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Preliminary Study On ELISA Method Of Bovine Viral Diarrhea Virus Nanobody

Posted on:2020-04-17Degree:MasterType:Thesis
Country:ChinaCandidate:Z H ZhouFull Text:PDF
GTID:2370330623960957Subject:The vet
Abstract/Summary:PDF Full Text Request
Bovine viral diarrhea virus(BVDV)can infect cows,sheep,pigs and other animals,and produce persistent infection,which is a long-term threat to the healthy development of animal husbandry and causes serious economic losses in China.Nanobodies have the advantages of strong water solubility,high stability,high affinity and weak immunogenicity.They are widely used in the detection and treatment of diseases,food safety analysis and other,becoming a hot spot in the field of life science research.Objective: The anti-BVDV nanobody was obtained by prokaryotic expression system,and the BVDV polyclonal antibody was screened and purified to establish a double-antibody-sandwich ELISA assay based on anti-BVDV nanobody.Methods: 1.In the preliminary work of our laboratory,two anti-BVDV Nanobody11 and Nanobody12 were successfully screened and obtained,and the nanobody genes were respectively obtained by gene synthesis.2.The prokaryotic expression vector was constructed to obtain and purify Nanobody11 and Nanobody12 proteins.3.The ability of the anti-BVDV Nanobody11 and Nanobody12 to bind to the BVDV antigen protein was verified by Western Blot and indirect ELISA.4.Immunized alpaca with BVDV inactivated virus,and purified polyclonal antibodies in immune serum with ?KTA avant protein purifier.Affinity chromatography was used to conjugate BVDV antigen protein(E0,E2,NS2-3,NS5A)with Ni+ SEPHAROSE 6 Fast Flow filler,and then specific polyclonal antibodies were elutriated by gravity column.5.Combine Nanobody11 with biotin to obtain Bio-nanobody11.Meanwhile,Nanobody12,E0 polyclonal antibody,E2 polyclonal antibody,NS2-3 polyclonal antibody,NS5 A polyclonal antibody and alpaca serum polyclonal antibody were respectively connected to horseradish peroxidase.6.Be used anti-BVDV Nanobody11,Nanobody12 and purified 5 BVDV polyclonal antibodies,and the optimal coating amount,optimal blocking solution,optimal sample dilution,optimal enzyme-labeled antibody dilution were determined in turn,a double-antibody sandwich ELISA method for detecting BVDV antigen.Results: 1.Successfully construct the prokaryotic expression system of anti-BVDV nanobodies Nanobody11 and Nanobody12,and obtain Nanobody11 and Nanobody12 proteins reactive with BVDV antigen protein at concentrations of 0.326 mg/m L and 0.317 mg/m L,respectively.2.Purification of BVDV alpaca serum polyclonal antibodies,as well as polyclonal antibodies against E0,E2,NS2-3 and NS5 A.The antibody concentrations were 6.744 mg/m L,3.301 mg/m L,3.383 mg/m L,1.834 mg/m L,and 2.733 mg/m L,respectively.3.Bio-Nanobody11 and E2 polyclonal antibodies were used to preliminarily establish a double-antibody-sandwich ELISA method based on BVDV nanobody.Conclusion: 1.Successfully purified the anti-BVDV Nanobody11,Nanobody12 and demonstrated their reactivity with the BVDV antigen E0 protein.2.Successfully obtained polyclonal antibodies against E0,E2,NS2-3 and NS5 A.3.Bio-Nanobody11 and E2 polyclonal antibodies were used to establish a double-antibody sandwich ELISA method based on anti-BVDV nanobody.
Keywords/Search Tags:bovine viral diarrhea virus, nanobody, polyclonal antibody, double-antibody sandwich ELISA
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