表达大鼠IL-2、B7-1基因CBRH7919细胞的建立  

Establishment of a CBRH7919 cell line expressing rat IL-2 and B7-1

作　　者：史瑶平[1] 郭燕[1] 李鹤平[1] 沈俊杰[1] 杨建勇[1] 

机构地区：[1]中山大学附属第一医院放射科,广东省广州市510080

出　　处：《世界华人消化杂志》2011年第25期2660-2663,共4页World Chinese Journal of Digestology

基　　金：国家自然科学基金面上资助项目;No.30600156;No.81071247;No.30870692~~

摘　　要：目的:研究携带大鼠IL-2、B7-1目的基因的CBRH7919细胞(即CBRH7919/IL-2/B7-1)体外表达目的基因的能力.方法:RT-PCR扩增Wistar大鼠IL-2、B7-1目的基因,分别克隆至重组逆转录病毒载体pBaBe-puro及pMSCV-neo,构建逆转录病毒载体pBaBe-puro-IL-2及pMSCV-neo-B7-1,重组质粒转染病毒包装细胞293FT(胚胎肾细胞),产生病毒后感染CBRH-7919,用抗生素puro/G418筛选出细胞抗性克隆,并用RT-QPCR、Westernblot及ELISA实验证实抗性细胞在转录水平和蛋白水平IL-2、B7-1的表达情况.结果:用RT-PCR方法成功扩增了Wistar大鼠IL-2及B7-1目的基因,经测序结果证实成功构建了pBaBe-puro-IL-2及pMSCV-neo-B7-1重组质粒.利用该重组质粒成功构建了CBRH7919/IL-2/B7-1细胞系,实时荧光定量PCR检测结果显示CBRH-7919/IL-2/B7-1细胞中IL-2和B7-1基因的平均表达量分别是CBRH-7919-pmscv-neo的4.153倍和17.040倍;Westblot检测结果显示CBRH-7919/IL-2/B7-1细胞B7-1表达是无基因修饰CBRH7919的3倍;ELISA法检测结果显示CBRH-7919/IL-2/B7-1细胞IL-2表达量是无基因修饰CBRH7919的190余倍.结论:成功建立了稳定高效表达大鼠IL-2、B7-1的CBRH7919/IL-2/B7-1细胞系,为下一步开展研究肝癌免疫基因治疗奠定了基础.AIM： To establish a CBRH7919 cell line CBRH7919 expressing rat IL-2 and B7-1 genes and to examine their ability to express the IL-2 and B7-1 genes in vitro.METHODS： The IL-2 and B7-1 genes were am- plified by RT-PCR and subcloned into retroviral vectors pBaBe-puro and pMSCV-neo, respectively, to obtain the recombinant retroviral vectors pBaBe-puro-IL-2 and pMSCV-neo-B7-1. The recombinant plasmids were then transfected into the 293FT packaging cells. The obtained infectious viruses were used to infect the CBRH7919 cell line, and puro/G418-resistant clones were acquired after puro/G418 selection. The expression of IL-2 and B7-1 was detected using Q-PCR, Western blot and ELISA.RESULTS： The rat IL-2 and B7-1 genes were successfully amplified by RT-PCR, and the recombinant plasmids pBaBe-puro-IL-2 and pMSCV-neo-B7-1 were successfully constructed and verified by direct sequencing. A CBRH7919 cell line （CBRH7919/IL-2/B7-1） expressing rat IL-2 and B7-1 was established. Q-PCR analysis showed that the expression levels of IL-2 and B7-1 mRNAs in CBRH-7919/IL-2/B7-1 cells were 4.15 and 17.04 times higher than those in CBRH-7919-pmscv-neo cells. Western blot analysis showed that the expression level of B7-1 protein in CBRH-7919/IL-2/B7-1 cells was 3 times more than that in unmodified ceils, while ELISA showed that expression level of IL-2 was 190 times more than that in unmodified cells.CONCLUSION： A CBRH7919 cell line stably and effectively expressing rat IL-2 and B7-1 genes was obtained and provides a good basis for further research of immuno-gene therapy of liver cancer.

关 键 词：白介素2 B7-1 CBRH7919 原发性肝癌 

分 类 号：R735.7[医药卫生—肿瘤]