摘要
应用基因重组技术构建了人可溶性FL基因逆转录病毒载体pLXSN FL ,经转染PA317细胞和G4 18筛选抗性细胞克隆 ,获得重组病毒液。NIH3T3细胞进行病毒滴度测定后 ,选择适当滴度的重组病毒 (5× 10 5CFU/ml)感染ECV30 4细胞 ;应用聚合酶链反应 (PCR )及反转录聚合酶链反应 (RT PCR )检测感染ECV30 4细胞FL的DNA及mRNA表达 ;应用ELISA测定rhFL在ECV30 4中的表达水平 ,用荧光标记和流式细胞仪检测转基因上清对单个核细胞来源DC的表型影响及3 H TdR掺入法测定DC对T细胞的促增殖作用。结果表明 ,外源性rhFL基因整合到ECV30 4细胞染色体DNA并有效地转录和翻译 ,稳定表达水平为 82 4ng (10 6细胞 /2 4h )的人FL。转基因ECV细胞培养上清能有效诱导人PBMC来源DC的分化和增强DC对T细胞的激发作用。外源性FL基因可以转移到ECV30 4细胞并稳定表达 。
With gene recombination technique,pLXSN FL was introduced into packaging cell PA317 and the cells were selected by G418,the supernatants containing viruses were collected and determined by NIH3T3 assay Exogenous gene rhFL was transfected into ECV304 at optimal titre of the recombinant virus (5×10 5 CFU/ml) DNA and mRNA of FL were analyzed from the transfected ECV304 using PCR and RT PCR Expression of rhFL in ECV304 was measured by ELISA Results showed that the exogenous rhFL gene was subcloned into retroviral vector pLXSN successfully,and it has been integrated into chromosal DNA of transfected ECV304 Results of RT PCR and ELISA showed that the exogenous rhFL gene was expressed in the transfected ECV304 and the expression level of stably soluble FL was 82 4 ng (10 6 cells/24 hrs) The supernatants from ECV FL transfected cells could efficiently induce differentiation of dendritic cells (DC) from PBMC and enhance DC to activate T lymphocytes Exogenous rhFL gene transfected into ECV304 and expressed stablly provides foundation for the further researches in the interaction between endothelial cells and immune cells in vitro
出处
《上海免疫学杂志》
CSCD
北大核心
2003年第1期6-9,共4页
Shanghai Journal of Immunology
基金
江苏省自然科学重点基金资助项目 (No BQ980 3 8)
国家卫生部基金资助项目 (No 98 1 3 2 4)
