Prokaryotic expression of human cTnI and preparation of anti-cTnI monoclonal antibody
- VernacularTitle:人心肌肌钙蛋白I的重组表达及其单克隆抗体的制备
- Author:
Neng YANG
;
Xia XU
- Publication Type:Journal Article
- Keywords:
Troponin I;
Antibodies;
Monoclonal
- From:
Chinese Journal of Laboratory Medicine
2003;0(11):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To prepare monoclonal antibody (mAb) against recombinant human cardiac troponin I(cTnI).Methods The full-length gene encoding human cardiac troponin I (cTnI) was synthesized chemically and inserted into expression plasmid pBV220 to construct recombinant plasmid p pBV220/cTnI. The recombinant plasmid was transformed into E.coli DH5? which then expressed cTnI. The immunological activity of the expressed cTnI was analyzed by Western blot. Recombinant human cTnI protein was used as antigen to immunize BALB/c mice. Monoclonal anti-bodies against cTnI were prepared by normal hybridoma technology. The relative affinity of mAbs was determined by ELISA. Specificity of mAbs was analyzed by Western blot.Results Human cTnI gene was synthesized and confirmed by DNA sequencing. Positive recombinant clones were identified by restriction enzyme digestion analysis and DNA sequencing. Western blot analysis showed that the cTnI protein could be recognized by an anti- cTnI antibody. Two hybridmas producing antibodies against cTnI were obtained. IgG isotypes of two mAbs were IgG2a and IgG2b. Western blot showed that the antibodies were specific for cTnI. Neutralisation test showed that these mAbs could be evidently neutralized by cTnI.Conclusion The recombinant expression plasmid of cTnI was constructed successfully and expressed in E.coli. The method of EL ISA established to test serum cTnI is to clinically useful. The cTnI mAb which using cTnI as antigen prepared in this paper can be used for cTnI immunoassay in vitro.
