摘要
目的:构建共表达TCRα12-2和TCRVβ7.1重组腺病毒载体Ad.TCRα12.2-IRES-Vβ7.1,并研究其杀伤肝癌细胞的作用。方法:提取外周血单核细胞(peripheral blood mononuclear cell,PBMC)总RNA,用RT-PCR的方法得到TCRα12-2基因。TCRVβ7.1基因由pCDN3.1-Vβ7.1扩增获得,将这两个基因以IRES相连克隆入腺病毒穿梭质粒pDC315中。穿梭质粒和腺病毒骨架质粒共转染HEK293细胞,包装出可同时表达TCRα12-2和TCRVβ7.1的重组腺病毒。然后提取病毒基因组DNA,对外源目的基因进行PCR鉴定;病毒感染宫颈癌细胞(HELA),48小时后提取细胞总RNA,通过RT-PCR鉴定目的基因表达;最后病毒感染PBMC,用流式细胞仪检测目的蛋白的表达。对鉴定正确的病毒进行大量扩增并测定滴度。用MTT比色法检测Ad.TCRα12-2-IRES-Vβ7.1感染的PBMC对肝癌细胞HEPG2和BEL-7402的杀伤作用。结果:从重组病毒基因组中扩增出目的基因TCRα12-2和TCRVβ7.1,RT-PCR和流式细胞仪检测表明目的基因可以有效的表达。Ad.TCRα12-2-IRES-Vβ7.1感染PBMC组对肿瘤细胞的杀伤率明显高于PBMC组和Ad-GFP感染组。结论:成功构建了双表达TCRα12-2和TCRVβ7.1基因的重组腺病毒载体Ad.TCRα12-2-IRES-Vβ7.1,且其感染后的PBMC可有效的杀伤肝癌细胞。
Objective：To construct the recombinant adenovirus Ad.TCRα12-2-IRES-Vβ7.1,and study the cytotoxicity in hepatoma cell.Methods：TCRα12-2 gene was obtained from peripheral blood mononuclear cell （PBMC） by RT-PCR,and TCRVβ7.1 gene was obtained from pCDNA3.1-Vβ7.1.Then,the two genes were cloned into the shuttle plasmid adenovirus pDC315,and the product was co-transfered into HEK-293 cell with adenovirus genomic plasmid.Through high efficiency site specific recombination,we obtained a replication- defective adenovirus Ad.TCRα12-2-IRES-Vβ7.1,which could simultaneously express TCRα12-2 gene and TCRVβ7.1 gene. The recombinant adenovirus was analyzed by PCR;The expression of Ad.TCRα12-2-IRES- Vβ7.1 in Hela cells was confirmed by reverse transcription polymerase chain reaction （RT-PCR）.The protein TCR was detected by flow cytometry after 48h.PBMC was infected by Ad.TCRα12-2-IRES-Vβ7.1.The recombinant adenovirus was propagated in HEK293 cell,and the titer was measured by TCID50. The effect of Ad.TCRα12-2-IRES-Vβ7.1 on BEL-7402 cells and HepG2 cell was measured by MTT assay.Rsults：The TCRα12-2 gene and TCRVβ7.1 gene was detected from genome of the recombinant adenovirus.The expression of TCRα12-2 gene and TCRVβ7.1 gene were detected by RT-PCR and flow cytometry.The killing rate of the PBMC infected by Ad.TCRα12-2-IRES-Vβ7.1 was obviously higher than the PBMC group and Ad.GFP group.Conclusion：The recombinant adenovirus,Ad.TCRα12-2-IRES-Vβ7.1,were constructed successfully,which can express TCRα12-2 and TCRVβ7.1.PBMC simultaneously and have strong killing effect on BEL-7402 cells and HepG2 cells.
出处
《现代生物医学进展》
CAS
2008年第11期2001-2004,共4页
Progress in Modern Biomedicine
基金
国家自然科学基金(30572124)
广东省自然科学基金(5002855)
广东省科技计划项目(2004B31201001)
广东药学院博士科研启动基金(43555014)
