鸡PD-1单克隆抗体的制备及生物学功能研究

doi:10.16431/j.cnki.1671-7236.2018.12.007

Abstract

Abstract:

This study was aimed to screen and prepare a monoclonal antibody against chicken PD-1,and preliminarily study the immunological characteristics,binding activity of the monoclonal antibody and its blocking effect on the activation of the chicken PD-1/PD-L1 signaling pathway.The hybridism cell lines was obtained using hybridism technique,the immunological activity of antibody was identified by ELISA method,the biological activity of Ig antibody subtype identification kit and Western blotting.The combination of corresponding monoclonal antibody with the PBMCs was detected by indirect immunofluorescence technique and flow cytometry.Furthermore,the monoclonal antibody was used to treat chicken PBMC cells after IBDV infected for 7 days,and the expressions of IL-2,IL-6 and IFN-γ were detected by Real-time quantitative PCR.The results showed that a hybridism cell line with specific and stable secretion of chicken PD-1 monoclonal antibody was obtained and named PD-1-D05.The subtype of monoclonal antibody belonged to IgG1,and the titers of hybridism cell culture supernatant and ascites were 1:2¹¹ and 1:2.048×10⁵,respectively.ELISA and Western blotting results showed that PD-1-D05 monoclonal antibody reacted specifically with immunogens,no cross reaction was found between PD-1-D05 and pET-28a(+),Rosetta strain protein extract supernatant and unrelated proteins.The indirect immunofluorescence and flow cytometry results showed that the obtained PD-1-D05 monoclonal antibody could specifically combine with the chicken PBMC,the expression of IL-2 significantly increased after PBMC treatment with PD-1-D05 after 7 days of IBDV infection (P<0.05),the expression of IFN-γ significantly decreased (P<0.05),the expression of IL-6 decreased compared with IBDV attack group,but there was no statistical difference (P>0.05).The results suggested that it successfully screened and prepared a cell line that secretes the monoclonal antibody to the extracellular domain of chicken PD-1 and obtained a PD-1 monoclonal antibody with good immunological activity.This monoclonal antibody could specifically identify the PD-1 molecule and combine the PBMCs.Moreover,it could also effectively inhibit and restore immune regulation related cytokines caused by activation of PD-1/PD-L1 signaling pathways induced by IBDV infection,such as the abnormal expression of IL-2 and IFN-γ.

Key words: chicken; PD-1; monoclonal antibody; biological function

CLC Number:

S852.4⁺3

WANG Lingling, SUN Guopeng, XU Ruonan, HE Haixun, YAN Zhanpeng, WANG Yaping, XING Ruilin, REN Pengju, LI Peng, ZHANG Yanfang, ZHU Yanping, YUE Feng, WANG Xuannian. Study on Preparation and Biological Function of Chicken PD-1 Monoclonal Antibody[J]. , 2018, 45(12): 3363-3370.

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Study on Preparation and Biological Function of Chicken PD-1 Monoclonal Antibody

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