Screening Of Nanobodies For Important Functional Proteins Of BVDV And Its Effect On Virus Replication

Bovine viral diarrhea virus/mucosal disease virus(BVDV/MDV)can cause acute,fever,and contact infectious diseases in cattle,sheep and pigs,called bovine viral diarrhea/mucosal disease.Most of them are recessive infections,but also cause persistent infections.The disease had brought huge economic losses to the global cattle industry.At present,there are no good treatment measures for the disease,and the focus is on prevention and control.In recent years,studies have shown that the important functional proteins E0,E2,NS3 and NS5A of BVDV participate in important biological functions such as virus replication and mutation,and are important target proteins for the development of anti-BVDV therapeutic drugs.Nanobodies have the advantages of small size,strong specificity,high temperature resistance,easy expression in prokaryotic systems,strong tissue penetration,high stability,and even oral absorption without being degraded.At present,there have been many studies on human virus nanobodies and have shown good antiviral effects,but there are few reports on animal viruses.The development of nanobody drugs has broad prospects in the diagnosis and treatment of diseases.Objective:Using phage display technology to screen specific nanobodies against important functional proteins of BVDV,obtain highly efficient and specific nanobodies and explore their antiviral activities and functions,and provide experimental evidence and material basis for the prevention and treatment of BVDV and the development of anti-BVDV nanobodies.Methods:(1)Immunization with inactivated virus BVDV alpaca,to extract total peripheral blood lymphocytes isolated an RNA,reverse transcription of the c DNA,nested PCR is then used Nanobody gene cloned into the expression vector p CANTAB-5E,and transformed into E.coli TG1 competent cells,bacterial liquid PCR to identify the target gene insertion rate and library capacity;the initial nanobody library was rescued and expanded by helper phage(M13K07).After three rounds of"adsorption-elution-amplification"affinity panning,the reactivity was identified by ELISA.(2)After sequencing and identification,the prokaryotic expression vector of the nanobody gene with p ET-30a was constructed,transformed into E.coli BL21(DE3),and induced by IPTG for expression,and passed through Ni-IDA+affinity Chromatographic column purification.The reactigenicity was detected by ELISA and WB.(3)Mix the BVDV virus and Nanobody Nb2,Nb3,and Nb-YT,respectively,and incubate them,and inoculate about 80%of the MDBK cells in the culture dish.After culturing for a certain period of time,total cell RNA was extracted,and q RT-PCR was performed to analyze the blocking effect of different concentrations of Nanobodies on BVDV virus.(4)After oral administration of BVDV virus in mice,BVDV nanobodies were taken orally,and tissue samples were taken for q RT-PCR to analyze the changes in body weight and the expression of BVDV copy number.Result:(1)A phage display library with an insertion rate of 92.8%,an initial library capacity of 1.52×10⁷CFU/m L,and a storage capacity of 1.84×10¹⁴CFU/m L after rescue by helper phage.(2)After three rounds of screening and panning,7 kinds of Nanobodies with different amino acid sequences were obtained by sequencing,including 3 Nanobodies that bind to E2 protein,1 Nanobody that binds to NS3protein,and 1 Nanobody that binds to NS5A protein.The ELISA results of the two kinds of Nanobodies that bind to E0 protein showed good reactogenicity.(3)The recombinant plasmid of Nb2?Nb3?Nb-YT protein was successfully constructed,and the recombinant nanobody protein Nb2?Nb3?Nb-YT were obtained,with a molecular weight of about 15 Ku,and the reactigenicity was detected by WB.(4)q RT-PCR verified that Nanobodies can neutralize BVDV virus.Conclusion:(1)A phage display library of BVDV Nanobodies was established,and the corresponding Nanobodies were screened for important functional proteins of BVDV.(2)The Nb2,Nb3,and Nb-YT nanobodies were successfully expressed using the E.coli system to provide biological materials for the study of the biological functions of anti-BVDV nanobodies.(3)Cell and animal experiments have confirmed that Nanobodies can block BVDV virus replication.