CONSTRUCTION OF EXPRESSION VECTORS AND STUDY ON SINGLE-CHAIN ANTIBODYAND RESHAPING SINGLE-DOMAIN ANTIBODY AGAINST CD3

Two vectors, pWAI80 and pROH80, for expression of single-chain Fv frag ments (ScFv) were constructed. The anti-CD3 VH and VL genes were ampli fied from UCHT1 cells by RT-PCR and sequenced; Both genes were cloned in pWA180 to express native ScFv and pROH80 for GST-ScFv fusion protei n expression. The expression products were analysed by ELISA and Weste rn blot. The combining site of OKT3 was modeled. Human Ig LS1 and Nd w ere selected as accepters of CDRs of OKT3 VL and VH to construct a, re shaping antibody against CD3. By comparing OKT3, LS1 and Nd with their own family sequences, some residues were changed and the reshaping VL and VH genes were designed. The full VH gene was assembled in three s teps with eight chemically synthesized oligonucleotide fragments using overlapping PCR and sequenced. The VH gene was expressed as active pr otein in pCOMB3 and as inclusion bodies in pGEX-4T-1 by ELISA and West ern blot analysis.