[Gene construction and screening of humanized single chain antibody library against VSTM1-v2 cytokine].
To rapidly select potent anti-VSTM1-v2 scFv (single-chain antibody fragment) by construction and screening of a humanized scFv library in which a murine VH-CDR3 library was grafted onto a human scFv framework. A murine VH-CDR3 library was amplified from anti-VSTM1-v2 murine cDNA and grafted on human scFv (VH3-VK1) framework. Anti-VSTM1-v2 scFv templates were selected and enriched through ribosome display, TA-cloned into expression vector, and transformed into BL21 (DE3) for soluble expression of target scFv. A total of 1000 clones were randomly picked. Positive ones were first identified using colony PCR, indirect ELISA, Western blotting and then verified with sequencing and dose response ELISA. At last an anti-VSTM1-v2 humanized scFv with good binding affinity (EC50 = 21.35 nmol x L(-1)) was selected from the humanized library of 10(12) members generated in this study. This scFv antibody might have potential applications. This study provides a new approach for rapid screening of humanized antibodies.
Sichuan Industrial Institute of Antibiotics, Chengdu 610052, China.
Xiao-jin
XJ
Fu
Chengdu Institute of Biological Products Co., Ltd., Chengdu 610023, China.
Yong-xia
YX
Zhang
Chengdu Institute of Biological Products Co., Ltd., Chengdu 610023, China.
Yun-jian
YJ
Dai
Chengdu Institute of Biological Products Co., Ltd., Chengdu 610023, China.
Ming-rong
MR
Wang
D000818:Animals; D022801:Complementarity Determining Regions / Q000235:genetics* / Q000276:immunology; D016207:Cytokines / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000276:immunology; D051379:Mice; D019151:Peptide Library; D011485:Protein Binding; D011971:Receptors, Immunologic / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D022801:Complementarity Determining Regions; D016207:Cytokines; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D019151:Peptide Library; D011971:Receptors, Immunologic; D057127:Single-Chain Antibodies; C550469:VSTM1 protein, human