Preparation and biological identification of monoclonal and functional antibody against human specific polypeptide FXYD6
10.3969/j.issn.1671-8348.2018.10.004
- VernacularTitle:抗人FXYD6特异性多肽单克隆功能性抗体制备及生物学作用鉴定
- Author:
Xiongfei CHEN
1
;
Junjian YUAN
;
Zhiquan ZHANG
;
Zhongjian GUO
;
Yao GUO
;
Ruhai LIU
;
Fengshan LI
Author Information
1. 河北省沧州市中心医院普外一科 061001
- Keywords:
FXYD6;
monoclonal antibodies;
preparation;
biological identification
- From:
Chongqing Medicine
2018;47(10):1309-1313
- CountryChina
- Language:Chinese
-
Abstract:
Objective To study and prepare the monoclonal antibody library against human FXYD6 functional region,to screen the hybridoma cell lines secreting the monoclonal antibodies against intracellular or extracellular region of human FXYD6,and to identify the biological function of monoclonal antibody against extracellular domain.Methods FXYD6 functional region recombinant protein which did not contain the transmembrane region was prokaryotically expressed,purified,and FXYD6 recombinant protein was used to immunize BALB/c mice.Then splenocytes after immunization were fused with myeloma cells SP2/0.After several rounds of screening and cloning,the hybridomas which secreted the antibodies against the extracellular domain or the intracellular domain of human FXYD6 were established.The antibody specificity and subtype were identified with indirect ELISA,western blot and immunohistochemistry.The monoclonal antibodies against the extracellular domain which recognized the native conformation were screened with flow cytometry.The antibody against extracellular region was prepared with the ascites revulsion method and purified.The affinity constants were measured with indirect ELISA.The function of extracellular monoclonal antibody was detected by HepG2 cell line with high expression of FXYD6.Results The hybridoma cell library which secreted the monoclonal antibody against extracellular domain or the intracellular domain of human FXYD6 was successfully obtained,and extracellular region monoclonal antibodies with the functional blocking were prepared.Conclusion The prepared anti-human FXYD6 extracellular monoclonal antibodies could inhibit HepG2 cell proliferation.
