The establishment of human anti-TROP2 antibody IgG and its inhibition function on pancreatic cancer cell proliferation
  
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DOI:10.46701/APJBG.2017042017045
KeyWord:TROP2, CHO dhfr- cell line, eukaryotic expression system, cell proliferation, pancreatic cancer
                    
AuthorInstitution
Huan Wang Department of Laboratory Medicine, The Affiliated Jiangning Hospital of Nanjing Medical University, Nanjing, Jiangsu, 211100, China;
Xiaojun Tang Key Laboratory of Antibody Technique of Ministry of Health, Nanjing Medical University, Nanjing, Jiangsu, 211166, China;
Xin Xu School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, Jiangsu, 212001, China;
Chu Chu Nanjing Maternity and Child Health Hospital, Nanjing, Jiangsu, 210004, China;
Siping Xiong Key Laboratory of Antibody Technique of Ministry of Health, Nanjing Medical University, Nanjing, Jiangsu, 211166, China;
Feng Zheng Huadong Medical Institute of Biotechniques, Nanjing, Jiangsu, 210002, China
Qisong Peng Department of Laboratory Medicine, The Affiliated Jiangning Hospital of Nanjing Medical University, Nanjing, Jiangsu, 211100, China.
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Abstract:
      On the basis of anti-TROP2 Fab antibody, this study seek to construct a eukaryotic expression system of human anti-TROP2 antibody IgG, and to analyse the inhibition function of human anti-TROP2 antibody IgG in the cell proliferation of pancreatic cancer. The heavy and light chain genes of anti-TROP2 antibody were amplified respectively to establish the recombinant expression vector of human anti-TROP2 antibody IgG, named pWS-anti-TROP2. The human anti-TROP2 antibody IgG was obtained through transfecting the plasmids into the CHO dhfr- cell line, selecting the monoclonal cell strains with high amounts of antibody expression by MTX screening and applying Protein G affinity in purification. The identification and immunologic activity of human anti-TROP2 antibody IgG were researched by Western Blot,SDS-PAGE, ELISA, immunofluorescence assay and flow cytometry method (FCM). MTT assay was conducted to analyse the inhibition effect of human anti-TROP2 antibody IgG on BxPC3 cell proliferation. The human anti-TROP2 antibody IgG eukaryotic expression system was established successfully to express human anti-TROP2 antibody IgG, in which the molecular weight of heavy chain and light chain were consistent with expectation, and it could specifically combine with TROP2 protein, the antibody titer reached 1:6,400. The MTT assay results indicated that human anti-TROP2 antibody IgG had a significant effect on inhibiting the proliferation of BxPC3 cell, and the inhibition function can be gradually increased with improved antibody dose and prolonged time. In the study, the human anti-TROP2 antibody IgG eukaryotic expression system was constructed successfully, the antibody could specifically bind to TROP2 protein on the surface of pancreatic cancer cells, and it is shown to have a significant inhibitory action in pancreatic cancer cell proliferation.
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