Cloning and expressing of tissue inhibitor of metalloproteinases I gene fragment and preparation of monoclonal antibodies against the recombinant protein.
- Author:
Jun HOU
1
;
Jian ZHANG
;
Jing-Xia GUO
;
Lin CHENG
;
Jing ZHAO
;
Jia LIU
;
Jun XU
;
Ai-Xia LIU
;
Yong-Ji SONG
;
Pan-Yong MAO
;
Bo-An LI
;
Yuan-Li MAO
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Antibodies, Monoclonal; immunology; Cloning, Molecular; Humans; Mice; Mice, Inbred BALB C; Recombinant Proteins; biosynthesis; immunology; isolation & purification; Tissue Inhibitor of Metalloproteinase-1; genetics; immunology
- From: Chinese Journal of Experimental and Clinical Virology 2013;27(3):231-233
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo prepare the monoclonal antibody (mAb) against tissue inhibitor of metalloproteinases I (TIMP-I) fusion protein.
METHODSTIMP-I gene was amplified from fibrotic human liver tissue by RT-PCR, then ligated with pQE31 to form recombinant plasmid pQE-TIMP-I and transformed into E. coli BL21. The protein induced by IPTG was purified by 6 x His-tag and used to immunize the BALB/c mice. The specific monoclonal antibodies (mAbs) were prepared by the cell fusion technique. Western Blot were used to detect specificity of mAbs.
RESULTSThe prokaryotic plasmid expressing the recombinant protein was constructed, and the TIMP-I recombinant protein was expressed and purified. Four hybridoma cell lines that secreted anti-TIMP-I mAbs were obtained. 3 of 4 mAbs were the IgG1 subtype. Western Blot indicated the mAbs showed specific combination with TIMP-I protein.
CONCLUSIONThe TIMP-I recombinant protein is highly purified and has strong antigenicity. The anti- TIMP-I mAbs were prepared successfully.
