新型基因工程抗CD20抗体片段F(ab')2的构建、表达和活性测定

从抗CD2 0 Fab’表达载体上利用PCR方法扩增并修饰其重链恒定区CH1C 末端的DNA序列 ,然后将修饰后的CH1DNA序列重组到原Fab’表达载体中 ,构建成抗CD2 0抗体片段F(ab’) 2 表达载体。将该载体转化宿主大肠杆菌 16C9,实现了抗CD2 0 抗体片段F(ab’) 2 在工程菌中的可溶性分泌表达。经分离纯化获得具有与抗原CD2 0 特异结合的F(ab’) 2 活性片段。竞争性免疫荧光实验的结果表明 :抗CD2 0 F(ab’) 2 片段具有比Fab’更强的竞争性抑制亲本鼠源性单克隆抗体HI4 7与Daudi细胞表面CD2 0 相结合的能力 ;用MTT法检测所得到的数据说明 :F(ab’) 2 比Fab’更为有效地抑制在体外培养的Daudi细胞的生长

Construction, Expression and Active Appraising of a Novel Antibody Fragment F(ab' )2 from a Gene Engineering Anti-CD20 Antibody

The plasmid expressing antibody fragment F(ab') 2 was obtained through remaking the DNA sequence of Fab'. Imitating the hinge domain structure of nature human antibody, the C term of a DNA sequence which coding the domain CH 1 of H chain was amplified and decorated with the PCR technology. The former DNA sequence was replaced by the new modified sequence and the expression vector plasmid of F(ab') 2 was formed. This plasmid was transferred into the E coli 16C9 and the solutable fragment F(ab') 2 was secreted into the periplasmic space. After affinity purification using streptococcal protein G at pH2.7 and the S200 HR size exclusion chromatography, the fragment F(ab') 2 was gained. The result of competing immuno fluorescent light experiment showed that F(ab') 2 could more violently inhibit the combining between the original monoclone antibody HI47 and the CD 20 of cell Daudi than Fab'. The other experimental consequence certificated that F(ab') 2 could more intensely restrain the growth of cell Daudi in vitro than Fab'.