The Construction And Expression Of Engineering Antibody Chimeric Fab Rat Against Human CD2

Aim:To clone Fd and light chain genes and V_H and V_L genes of monoclonal antibody OX34 against human CD2 and verify their accuracy and liability. And then to construct pComb3-displaying OX34-Fab and pComb3C-dispIaying chimeric Fab antibody by use of cloned Fd and light chain genes , V_h and V_l genes respectively. Finally, the gIII genes of the recombinant plasmids were cut out to construct a secretory expression vectors, to confirm the relative antigen binding ability of two kinds of expressed engineering antibody and make a affinity comparison between them and parental mouse antibody. Methods:1. Cloning of the Fd and light chain genes of mAb OX34 rat against human CD2 The total RNA was extracted from hybridoma cell, and the Fd and light chain genes and V_h and V_l genes of mAb OX34 were amplified by RT-PCR. After the cloned genes were inserted into cloning vector pMD18-T, DNA sequencing was done. Then, all obtained DNA sequences were analyzed by corresponding software.2. Construction and expression of OX34-Fab antibody against human CD2 The light chain and Fd genes were sequentialy cloned into pComb3 to construct displaying vector pComb3/Fab-gIII which could be used to display Fab on the surface of phages. Moreover, this vector was transformed into E.coli XL1-blue and rescuing was done with helper phage M13K07. Phages were prepared by...