Font Size: a A A

Preparation Of Anti-bovine PD-1 Antibody And Effect Of PD-1 Polyclonal Antibody Blockade On Proliferation And Apoptosis Of BVDV-infected PBL Cells

Posted on:2020-07-05Degree:MasterType:Thesis
Country:ChinaCandidate:S S LiuFull Text:PDF
GTID:2370330572497570Subject:Prevention of Veterinary Medicine
Abstract/Summary:PDF Full Text Request
Bovine Viral Diarrhea Virus(BVDV)infects ruminants around the world and causes an economic loss of approximately $2 billion annually.BVDV infection results in a severe reduction in lymphocyte population,causing immunosuppression,and the degree of lymphocyte exhaustion is related to the virulence of the BVDV strain and reversible.Programmed cell death protein-1(PD-1)is known to cause peripheral immune tolerance and pathogen-specific immunosuppression,and lead to "depletion" of T cells.Studies have shown that during chronic viral infection,PD-1 is highly expressed on depleted T cells,and can restore T cell function by blocking the binding of PD-1 to PD-L1.However,it is not clear whether blocking the PD-1/PD-L1 pathway can restore lymphocyte levels during acute BVDV infection.In this study,the bovine PD-1 extracellular protein was cloned and expressed to prepare the polyclonal antibody and monoclonal antibody,and determined the effect of PD-1 polyclonal antibody blockade on proliferation and apoptosis of BVDV-infected PBL cells.Firstly,this study,the extracellular domain gene of bovine PD-1 was cloned by PCR,and linked it to the prokaryotic expression vector p ET-28 a to construct the recombinant plasmid p ET-28a-PD-1.And the recombinant p ET-28a-PD-1 was transformed into E.coli BL21(DE3)competent cells to induce expression of bovine PD-1 protein by IPTG.SDS-PAGE and Western-blot analysis were used to determine the expression of the products.The results showed that the bovine p ET-28a-PD-1 recombinant plasmid was successfully constructed,and the sequence alignment showed 100% homology with the bovine PD-1 sequence released on NCBI.A large amount of about 19 k Da bovine PD-1 protein was induced at 37 °C and 0.8 mmol·L-1IPTG,which had good reactogenicity.Secondly,the purified recombinant protein was emulsified with an equal amount of Freund's adjuvant to prepare an immunogen,and the BALB/C mice were immunized to prepare polyclonal antibody.Bovine PBL cells were isolated and infected with CP and NCP type BVDV in vitro,and PD-1/PD-L1 pathway was blocked by polyclonal antibody.BVDV virus copy number,lymphocyte proliferation and apoptosis were detected.The results showed that bovine PD-1 polyclonal antibody recognizes the extracellular domain of bovine PD-1 protein andrestores lymphocyte proliferation,inhibits apoptosis,and reduces BVDV virus copy number.Multi-antibody blockade has a more significant recovery effect on cell proliferation induced by CP-type BVDV infection than NCP type.Finally,the mices were immunized to prepare the bovine PD-1 monoclonal antibody by hybridoma cell fusion technique.The titer and specificity of monoclonal antibody were detected by ELISA,SDS-PAGE,Western blot and indirect immunofluorescence.The results showed that the monoclonal antibody titer reached 1:26,and the obtained monoclonal antibody was able to recognize the exogenous bovine PD-1 protein.In summary,we obtained the recombinant protein of bovine PD-1 extracellular domain,polyclonal antibodies and monoclonal antibodies against the extracellular domain of bovine PD-1 were developed.It was confirmed that the anti-bovine PD-1 polyclonal antibody blocked the lymphocyte proliferative ability of BVDV acute infection,reduced apoptosis,and improved the ability of lymphocytes to clear BVDV virus.To lay the foundation for understanding the role of PD-1 antibodies in acute BVDV infection.
Keywords/Search Tags:bovine viral diarrhea virus, programmed cell death protein-1, prokaryotic expression, polyclonal antibody, monoclonal antibody
PDF Full Text Request
Related items