【摘要】 目的:B7-H3作为B7家族的最新成员,其受体在活化的T细胞表面可被诱导表达,B7-H3对CD4+和CD8+细胞的生成均具有增加作用,并可选择性增加IFN-γ的生成。为将B7-H3基因转染入舌鳞癌细胞Tca8113中制备瘤苗,克隆人共刺激分子B7-H3基因,构建其真核表达载体,并检测B7-H3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B7-H3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中,构建成最终的表达载体pEGFP-C1-B7-H3。运用脂质体方法将pEGFP-C1-B7-H3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检测B7-H3在该细胞中的表达。结果:从淋巴细胞中提取的RNA质量较好,经RT-PCR扩增出目的基因B7-H3,其全长大小为951bp。测序鉴定,该序列与GenBank中序列相同。转染pEGFP-C1-B7-H3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B7-H3的一个215bp大小的cDNA扩增产物。结论:酶切及RT-PCR证实成功构建人B7-H3的真核表达载体pEGFP-C1-B7-H3,将该载体转染到Tca8113中,RT-PCR鉴定B7-H3基因在该细胞中表达。更多还原

【Abstract】 PURPOSE:B7-H3 is a new member of B7 family and the expression of B7 receptor could be induced on activated T cells.The production of CD4+ and CD8+ was stimulated by B7-H3,and the progress of IFN-γwas also selectively activated by B7-H3.For making tumour vaccine,in this study,human costimulating molecule B7-H3 was cloned,and an eukaryotic expression vector,pEGFP-C1-B7-H3 was constructed and the expression of B7-H3 was detected in tongue squamous cell carcinoma Tca8113 cells.METHODS:The total RNA was isolated from PBMC lymph-cell and the B7-H3cDNA was amplified from the mRNA by RT-PCR,and then the PCR products of B7-H3 were inserted into pEGFP-C1,to form the expression vector,pEGFP-C1-B7-H3.After 24 hours of transferring of pEGFP-C1-B7-H3 on Tca8113 cell lines by lipofectamine 2000,the expression of GFP protein and B7-H3 were detected by fluorescence microscopy and RT-PCR,respectively.RESULTS:The coding sequence of B7-H3,951bp,was confirmed by sequencing which was identical with the sequence of human B7-H3 mRNA in GenBank.The recombined expression vector pEGFP-C1-B7-H3 was determined by restriction enzyme digestion and PCR assay.Green fluorescence protein was expressed in Tca8113 cells that transferred with pEGFP-C1-B7-H3.A 215 bp fragment of RT-PCR product was detected by using the B7-H3 specific primers.CONCLUSION:An eukaryotic expression vector,pEGFP-C1-B7-H3 was constructed.B7-H3 was expressed in targeted cells.Supported by Foundation of Shenzhen Bureau of Science Technology and Information(Grant No.200404099).更多还原