Construction of expression vectors and study on single-chain antibody and reshaping single-domain antibody against CD3 |
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Authors: | LIU Xifu XIAO SaGU ZhengWANG YongZHANG WeiguoCHEN AiLIN QingHUANG HualiangSUN Jian CHEN RunshengSHEN Beifen CHEN Xing |
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Institution: | (1) Institute of Genetics, Chinese Academy of Sciences, 100101 Beijing, China;(2) Institute of Biophysics, Chinese Academy of Sciences, 100101 Beijing, China;(3) Institute of Basic Medicine, Academy of Military Medicine Sciences, 100850 Beijing, China |
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Abstract: | 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.
Project supported by the National Natural Science Foundation of China and “863” Plan. |
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Keywords: | expression vector single-chain Fv fragment (ScFv) molecular modeling reshaping antibody glutathione s-transferase (GST) |
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