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AIM:To express chimeric Fd (cFd) and chimeric light chain(cL) in E.coli respectively and refold them into chimericFab (cFab) antibody.METHODS:cFd and cL genes were respectively insertedinto the prokaryotic expression vector pET32a to constructrecombinant vectors pET32a/cFd and pET32a/cL.Then,the competent E.coli cells were transformed by therecombinant vectors and induced by IPTG.Moreover,alarge quantity of cFd and cL expression products wereprepared and mixed with equal molar to refold into cFabby gradient dialysis.The refolded products were identifiedand analyzed by sodium SDS-PAGE,Western blotting,ELISA and HPLC.RESULTS:High efficient prokaryotic expressions of bothcFd and cL in the form of non-fusion protein were obtainedwith the expression levels of 28.3% and 32.3% of totalbacteria proteins,respectively.Their relative molecularmasses were all 24 ku or so,and both of them mainlyexisted in the form of inclusion bodies.In addition,cFdand cL were successfully refolded into cFab by gradientdialysis,with about 59.45% of recovery when the startingtotal protein concentration was 100μg/mL.The renaturedcFab could specifically bind to related antigen with highaffinity.CONCLUSION:The cFab antibody against humanhepatoma was highly and efficiently expressed andrefolded,which laid a solid foundation for studying itsapplication in the treatment of hepatoma.
AIM: To express chimeric Fd (cFd) and chimeric light chain (cL) in E. coli respectively and refold them into chimeric Fab (cFab) antibody. METHODS: cFd and cL genes were respectively inserted into the prokaryotic expression vector pET32a to construct recombinant vectors pET32a / cFd and pET32a / cL.Then, the competent E. coli cells were transformed by therecombinant vectors and induced by IPTG. Moreover, a large quantity of cFd and cL expression products wereprepared and mixed with equal molar to refold into cFabby gradient dialysis.The refolded products were identified and analyzed by sodium SDS-PAGE, Western blotting, ELISA and HPLC .RESULTS: High efficient prokaryotic expressions of both cFd and cL in the form of non-fusion protein were obtained with the expression levels of 28.3% and 32.3% of totalbacteria proteins, respectively Their relative molecularmasses were all 24 ku or so, and both of them mainlyexisted in the form of inclusion bodies. In addition, cFd and cL were successfully refolded into cFab by gra dientdialysis, with about 59.45% of recovery when the starting total protein concentration was 100 μg / mL. The renaturedcFab could specifically bind to related antigen with highaffinity. CONCLUSION: The cFab antibody against human hepatoma was highly and efficiently expressed and removed, which laid a solid foundation for studying itsapplication in the treatment of hepatoma.