Screening Of Nano Antibody Against BVDV NS5A/E2 Protein And Its Effect On Virus Replication

Bovine viral diarrhea virus(BVDV)is a viral infectious disease that can cause diarrhea,abortion,fever,fetal malformation and other symptoms.Due to the complexity of disease pathogenesis,there is no effective means to control and treat BVDV.Therefore,effective antiviral strategies must be specified to fight BVDV infection in animal husbandry.Studies have shown that the neutralizing antibody produced by BVDV-E2 protein can effectively inhibit virus infection.NS5A protein and NS5B protein together constitute virus replicase,and NS5B antibody can inhibit BVDV replication.Therefore,these two proteins can become potential targets of BVDV prevention and treatment drugs.At present,many studies have shown that nano antibodies have good antiviral effects on a variety of viruses,such as HIV,HCV,SARS-Co V-2,but there are few studies on BVDV.The development of BVDV nano antibody is conducive to disease prevention and control.Objective:The purpose of this study is to screen nano antibodies against bvdv-ns5a/E2 protein,obtain high specific nano antibodies,explore their impact on virus replication,and provide experimental materials for the development of anti BVDV biological agents.Methods:(1)The recombinant plasmid p ET30a-E2/NS5A was transformed into BL21(DE3)competent cells.After induction,expression and purification,the recombinant protein was obtained and its reactivity was detected.(2)Alpacas were immunized with recombinant NS5A and E2 proteins,lymphocytes were isolated,VHH gene was amplified by nested PCR,recombinant plasmid pcantab-5e was obtained,TG1 competent cells were transformed,and the insertion rate and library capacity of target gene were identified;The binding ability of phage display library was verified by three rounds of solid-phase panning and ELISA.(3)By analyzing and selecting better nano antibody sequences,the recombinant expression vector was constructed,transformed,induced,expressed and purified.Test its reactivity.(4)BVDV virus was incubated with nano antibody and inoculated into MDBK cells.The blocking ability of different concentrations of nano antibody to BVDV virus replication was analyzed and evaluated by Q-PCR.Result:(1)The recombinant proteins NS5A and E2 were obtained with molecular weights of about 57and 39 k Da respectively.The reactivity was verified by WB.(2)The insertion rate was 88.5%and the library capacity was NS5A nano antibody display library with 3.42×10~6CFU/m L had an insertion rate of83.3%and a library capacity of E2 nanoantibody display library of 8.4%×10~5CFU/m L.(3)After three rounds of panning,the reaction of crude extract of nano antibody was verified by ELISA,and 12 nano antibodies were obtained,including 5 nano antibodies binding to E2 protein and 7 nano antibodies binding to NS5A protein.(4)Two specific nano antibodies Nb2 and NB8 were selected,and the recombinant nano antibody proteins Nb2 and NB8 with molecular weight of about 15 k Da were obtained.The reactivity was good by ELISA.(5)The cell test showed that the nano antibodies Nb2 and NB8 had a certain neutralization ability to the virus.The nano antibody Nb2 reduced the copy number of BVDV by more than 10 times compared with the positive control group in a short time.Conclusion:(1)The recombinant proteins NS5A and E2 were successfully expressed in prokaryotic cells,providing biomaterials for the establishment of nano antibody phage display library.(2)NS5A/E2phage display libraries were constructed respectively to obtain specific nano antibodies.(3)It was confirmed by cell test that nano antibody could block BVDV virus replication.