected into 16 HBE cells using Lipofectamine 2000.The results of transient transfection were observed using fluorescence microscopy.The transfected 16 HBE cells were screened with 0.6 mg/ml of G418 and were maintained with 0.4 mg/mL of G418 for two weeks.The expression of fluorescent protein in 16 HBE cells transfected with pEGFP-C2-hTim-3and pEGFP-C2 was observed using fluorescence microscopy. Results 1.PCR results indicated that hTim-3was expressed in monocytes from human peripheral blood and in 16 HBE,GLC-82,and A549 cells.2.DNA sequencing indicated that the sequence of the constructed plasmid（pEGFP-C2-hTim-3）was in accordance with that of GenBank accession nos.BC063431 and AF450242.3.After transient transfection,fewer than 8% of 16 HBE cells transfected with the plasmid pEGFP-C2-hTim3 expressed green fluorescent protein.Fewer than 15% of the 16 HBE cells transfected with the plasmid pEGFP-C2 expressed green fluorescent protein.After screening with G418,fluorescence microscopy revealed that green fluorescence in 16 HBE cells transfected with the plasmid pEGFP-C2-hTim3 was located on the cell membrane,suggesting that human fl-Tim3 was expressed on the membrane.The green fluorescence of 16 HBE cells transfected with the plasmid pEGFP-C2 was mainly located in the cell cytoplasm. Conclusion HTim-3 was expressed in monocytes from human peripheral blood and in 16 HBE,GLC-82,and A549 cells,suggesting that Tim-3may be associated with respiratory tract diseases.The eukaryotic expression vector pEGFP-C2-hTim-3was constructed successfully.A 16 HBE cell line that was stably transfected with the vector pEGFP-C2-hTim-3was successfully established.