Construction, expression and in vitro biological effects of idiotype Ig Fab fragment of B-chronic lymphocytic leukemia.
The purpose of this study was to construct expression vectors of idiotype (Id) SmIg in patients with B-chronic lymphocytic leukemia and to express them in E.coli to obtain recombinant Id, and to investigate the effect of the protein on the proliferation and secretion of IL-2 and IFN-gamma of stimulated peripheral blood mononuclear cells (PBMC) in vitro. Light chain gene and Fd fragment of heavy chain gene were inserted into fd-tet-DOG2 vector to construct fd-tet-DOG2-Fab. Fab gene was further cloned into expression vector pHEN2 to construct the soluble expression vector pHEN2-Fab. After induction by IPTG, Fab protein was purified by Ni-NTA-chromatography. MTT was used to determine the effects of purified protein on the proliferation of stimulated PBMC in vitro and the concentrations of IL-2 and IFN-gamma in the culture supernatants were detected by ELISA. The results showed that recombinant pHEN2-Fab expression vector was constructed successfully. Fab protein was expressed in positive clone after induced by IPTG and two specific bands at 24-25 kD position were observed by SDS-PAGE electrophoresis. Proliferation of PBMC could be induced by purified Fab and the concentrations of IL-2 and IFN-gamma in culture supernatants were increased significantly after induction. It was suggested that the expression vector of SmIg Fab fragment was constructed successfully, and expressed and secreted from E. coli. The Fab protein could induce proliferation of PBMC and promote secretion of IL-2 and IFN-gamma.
Wang
Feng
F
Department of Immunology, School of Medical Sciences, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. biowangfeng@163.com
Lei
Ping
P
Hu
Ping
P
Zhu
Lijuan
L
Zhu
Huifeng
H
Zhang
Yue
Y
Yang
Jing
J
Shen
Guanxin
G
D049109:Cell Proliferation; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics; D007130:Immunoglobulin Idiotypes / Q000096:biosynthesis* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics*; D007371:Interferon-gamma / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000276:immunology* / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
C508594:IL2 protein, human; D007140:Immunoglobulin Fab Fragments; D007130:Immunoglobulin Idiotypes; D007135:Immunoglobulin Variable Region; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D007371:Interferon-gamma