人共刺激分子4-1BBL表达载体的构建及转染Tca8113细胞的研究

Construction of expression vector with human costimulatory molecules 4-1BBL and its stable expression in TcaS113 cells

目的:克隆人肿瘤坏死因子配体超家族(tumor necrosis factor superfamily,TNFSF)成员4-1BBL基因,构建其真核表达载体,并检测4-1BBL基因在Tca8113中的表达。方法:从淋巴细胞中提取RNA,用RT-PCR方法将4-1BBL的编码序列cDNA扩增。然后将该基因的编码序列插入真核荧光蛋白载体pEGFP-C1质粒中,构建成最终的表达载体pEGFP-Cl-4-1BBL。运用脂质体方法将pEGFP-Cl-4-1BBL转染Tca8113细胞,转染24h后,荧光显微镜下观察绿色荧光蛋白的表达,RT-PCR和免疫印迹检测4-1BBL在该细胞中的表达。经G418筛选后,用有限稀释法建立稳定高表达的带有4-1BBL基因的Tca8113细胞系。结果:从淋巴细胞中提取的RNA经RT-PCR扩增出目的基因4-1BBL,其全长大小为768bp。测序鉴定该序列与GenBank中的序列相同。转染pEGFP-Cl-4-1BBL载体的靶细胞Tca8113中,荧光显微镜下可见其表达绿色荧光蛋白,RT-PCR方法检测到目的基因4-1BBL的7686p大小的cDNA扩增产物。裂解的4-1BBL/Tca8113基因转染细胞膜蛋白中相对分子质量约27.5Kd的特异性条带。结论:转染4- 1BBL基因细胞株的建立,为该基因功能的后续研究和单克隆抗体的研制奠定了基础。

PURPOSE： To clone human 4-1BBL cDNA,which is a member of the tumor necrosis factor （TNF） ligand superfamily,construct an eukaryotic expression vector,pEGFP-C1-4-1BBL, and detect the expression of 4-1BB ligand（4- 1BBL） in Tca8113 cells. METHODS： Total RNA was isolated from PBMC lymph-cell, 4-1BBL cDNA was amplified by RT-PCR, PCR products of 4-1BBL were inserted into pEGFP-C1 to form the expression vector of pEGFP-C1-4-1BBL. Twenty-four hours after transfection of PEGFP-C1-4-1BBL into Tca8113 cell line by lipofectamine 2000, the expressions of GFP protein and 4-1BBL were detected by fluorescence microscopy, RT-PCR and Western blot, respectively. Transfected cells were selected in medium containing G418 （400μg/ml） and termed as Tca8113/4-1BBL, using definite dilution method, the cell line of Tca8113/4-1BBL was constructed. RESULTS： The sequence of 4-1BBL,768 bp,was confirmed by sequencing which was identical to be the human 4-1BBL mRNA in GenBank. The recombined expression vector PEGFP-C1-4-1BBL was determined by restriction enzyme digestion and PCR assay.Green fluorescence protein was expressed in Tca8113 cells that transferred by PEGFP-C1-4-1BBL. A 271 bp fragment of RT-PCR product was detected using the 4-1BBL specific primers. Western blot showed a belt about 27.5 kD protein. CONCLUSION： Construction of human 4-1BBL transfectant cell line has a great value for further study of 4-1BBL, which provides preparation of monoclone antibody of 4-1BBL. Supported by National Natural Science Foundation of China （Grant No.30672335）, Guangdong Natural Science Foundation （Grant No.06027977） and Foundation of Shenzhen Bureau of Science, Technology and Information （Grant No. 200601008）.