Biological Characteristics Of Nanobody To Bovine Viral Diarrhea Virus (BVDV) And The Establishment Of ELISA Detection Methods

Bovine viral diarrhea virus(BVDV)is a single stranded RNA virus belonging to the Flaviviridae Pestivirus.BVDV is not only infected with cattle,but also persistent infections of goats and sheep.And cause huge economic losses to livestock breeding industry in our country and the global.One important reason that BVDV cannot be eliminated is the existence of persistent infection(PI)animals.Considering that PI animals provide important conditions for the transmission of the BVDV,the key factor is identify and eliminate PI animals to control BVDV.Since 1993,Hamers-Casterman in camel found a lack of light chain of antibody,also known as nanobody.Nanobodies has many advantages but traditional antibodies do not possess,such as high water solubility,strong stability and antigen recognition ability,ow immunogenicity,strong tissue penetration and so on,and the nanobody has been widely used in the field of biology.Objective:To establish an ELISA detection method for bovine viral diarrhea virus(BVDV).Methods:(1)The BVDV-E0 expression vector was constructed,and BVDV-E0recombinant protein was used as coating antigens,and on the 96-microporous plate to conduct the checkerboard titration experiment and optimize the indirect ELISA condition.(2)recombinant BVDV E0 protein as a target antigen,three rounds affinity screening of"dsorption-eluting-amplification"for phage display libraries of propagation,random pick monoclonal by ELISA detection and sequence analysis.(3)the prokaryotic expression vector pET-30a-Nb1 and pET-30a-Nb2 was constructed,and transformed into e.coli BL21(DE3),IPTG induced expression and obtained Nb1 and Nb2 prokaryotic expression protein.(4)the binding capacity of Nbs with recombinant protein E0 and BVDV virus was verified by WB,and its affinity and activity were detected by ELISA.The BVDV virus was inoculated into MDBK cells to explore toxicity on cell.(5)with purified recombinant protein E0 immune rabbit and alpaca,rabbit serum and alpaca serum were obtained respectively.Then,it was assembled into double antibody sandwich ELISA system with nanobody Nb1 to detect BVDV antigen.Results:(1)use establish an indirect ELISA method for 88 serum samples collecte for testing,testing result and import the kits positive results coincidence rate reaches 84%.(2)The results showed that titer of the rescued phage display library was 4.3×10~8 cfu/mL,after three rounds affinity screening of"dsorption-eluting-amplification",the two monoclonals were tested positive by ELISA.(3)the prokaryotic expression vector pET-30a-Nb1 and pET-30a-Nb2 was successfully constructed,and obtain recombinant protein Nb1and Nb2(4)Nb1 and Nb2 can specific binding BVDV virus.On the other hand,cell experiments show that Nb1 and Nb2 are toxic to MDBK cells.(5)based on nanobody detection method were established,some problems still exist in this method.Conclusion:An indirect ELISA was established to detect BVDV antibodies,using recombinant protein BVDV-E0.In this study,two positive nanobodies were successfully found,and the prokaryotic expression vector was constructed.Some biological characteristics are verified,and the detection method based on nanobody is preliminarily explored,and the method is still to be improved.