title: [Gene cloning,  expression and  binding activity  of anti-CD86 scFv].
abstract: OBJECTIVE
  To construct and express the single chain variable fragment (scFv) against human CD86 in CHO cells, and observe its biological functions of binding with antigen.
  
  
  METHODS
  The V(H); and V(L); genes were cloned by RT-PCR from a murine hybridoma cell line 1D1, which produced the monoclonal antibody (mAb) against human CD86. The CD86-scFv gene was integrated into eukaryotic expression vector to construct pIREST2-EGFP/scFv. CHO cells were transfected by pIREST2-EGFP/scFv plasmid with Lipofectamine(TM); 2000 and then were selected by G418. We used IMAC affinity chromatography to purify CD86-scFv and quantified its concentration by BCA method. Then the identification of CD86-scFv binding to membrane CD86 was performed through competitive inhibition assay. In addition, the growth inhibition effect of CD86-scFv on Raji cells was detected via MTT assay.
  
  
  RESULTS
  One cell line stably expressing CD86-scFv was obtained. The CD86-scFv could identify CD86 molecules on L929-CD86, Raji and Daudi cells, and the positive rates were 67.0%, 72.3% and 80.5%, respectively. CD86-scFv showed the competitive binding to murine parent antibody 1D1. Moreover, CD86-scFv inhibited the growth of Raji cells and the inhibition rate was 28.3% 72 h after 20 μg/mL CD86-scFv was added into Raji cells.
  
  
  CONCLUSIONS
  CD86-scFv has been successfully expressed in CHO cells (named SA-IV) and the antibody shows a good biological function of recognizing CD86 and inhibiting the growth of Raji cells.
authors: Department of Immunology, Medical College, Soochow University, Suzhou, China.
authors: Meng
authors: M
authors: An
authors: Zhiqiang
authors: Z
authors: Wang
authors: Huating
authors: H
authors: Zhu
authors: Yong
authors: Y
authors: Kong
authors: Jing
authors: J
authors: Wang
authors: Lijun
authors: L
authors: Shen
authors: Lianhua
authors: L
authors: Han
authors: Yongjie
authors: Y
authors: Xia
authors: Yuhua
authors: Y
authors: Qiu
mesh_terms: D000818:Animals; D051940:B7-2 Antigen / Q000235:genetics*; D001665:Binding Sites; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D057127:Single-Chain Antibodies / Q000235:genetics*
chemical_list: D051940:B7-2 Antigen; D057127:Single-Chain Antibodies