Whole Genome Sequencing Of Bovine Enterovirus HN1817 Strain,Expression Of VP1 Protein And Preparation Of Egg Yolk Antibody

Bovine enterovirus(BEV)is a member of the genus Bovine enterovirus of the family Micro RNA Viruses.Its genome is about 7500 bp in lenth and single-stranded RNA.In this study,the clinical isolates of BEV HN1817 were studied by whole genome sequencing and genetic evolution analysis.Based on this,the expression of VP1 protein and the preparation of egg yolk antibody were carried out to provide material basis and technical support for further in-depth study of the pathogenesis of BEV,prevention and development of new prevention and treatment measures.The conclusions are as following:1.According to the conservative sequence of BEV HN1817 strain,9 pairs of specific primers for HN1817 strains were designed,9 gene fragments were amplified by RT-PCR method,and the 3'end of the gene was amplified by the 3'RACE method,and the whole genome sequence of BEV HN1817 strain was obtained after the sequence splicing and proofreading,the total length of the virus genome was 7 446 bp,and the total length of the virus genome coding area was 6 525 bp.The 5'non-coding region(5'UTR)was 814 bp and the 3'non-coding region(3'UTR)was 105 bp.Genetic evolution analysis of the whole genome,VP1,ORF and 5'UTR sequences showed that the whole genome,VP1,ORF and 5'UTR of HN1817 strain were in the same branch BEV-GX1902 E strain on the evolutionary tree,and the nucleotide homology was highest with 84.50%,81.90%and 84.60%identity respectively,while the He N-A12 as BEV F reference strain were in different branches on the evolutionary tree and the nucleotide homology was relatively low.Amino acid sequence analysis revealed mutations and deletions of some amino acids in BEV HN1817 strain.Therefore,it can be presumed that the HN1817 strain is genetically and evolutionarily belonging to the E-type enterovirus,and the strain also has a large variation during the long-term epidemic.HN1817 strain showed a viral titer of 1×10^8.5TCID50/m L on MDBK cells.2.According to the complete genome sequence of bovine enterovirus HN1817strain,The gene fragment containing VP1(858bp)was amplified by RT-PCR method.The amplified product was cloned into p MD-18T vector and the target fragment was attached to the expression vector p ET-32a.The recombinant plasmid was transformed into Escherichia coli B21(DE3)to obtain the recombinant expression vector BEV-VP1-p ET-32a.After IPTG induction,the SDS-PAGE results showed that the recombinant protein was soluble and highly expressed in Escherichia coli.Different induction conditions were explored in this experiment.It was found that different IPTG concentration,induction time,induction temperature and induction speed had no obvious effect on the expression of target protein.Therefore,1mmol/L IPTG,16?and 24 h were finally used as the best induction conditions for target protein.The purified VP1 protein showed a single target band on PVDF membrane by Western blot,which was the same as the target band in SDS-PAGE,indicating that the purified VP1 protein has good specificity and can be used for animal immunity.3.At present,there is no effective specific drug for the treatment of BEV,and the preventive and therapeutic effects of yolk antibody on viral diseases have been confirmed effectively.Based on this,this study prepared bovine enterovirus inactivated vaccine immune laying hens,and a total of 4 immunizations were carried out.The egg yolk antibody was extracted by water dilution-ammonium sulfate secondary precipitation method,and the egg yolk antibody was purified.The SDS-PAGE purity of the prepared yolk antibody was determined.The gel results showed that there were two chains with sizes of 65 k Da and 33 k Da respectively,which were consistent with the known heavy chain and light chain sizes of the yolk antibody.The protein content of yolk antibody showed that with the increase of immunization times,the protein content of yolk antibody showed an increase trend.After the fourth immunization,the protein content reached the highest value,and the calculated concentration was 50.36 mg·L^-1.The neutralization test results of chicken serum serum and yolk antibody showed that the neutralization titer of Ig G and Ig Y increased with the increase of immunization times.The antibody level in the third immunization serum reached the maximum with a titer of 1:1024,and the antibody level in the fourth immunization reached the maximum with a titer of 1:512.These works provide further evidence for animal experiments.