Isolation And Identification Of Bovine Viral Diarrhea Virus HB-1 Strain And Establishment Of Indirect ELISA For Detection

Bovine viral diarrhea(BVD),also known as mucosal disease,is a worldwide infectious disease caused by bovine viral diarrhea virus(BVDV),which is characterized by intestinal mucosal shedding.Early diagnosis and detection is an important link in the prevention and control or purification of the disease.NS3 protein is an important non-structural protein encoded by BVDV,which is highly conserved and is the immune dominant protein of BVDV.Therefore,it is of great significance to establish a BVDV detection method using NS3 protein.The main research contents are as follows:1.Isolation and identification of BVDV strainNine samples of infected cattle from 2019 to 2020 were collected and identified by RT-PCR.The virus was isolated from the positive samples.After 5 generations of blind transmission,the virus was verified by RT-PCR.After verification,the virus was passed on for 5 generations.Finally,a NCP BVDV strain was successfully isolated and named HB-1.The whole genome sequence of HB-1 was obtained by designing the whole genome primers and amplifying the gene sequence.The homology analysis of nucleotide sequence of the whole genome showed that HB-1and ZM-95 were the highest,86.9%;phylogenetic tree analysis showed that HB-1and BVDV-1m were in the same branch,which proved that HB-1 isolate was BVDV-1m subtype.The results of amino acid sequence analysis of E0,E2 and NS3 proteins were consistent with the results of whole genome typing.2.Establishment of indirect ELISAIn this study,the NS3 gene of HB-1 was amplified by RT-PCR,and the recombinant expression plasmid p ET-32a-NS3 was constructed.The recombinant plasmid was transformed into E.coli BL21(DE3)for expression.The expression of NS3 protein in the supernatant was identified by SDS-PAGE.Western blot analysis showed that NS3 protein had reactivity.Then,the purified and concentrated NS3 protein was used as the coating antigen to establish an indirect ELISA method for BVDV antibody detection.NS3 recombinant protein only reacted specifically with BVDV positive serum,but not with BRV and IBRV positive serum,indicating that the method had good specificity;the sensitivity test of this method showed that BVDV positive serum was still positive at 1:12800 dilution,indicating that the method had high sensitivity;the intra and inter assay coefficients of variation of this method were less than 10%,indicating the methods had good repeatability.The total positive rate of immune serum was 95.59%,and the total positive rate of non-immune serum was 86.16%.The established detection method provides technical support for the detection of BVDV antibody in China.