Résumé
Objective:To highly express the secretory gene engineering anti CD20F(ab') 2 in the E.coli,simplify working processes and enhance the bioactivity of the antibody fragment.Methods:Using the single factor analysis to optimize the ferment parameters.A culture method which most suit for E.coli secreting anti CD20F(ab') 2 was selected.The anti CD20F(ab') 2 was extracted from periplasm then purified it using the strptococal protein G affinity colume and the S200 HR size exclusion chromatography.The activity of anti CD20F(ab') 2 inhibiting the growth of cell Daudi in vitro was eamined using MTT.Results:Using the optimized culture method,the yield of anti CD20F(ab') 2 was distinctly enhanced from 1.9～2.2 mg/L to 3.7～4.3 mg/L and the proportion of anti CD20F(ab') 2 in all the extract also was improved from 9.7～13.2% to 38.1%～46.8%.After the S200 HR size exclusion chromatography,the purity of anti CD20F(ab') 2 could exceed 85%.The outcome of MTT exmiination showed the IC 50 of anti CD20F(ab') 2 and Fab' were 14.6 ?g/ml and 39.5 ?g/ml,respectively.Conclusion:The gene engineering anti CD20F(ab') 2 could highly express in the E.coli by using the optimized culture method.The anti CD20F(ab') 2 inhibited the growth of Daudi cells in vitro more strong than anti CD20Fab'.
Résumé
Background and purpose:Bispecific antibody is a special pattern of genetic engineering antibody.It possesses 2 antigen binding sites, one directed at the effecter cells and the other at the target cells which are always the tumor cells.This enables the effecter cells to recruit to the targets efficiently and exert cytotoxic effect.The experiment was aimed to construct the expression vector of anti-CD3?anti-CD19 bispecific antibody, purification and analyzing the activity of the diabody.Methods:Applyed PCR and overlap PCR to construct the expression vector of anti-CD3?anti-CD19 bispecific antibodies, transformed it into E.coli 16C9 for expression.Purified the diabody by His-tag affinity chromatography and confirmed by 12% SDS-PAGE and Western blot.Antigen binding activity of the diabody was analyzed by FACS.Results:The DNA sequence of the expression vector containing anti-CD3?anti-CD19 fragment was comfirmed.The yield of purified diabody was up to 5 mg/L.The molecular weight of diabody was correctly indicated by SDS-PAGE and Western blot.The diabody could specifically bind to CD19+ Raji cells and CD3+ Jurkat cells while competitively inhibit binding of HIT3a and HIT19a to the cells.Conclusion:Successfully constructed the expression vector of anti-CD3?anti-CD19 bispecific antibodies, the diabody acquired by the method could be produced with high level expression and also had high specific affinity with CD19+ Raji cells and CD3+ Jurkat cells.