Résumé
Objective:To obtain antibodies against amylin from a 'naive' human Fab fragment antibody phage diasplay library and to analyze the specificity of antigen binding activity.Methods:Panning and screening Fab antibody from the antibody library,the positive clones with well reactivity to amylin were selected after five times selection of 'adsorption-elution-enrichment'.Then the plasmid DNA which was extracted from the clones,was digested with Spe Ⅰ and Nhe Ⅰ to delete gⅢ (about 660 bp).The digested 47 000 bp DNA which was purified after separation of bands from agarose gel was ligated with T4-DNA ligase.The constructed expressing phagemids were transformed to the BL21(DE3)pLysS,soluble Fab was expressed in it by the induction of IPTG and its characteristics and specificity were determined by ELISA and Western blot.Results:Soluble Fab antibodies were expressed in E.coli.According with molecular weight of IgG Fab,protein band of about 47 kD was shown by SDS-PAGE.Western blot using the goat anti human IgG-HRP showed their binding activities.ELISA showed their specificity with amylin antigens and they did not react with bovine serum albumin.Conclusion:The high level expression and identification of the soluble human anti- amylin Fab fragment antibodies has been obtained successfully,which lays a solid foundation for further researching about the biological and pathological activities of amylin.
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Objective To investigate the best fermentation and induction models of the engineered Escherichia coli, in order to obtain the highest expression level of humanized anti HBsAg Fab. Methods The optimal condition governing the growth of the Escherichia coli, with the flask shaking method and the best fermentation and induction conditions to yield the highest production level were explored, and then E coli were grown in fermentor using the fed batch method following the same principle under flask shaking condition to ensure the best production. Results The data obtained from flask shaking conditions showed that when the induction procedure started the amount of anti HBsAg Fab would reach the highest level at mid log growth phase under the induction condition of 25℃ and 0 2% arabinose. Using the DO stat fed batch method, the OD 600 value of the culture would reach 55 2, which corresponded to 110g/L bacterial wet weight. The biological activity of Fab was proved to have well preserved. Conclusion We established the optimal production technic of HBsAg Fab, and lay a foundation to produce HBsAg Fab on a large scale
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Objective To prepare human Fab fragment antibodies against Interleukin-2 and identify their antigenic specificity and combining activities with antigens. Methods The specific Anti-interleukin-2 clones were screened from a natural human Fab fragment antibodies phage display library against the immobilized interleukin-2 antigens. Then the phagemid DNA from the specific clones was digested with Spe I and Nhe I to delete gⅢ (about 660bp). The digested 4.7kb DNA, which was purified after separation of bands from agarose gel using purification kit, was ligated with T4-DNA ligase and the ligated reaction mixture were transformed to the BL21 (DE3) pLysS. Positive clones on the LB agar plates were inoculated to liquid LB culture medium, and when the bacteria were grown to OD600≈0.5 at 37℃ with continuous shaking, IPTG was added to induce the expression of soluble Fab fragment antibodies at 30℃. The expressed products containing Fab fragment antibodies were determined by SDS-PAGE, Western blot and ELISA. Results The soluble products were identified as containing human Fab fragment antibodies against Interleukin-2 by Western blot and formed a Mr 47?103 band under non-reducing condition on SDS-PAGE. The band was then proved as anti-human Fab fragment antibodies by Western blotting. ELISA demonstrated that Fab fragments possessed good antigenic specificity as well as excellent combining activity with interleukin-2 antigens, and the fragments did not react with bovine serum albumin and IL-4 in ELISA. Conclusions The soluble human anti-interleukin-2 Fab fragment antibodies have been highly expressed and successfully identified, and an effective way has been searched out for constructing the engineering antibodies. All of the results may lay a potentially good foundation for engineering human Fab antibodies, and for the clinical application of the antibodies on the immunotherapy of tumor diseases.
Résumé
Objective To assess functional affinity of humanized anti-HBsAg Fab in order to provide theoretical basis for the radioimmunotherapy of liver cancer bearing HBV. Methods With cognizance of the advantages of solid phase method in time consumption and convenience, we assessed the functional affinity of engineered anti-HBsAg Fab with non-competitive ELISA method. After a series of trials affirming the best concentration and the best time of HBsAg to cover the plate and the proper binding time of Fab to HBsAg to reach an equilibrium, we plotted the OD standard curve of the binding reaction of Fab and HBsAg using four grades of concentration of HBsAg and a series of consistency of Fab. We got the consistency of Fab at OD50% through the standard curve, and then calculated the affinity by Law of Mass Action. Results The affinity of anti-HBsAg Fab is between 10 7 -10 8 M -1 , which was only smaller by 10 times than that of anti-HBsAg IgG. Conclusion The Fab which we constructed in our lab, can strongly bind to the target antigen, thus providing a theoretical basis for its clinical use.