| By adding a sequence encoding a small peptide, (CPP)3, to the base sequence of pYZFcd20, a new phasmid pYZcpp3 was constructed. Using the pYZcpp3 to transform E.coli. 16c9, the genetically engineered bacteria 10916# was obtained. 10916# can secret the soluble chimeric antibody fragment anti-CD20 F(ab')2 into periplasmic. Without denaturation and renaturation, the F(ab')2 has possessed the native three-dimensional structure. Through the purification of protein G affinity chromatography and S200 size exclusion chromatography, the purity of F(ab')2 was up to 90%.The F(ab')2 can distinguish and bind to the CD20+ B lymphoma cell. Under the same level, F(ab')2 could compete HI47 binding to the CD20 more efficaciously than Fab'. F(ab')2 could inhibit the proliferation of CD20+ B lymphoma cell more intensely in vitro than Fab'. IC50 of the inhibiting Raji and Daudi proliferation were 22.8 μ g/ml, 14.6 μg/ml for F(ab')2 and 45. 9 μg/ml, 39.5 μg/ml for Fab', respectively. F(ab')2 could induce apoptosis of CD20+ B lymphoma cell. The tumor growth of Raji cells xenograft on BALB/c nude mice can be inhibited by F(ab')2. The rate of F(ab')2 inhibiting xenograft tumor was 60%. Using the statistics software SAS 6.12 to analyze our data, a conclusion was drawn: there is a significant difference in the growth of xenograft tumor between the treated group and the control.By optimized the fermentation technology in shaking flask, the yield of chimeric antibody fragments has been improved from 2mg/L to 4mg/L, the percent of F(ab')2 in the chimeric antibody fragments has also been enhanced from 15% to 45%. Based on shaking flask fermentation data, the high density fermentation technology was developed. The OD600 of...
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