Evaluation Of Neutralization Effectiveness Of Chicken Egg Yolk Prepared On Four Different Antigens Of Classical Swine Fever Virus

Classical swine fever (CSF) is a highly contagious, acute, hyperthermic, multi-systemic hemorrhagic viral disease of pigs, which caused by classical swine fever virus (CSFV), a member of the genus Pestivirus of the Flaviviridae. The main pathological features are multi-hemorrhagic of internal organs, infarction and necrosis. Because morbidity and mortality rate are very high, so CSF has the great harm and it has caused serious economic losses to the pig industry. Someone has used interferon or Chinese herbal medicine for the prevention and treatment of CSF, but there is no success. At present, Swine Fever Vaccine is adopted for the prevention of the disease, but the phenomenon of immunization failure has often appeared, and there is no comprehensive and effective prevention for CSF. Egg yolk antibody as a kind of effective alternative to antibiotics in biological control showed the obvious effect and also opened a new way in the emergent prevention against CSF.In this study, a truncated E2gene located in the position of754to880amino acid in E2and named tE2encoding the major antigenic regions of E2protein that containing neutral antigenic domain of the A1/A2and higher antigenic in B and C was cloned, nucleotide sequences of two linear neutralizing epitopes of CSFV were synthesized, CKEDYRYAISSTNEIGLLGAGGLT is located in the position of693to716amino acid in E2and named P1, QTAVSPTTLRTEVVK is located in the position of693to716amino acid in E2and named P2. P1and P2epitopes were repeated in pUC57for four times. The recombinant proteins tE2,4P1and4P2which had neutralizing epitope were obtained respectively were expressed in E.coli after digesting and connecting into the expression vectors. The recombinant proteins were highly immunogenic by Western blot. Then the three different proteins and Swine Fever Vaccine mixed white oil were used as immunogen to immunize the nine months old Highland laying hens for three times. Each time interval was two weeks. The dose of tE2recombinant protein group of three immunity times was0.50mg per chicken,1.00mg per chicken and1.50mg per chicken respectively,4P1and4P2recombinant proteins groups of three immunity times were0.40mg per chicken,0.60mg per chicken and0.80mg per chicken respectively and Swine Fever Vaccine group of three immunity times was all10pigs dose per chicken.One week after the second immunization, the eggs were collected and the yolk antibodies were extracted, then the titers of the yolk antibodies were detected by indirect ELISA established. The highest titer of Swine Fever Vaccine group was1:1024against coating antigen tE2. The highest titer of the tE2recombinant protein group was1:4096against coating antigen tE2. The highest titer of the4P1recombinant protein group was1:4096against coating antigen4P1. The highest titer of the4P2recombinant protein group was1:1024against coating antigen4P2. Neutralization test in vivo by indirect immunofluorescence showed that anti-Swine Fever Vaccine antibody, anti-tE2recombinant protein antibody, anti-4P1recombinant protein antibody and anti-4P2recombinant protein antibody could neutralize HCLV in varying degrees. Anti-Swine Fever Vaccine antibody and anti-4P1recombinant protein antibody have strongly in neutralizing HCLV, each antibody mixed virus, then accessed PK-15, and immunofluorescence test showed there was no fluorescent appeared after24h and48h. But anti-tE2recombinant protein antibody and anti-4P2recombinant protein antibody mixed virus, then accessed PK-15, and immunofluorescence test showed there was some fluorescent appeared after24h and48h, however the number of fluorescent was significantly less than the positive control’s. Test of rabbit postchallenge result showed anti-Swine Fever Vaccine antibody and anti-4P1recombinant protein antibody can delay and reduce the fever of rabbits. The two antibodies have certain in neutralizing HCLV.Results of this study indicated that in varying degrees, four egg yolk antibodies produced by tE2,4P1and4P2recombinant proteins could neutralize HCLV. However anti-Swine Fever Vaccine antibody and anti-4P1recombinant protein antibody have strongly in neutralizing HCLV. So this study has further certified that the epitopes we selected can induce the production of neutralizing antibodies, also had potential applications in research of epitope vaccine and preparation of neutralizing egg yolk antibodies.