摘要
利用基因重组、PCR扩增、定点突变及转译优化等程序,构建了人白细胞介素6重组表达载体CSV-IL-6(1)和CSV-IL-6(2);并利用DEAE-Dextran法转染猴细胞系COS7获得了有生物活性IL-6的暂时性高表达。IL-6ELISA定量测定表达水平:转染后48小时收集的上清CSV-IL-6(1)2573pg/ml,72小时1476pg/ml;CSV-IL-6(2)分别为8261Pg/ml与7101pg/ml。利用IL-6依赖的杂交瘤细胞株7TD1检测转染后48小时收集的培养上清的IL-6生物活性结果为:CSV-IL-6(1)10~4U/ml、3.9×10~9U/mg;CSV-IL-6(2)6.3×10~4U/ml,7.6×10~9U/mg。
In this study, two expression vectors for recombinant human interleukin-6(rhIL-6):CSV-IL-6(1) and (2) were successfully constructed by routine gene manipulation, PCR amplification, sitespecific mutagensis, and optimionzation of translation. The constructed expression vectors for rhIL-6 were introduced into a simian cell line COS7 using gene transfer by DEAE-Dextran procedure. The expression level of rhIL-6 was determined by EL-6 ELISA using ELISA KIT for quantification of Human Interleukin-6, and the biological activity of rhIL-6 was assayed by 50% maximum incorporation of ~3H-TdR into 7TD1 cells, an IL-6-dependent hybridoma cell line. At 48h after transfection, the supernatant collected from the culture of COS7 cells transfected with CSV-IL-6 (1) contained rhIL-6 2573pg/ml, and at 72h after transfection it containde 1467pg/ ml; that for CSV-IL-6 (2)were 8261pg/ml and 7101pg/ml, respectively. The biological activity (hybridoma growth factor, HGF) at 48h after transfection from COS-CSV-TL-6 (1) was 10~4U/ ml corresponding to 3.9×10~9U/mg; and that from COS-CSV-IL-6 (2) was 6.3×10~4U/ml mcorresponeding to 7.6×10~9U/mg.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
1991年第6期337-342,共6页
Chinese Journal of Immunology
关键词
白细胞介素6
PCR扩增
基因转移
Interleukin-6
PCR amplification
Site-specific mutation
Gene transfer
COS7
