Preparation of an antifibrin thrombus-specific murine/human chimeric monoclonal antibody Fab fragment in Escherichia coli.
We have successfully developed a murine antifibrin monoclonal antibody designated SZ-63 with property of binding specifically with thrombus both in vitro and in vivo. In order to reduce its immunogenicity and molecular weight, a murine/human chimeric Fab fragment of the antibody was prepared. mRNA was selected on oligo (dT) cellulose from total RNA isolated from SZ-63 hybridoma cells. cDNAs coding for heavy and light variable regions were amplified by reverse transcriptase polymerase chain reaction. The amplified fragments were cloned and sequenced. The nucleotides of SZ-63 VH and VL are 354 and 321 respectively. The variable genes were then linked with human IgG gamma1 CH and kappa CL genes. Expression vector pHEN1-63 Fab/Hu was thereby constructed and chimeric Fab fragment was expressed in E. coli HB2151 cells in soluble form. Western blot and ELISA results showed that it remained the same capability of binding with cross-linked fibrin as the murine SZ-63 antibody, the content in culture is about 125 ug/L.
Xia
L
L
Jiangsu Institute of Hematology, Thrombosis and Haemostasis Research Unit, Suzhou Medical College, China.
Gu
J
J
Zhang
X
X
Liu
Y
Y
Wan
H
H
Li
P
P
Ruan
C
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005337:Fibrin / Q000276:immunology*; D006801:Humans; D006825:Hybridomas; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis*; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013927:Thrombosis / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins; D005337:Fibrin