Construction of expression vectors and study on single-chain antibody and reshaping single-domain antibody against...

Two vectors, pWAI80 and pROH80, for expression of single-chain Fv fragments (ScFv) were constructed. The anti-CD3 VH and VL genes were amplified from UCHT1 cells by RT-PCR and sequenced. Both genes were cloned in pWAI80 to express native ScFv and pROH80 for GST-ScFv fusion protein expression. The expression products were analysed by ELISA and Western blot. The combining site of OKT3 was modeled. Human Ig LS1 and Nd were selected as acceptors of CDRs of OKT3 VL and VH to construct a reshaping 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 steps with eight chemically synthesized oligonucleotide fragments using overlapping PCR and sequenced. The VH gene was expressed as active protein in pCOMB3 and as inclusion bodies in pGEX-4T-1 by ELISA and Western blot analysis.