The Development Of Anti-BVDV Monoclonal Antibodies And The Establishment Of Capture ELISA Detection Method For BVDV

Bovine viral diarrhea(BVD),also known as mucosal disease(MD),is a fever infectious disease of cattle caused by bovine viral diarrhea virus(BVDV).Widely distributed in the world scope,the disease can cause immune suppression and persistent infections(PI),spirit is depressed,concurrent respiratory infections,diarrhea,the digestive tract mucosa erosion,inflammation and necrosis,miscarriage or newborn calf severe diarrhea cause death,the cow milk production decline as the main clinical features,serious influence on the performance of the animal production and caused huge economic losses to the cattle industry.The BVDV genome encodes four structural proteins,namely C,Erns(E0),E1 and E2.Among them,C protein constitutes BVDV nucleocapsid;E0,E1,and E2 are glycosylated proteins that form the BVDV capsule.In this study,the whole BVDV virus was used as the immunogen to screen the monoclonal antibodies against BVDV structural proteins,aiming to establish a simple,rapid and accurate antigen capture ELISA method for the detection of BVDV.This research is mainly divided into two parts,as follows:In the first part,the preparation of monoclonal antibody against BVDV structural protein: CP type BVDV ZD-2018 strain was propagated by bovine testis(BT)primary cells,and purified by superionization and sucrose density gradient centrifugation.Balb /c mice were immunized.When the serum antibody titer was >1:12800,spleen cells of immunized mice were collected and fused with myeloma cells SP2/0,and the positive hybridioma cells were screened by ELISA.The identified positive hybridoma cells were subcloned continuously,and six hybridoma cell lines with stable expression of anti-BVDV antibodies were successfully screened out,which were named as6D2F2,4H6A6,1D8C7,1G8D8,1C7E7 and 3A8E1,respectively.The results of indirect immunofluorescence assay showed that the above monoclonal antibodies could specifically bind to BVDV.Prokaryotic soluble recombinant Escherichia coli expressing C,E0,E1 and E2 proteins were constructed and named as p Cold-tf-C /BL21,p Cold-tf-E0 /BL21,p Cold-tf-E1 /BL21 and p Cold-tf-E2 /BL21,respectively.The expression conditions of target proteins were optimized.Objective the protein was digested by 3C Protease and purified by TALON Metal Affinity Resin.The specificity of 6 monoclonal antibodies against C,E0,E1 and E2 proteins was detected by Western blot.The results showed that monoclonal antibodies 1C7E7 and 3A8E1 could specifically recognize BVDV E0 protein,and monoclonal antibodies 6D2F2,4H6A6,1D8C7 and 1G8D8 could specifically recognize BVDV E2 protein.In the second part,the establishment of BVDV capture ELISA detection method: firstly,E2 protein was prepared by recombinant Escherichia coli p ET-28a-E2/Rosseta,which expressed BVDV E2 protein constructed by our research group in the early stage.After purification by Ni column,rabbits were immunized to prepare rabbit anti-E2 protein polyantiserum.On this basis,the rabbit anti-E2 protein polyclonal antibody and mouse anti-E2 protein monoclonal antibody 6D2F2(purified from ascites)were used as detection antibodies.After optimization,the BVDV capture ELISA method was established.The results showed that the established detection method had good specificity,sensitivity and stability.Deals with the method of establishing 1017 cattle diarrhea feces samples,358 samples of blood and imports bovine serum samples of 200,testing,test results showed that positive rate were 37.95%,24.86% and 0,re-examination by rt-pcr method at the same time,the diarrhea feces samples and a blood sample results coincidence rate were greater than 95%,import bovine serum samples results coincidence rate 100%.In summary,in this study,the whole BVDV virus was used as immunogen to screen out 2monoclonal antibodies 1C7E7 and 3A8E1,and 4 monoclonal antibodies 6D2F2,4H6A6,1D8C7 and 1G8D8,which specifically recognized E2 protein.On this basis,a capture ELISA method for the detection of BVDV was established by using rabbit anti-E2 polyclonal antibody serum and mouse anti-E2 monoclonal antibody 6D2F2.The specificity,sensitivity and stability of this method were good,which provided technical support for the rapid and accurate detection of BVDV.