共表达TCRα12-2和TCRVβ7.1重组腺病毒载体的构建及其杀伤肝癌细胞作用的研究
摘要
目的:研究双表达TCRα12-2和TCRVβ7.1基因的重组腺病毒载体感染的PBMC对肝癌细胞的杀伤作用,为以后的TCR抗肿瘤研究奠定基础。
方法:主要包括三方面:一、腺病毒穿梭质粒pDC315-TCRα12-2-IRES-Vβ7.1的构建。提取外周血单核细胞(peripheral blood mononuclear cell,PBMC)总RNA,RT-PCR得到TCRα12-2基因。TCRVβ7.1基因由pCDNA3.1-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中得到pDC315-TCRα12-2-IRES-Vβ7.1。
二、重组腺病毒Ad.TCRα12-2-IRES-Vβ7.1的包装及鉴定。穿梭质粒pDC315-TCRα12-2-IRES-Vβ7.1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基因的重组腺病毒。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,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。鉴定正确的病毒命名为Ad.TCRα12-2-IRES-Vβ7.1并进行大量扩增,用TCID50法测定滴度。
三、MTT和流式细胞仪检测Ad.TCRα12-2-IRES-Vβ7.1感染的PBMC对肝癌细胞的杀伤作用。用包装好的病毒Ad.TCRα12-2-IRES-Vβ7.1和对照病毒Ad-GFP分别感染PBMC,感染24小时后和未处理的PBMC分别作用于两株肝癌细胞BEL-7402和HepG2。48小时后用MTT和流式细胞仪检测杀伤效果。
结果:酶切和测序结果证明腺病毒穿梭质粒pDC315-TCRα12-2-IRES-Vβ7.1构建正确并且包装成功。PCR从重组病毒基因组中扩增出目的基因TCRα12-2和TCRVβ7.1,说明目的基因整合到病毒中。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单独使用PBMC组和Ad-GFP感染PBMC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。Ad.TCRα12-2-IRES-Vβ7.1腺病毒感染后的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 its cytotoxicity to hepatoma cell. The use of Ad.TCRα12-2-IRES-Vβ7.1 could perhaps be potent tools for cancer gene therapy.
Methods First, construct the shuttle plasmid pDC315-TCRα12-2-IRES-Vβ7.1. 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 connected by IRES and then cloned into the shuttle plasmid of pDC315.
Second,package and identify the recombinant ahenovirus Ad.TCRα12-2-IRES- Vβ7.1. pDC315-TCRα12-2-IRES-Vβ7.1 and backbone plasmid of adenovierus were cotransfered into HEK293 cells using Lipofectamine2000 to generate recombinant adenovirus.A replication-defective adenovirus Ad.TCRα12-2-IRES- Vβ7.1 could be made by homologous recombinant,which can simultaneously express TCRα12-2 gene and TCRVβ7.1 gene.The recombinant adenovirus was analyzed by PCR to test target genes;The expression of Ad.TCRα12-2-IRES-Vβ- 7.1 in Hela cells was confirmed by reverse transcription polymerase chain reaction procedure(RT-PCR).The protein TCRVβ7.1 was detected by flow cytometry in PBMC which was infected by Ad.TCRα12-2-IRES-Vβ7.1 in 48hs.The recombinant adenovirus were propagated on HEK293 cells,and viruses were tittered useing TCID50 method.
Third,the effect of Ad.TCRα12-2-IRES-Vβ7.1 on BEL-7402 cell and HepG2 cell was measured by MTT and flow cytometry. PBMC ,which was infected by Ad.TCRα12-2-IRES-Vβ7.1and Ad-GFP,was added into the BEL-7402 cells and HepG2 cells . The killing effect on BEL-7402 cells and HepG2 cells was assayed by MTT and flow cytometry after 48 hours.
Results The TCRα12-2 gene and TCRVβ7.1 gene were detected from genome of the recombinant adenovirus.Both the RT-PCR and flow cytometry had detected the expression of TCRα12-2 gene and TCRVβ7.1 gene.The killing effect on tumor cells in the group,in which PBMC was infected by Ad.TCRα12-2-IRES-Vβ7.1 was obviously higher than the PBMC group and Ad.GFP group.
Conclusion Successfully construct the recombinant adenovirus, Ad.TCRα12-2-IRES -Vβ7.1,which can simultaneously express TCRα12-2 and TCRVβ7.1 genes.PBMC infected by TCRα12-2 and TCRVβ7.1 genes had strong killing effect on BEL-7402 cells and HepG2 cells.
