Construction of CSV-IL-6 and its expression in COS7 cells
- VernacularTitle:CSV-IL-6的构建及其在COS7细胞中的表达
- Author:
Jiaxi WANG
- Publication Type:Journal Article
- Keywords:
Interleukin-6;
PCR amplification;
Site-specific mutation;
Gene transfer;
COS7
- From:
Chinese Journal of Immunology
1985;0(06):-
- CountryChina
- Language:Chinese
-
Abstract:
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.
