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Isolation And Antiviral Function Research Of Bovine Viral Diarrhea Virus NS5B-specific Nanobodies

Posted on:2017-11-12Degree:MasterType:Thesis
Country:ChinaCandidate:H DuanFull Text:PDF
GTID:2370330485480754Subject:Veterinary Medicine
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Bovine viral diarrhea-mucosal disease?BVD/MD?,caused by Bovine viral diarrheamucosal virus?BVDV/MDV?,is infectious disease affecting the cattle industry worldwide.Its main clinical feature include:fever,diarrhea,mucous inflammation,erosion,necrosis,reproductive failure of cow,malformed fetus,immunosuppression etc.The nonstructural protein NS5B is located in the 3 'end of BVDV genome,which has RNA dependent RNA polymerase?Rd Rp?activity.NS5B protein plays an important role in viral replication.Although monoclonal antibodies have been used for the diagnosis and treatment of disease,because of its large size,poor stability,its application is limited.However,nanoantibody?Nb?has excellent performance,including small size,high specificity and solubility,strong penetrating power,high stability.In view of these advantages of Nb,Nb has been applied to clinical diagnosis and treatment.The objective of the present study was to isolate NS5Bspecific nanobodies via phage display,and study the antiviral function of the nanobodies,to provide experimental bases and materials for developing novel antiviral treatments for BVDV infection.The main works and results were as follows:1.Bactrian camel was immunized using the NS5B recombinant protein.The Ig G titer of the serum raised against NS5B recombinant protein reached 1: 128,000 after 6 rounds of immunization.A phage display VHH library,which contains approximately 4.2×108 individual colonies,was constructed from B lymphocyte c DNA encoding VHHs.8 various NS5B-specific nanobodies were screened via phage display,Nb1?Nb45 and Nb100 showed the highest binding activity against NS5B.2.VHH gene was cloned into p Trip CMV-puro vector via Xba I and Eco R I enzyme sites to construct p Trip-CMV-NbsEGFP-puro eukaryotic expression vector.293 T cell was transfected with ps PAX2 ? p MD2.G and p Trip-CMV-NbsEGFP-puro three plasmids.The recombinant Lentivirus expressing VHH was harvested in the culture supernatant.Then MDBK cells lines stably expressing nanobodies were established using Lentivirus.3.The cell lines were infected with BVDV,and the antiviral function of the nanobodies was analyzed.The results showed that intracellularly expressed Nb1 could fully block BVDV replication after incolating an MOI of 0.05 BVDV.Further studies indicated that,intracellularly expressed Nb1 could interact with BVDV-encoded NS5B.In summary,we isolated BVDV NS5B-specific nanobodies from a camel VHH library,and demonstrated its antiviral activity.These results provide useful information for further researching structure and function of BVDV NS5B protein and pave the way for future development of new stratigis to prevent BVDV infection.
Keywords/Search Tags:BVDV, NS5B, nanobody, antiviral function
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