方法:主要包括三方面:一、腺病毒穿梭质粒pDC315-TCRα12-2-IRES-Vβ7.1的构建。提取外周血单核细胞(peripheral blood mononuclear cell,PBMC)总RNA,RT-PCR得到TCRα12-2基因。TCRVβ7.1基因由pCDNA3.1-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中得到pDC315-TCRα12-2-IRES-Vβ7.1。
二、重组腺病毒Ad.TCRα12-2-IRES-Vβ7.1的包装及鉴定。穿梭质粒pDC315-TCRα12-2-IRES-Vβ7.1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基因的重组腺病毒。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,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。鉴定正确的病毒命名为Ad.TCRα12-2-IRES-Vβ7.1并进行大量扩增,用TCID50法测定滴度。
三、MTT和流式细胞仪检测Ad.TCRα12-2-IRES-Vβ7.1感染的PBMC对肝癌细胞的杀伤作用。用包装好的病毒Ad.TCRα12-2-IRES-Vβ7.1和对照病毒Ad-GFP分别感染PBMC,感染24小时后和未处理的PBMC分别作用于两株肝癌细胞BEL-7402和HepG2。48小时后用MTT和流式细胞仪检测杀伤效果。
结果:酶切和测序结果证明腺病毒穿梭质粒pDC315-TCRα12-2-IRES-Vβ7.1构建正确并且包装成功。PCR从重组病毒基因组中扩增出目的基因TCRα12-2和TCRVβ7.1,说明目的基因整合到病毒中。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单独使用PBMC组和Ad-GFP感染PBMC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。Ad.TCRα12-2-IRES-Vβ7.1腺病毒感染后的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 its cytotoxicity to hepatoma cell. The use of Ad.TCRα12-2-IRES-Vβ7.1 could perhaps be potent tools for cancer gene therapy.
Methods First, construct the shuttle plasmid pDC315-TCRα12-2-IRES-Vβ7.1. 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 connected by IRES and then cloned into the shuttle plasmid of pDC315.
Second,package and identify the recombinant ahenovirus Ad.TCRα12-2-IRES- Vβ7.1. pDC315-TCRα12-2-IRES-Vβ7.1 and backbone plasmid of adenovierus were cotransfered into HEK293 cells using Lipofectamine2000 to generate recombinant adenovirus.A replication-defective adenovirus Ad.TCRα12-2-IRES- Vβ7.1 could be made by homologous recombinant,which can simultaneously express TCRα12-2 gene and TCRVβ7.1 gene.The recombinant adenovirus was analyzed by PCR to test target genes;The expression of Ad.TCRα12-2-IRES-Vβ- 7.1 in Hela cells was confirmed by reverse transcription polymerase chain reaction procedure(RT-PCR).The protein TCRVβ7.1 was detected by flow cytometry in PBMC which was infected by Ad.TCRα12-2-IRES-Vβ7.1 in 48hs.The recombinant adenovirus were propagated on HEK293 cells,and viruses were tittered useing TCID50 method.
Third,the effect of Ad.TCRα12-2-IRES-Vβ7.1 on BEL-7402 cell and HepG2 cell was measured by MTT and flow cytometry. PBMC ,which was infected by Ad.TCRα12-2-IRES-Vβ7.1and Ad-GFP,was added into the BEL-7402 cells and HepG2 cells . The killing effect on BEL-7402 cells and HepG2 cells was assayed by MTT and flow cytometry after 48 hours.
Results The TCRα12-2 gene and TCRVβ7.1 gene were detected from genome of the recombinant adenovirus.Both the RT-PCR and flow cytometry had detected the expression of TCRα12-2 gene and TCRVβ7.1 gene.The killing effect on tumor cells in the group,in which PBMC was infected by Ad.TCRα12-2-IRES-Vβ7.1 was obviously higher than the PBMC group and Ad.GFP group.
Conclusion Successfully construct the recombinant adenovirus, Ad.TCRα12-2-IRES -Vβ7.1,which can simultaneously express TCRα12-2 and TCRVβ7.1 genes.PBMC infected by TCRα12-2 and TCRVβ7.1 genes had strong killing effect on BEL-7402 cells and HepG2 cells.
引文
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2.孙硕, 李官成. 人类肿瘤抗原的鉴定及应用进展[J]. 国外医学肿瘤学分册, 2005, 32(10): 723-726.
3.董国芳, 盛颖, 宗春华. 甲胎蛋白检测对胃癌诊断的意义[J]. 宁夏医学杂志, 2003, 25 (6) : 354-355。
4.Robert HV, CarlH. A translational bridge to cancer immunotherapy-exploiting costimμlation and target antigens for active and passive T cell immunotherapy[J]. Immun Res, 2003,27 (2-3): 341-355
5.BelloneM, Iezzi G, ImroMA, et al. Cancer immunotherapy synthetic and natural pep tides in the balance [ J ]. Immunol Today, 1999,20 (10) : 457 - 462
6.府军. 克癌致胜[M].中国医药科技出版社, 1992:161
7.郭宝强, 杨金平, 李公宝, 等. 黏附性淋巴因子活化的杀伤细胞抗自体白血病细胞作用的研究[J]. 中国肿瘤临床, 1996, 23(7): 479
8.吴军, 王晓怀, 杨太成, 等. 植物血凝素及淋巴因子激活的杀伤细胞体外抗肿瘤作用的 MTT 比色法分析[J]. 第一军医大学学报, 1999, 21(11): 841
9.徐琪, 安秀梅, 邵莹, 等. 肿瘤患者自体 CD3AK 与 LAK 细胞生物学特性研究[J]. 肿瘤免疫学杂志, 2000, 16(10): 547
10.Haas GP, Solomon D, Rosenberg S A. Tumor infiltrating lympho-cytes from nonrenal urological malignancies. Cancer Immunol Immunother ,1990 , 30 (6) : 342
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