Preliminary Study Of Egg Yolk Antibody Additive Against Pig Diarrhea

Yolk antibodies against specific pathogens can not only play a very good prevention effect, and because the yolk antibody itself contains rich nutrients can be used as feed additives to improve the growth performance of animals. Thus, by way of an antibody protein feed additives to control certain viral regarded as a solution. A porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is a porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) caused by vomiting, diarrhea and dehydration as the main clinical features highly contagious and highly lethal infection disease, is a major threat to the health of piglets, and poor routine vaccination of sows protective effect on piglets. Therefore, the development of egg yolk antibodies against porcine diarrhea feed additives has become one of the new hotspot.From this study with porcine epidemic diarrhea piglets intestinal tissue cloned pig epidemic diarrhea virus S gene antigen index is higher and contain neutralizing antigen domian of 499-638aa(COE gene) and 636-789aa(PEDVS534). Using prokaryotic expression technology will COE and PEDVS534 two neutralizing antigen epitope on cloning to carrier pMD18-T, respectively, after enzyme digestion connect-ion pET-32a expression vector expressed in escherichia coil, respectively with neutralizing antigen epitope COE recombinant proteins and PEDVS534 recombinant protein. The above COE prokaryotic expression, PEDVS534 two recombinant proteins and whole virus inactivated porcine epidemic diarrhea vaccine were used white oil emulsion adjuvants, immune grouping Hy-Line Brown laying hens 9 months of immunity three times, each time interval two weeks. One free PEDVS534 and COE dose of recombinant proteins by immunohistochemistry were 200ug/only, two free doses of 200p,g/only three free dose of 250μg/only; porcine epidemic diarrhea virus by immunohistochemistry of a full free dose of 0.5mL, two free dose of 1.0mL, three free dose of 1.5mL. After three free days began to collect the eggs, egg yolk antibodies extracted and purified using a gradient of ammonium sulfate precipitation method, SDS-PAGE analysis of the purity of the purified yolk antibodies. Virus neutralization assay using egg yolk antibodies neutralizing activity, Selection and the role of strong anti-COE recombinant protein yolk antibodies in clinical trials the incidence of swine, clinical trial results showed that the mortality rate of feeding egg yolk antibody group was significantly lower than the control group.At the same time of enkaryotic expression COE gene cloning technology to pichia expression vector pPIC9K, expressed in pichia, COE for eukaryotic expression recombinant protein. Western blot to identify three types of recombinant proteins have antigenicity. COE expression of recombinant proteins in eukaryotic emulsified with Freund’s adjuvant immunized mice after total free twice at intervals of two weeks, the first free dose of 60μg/only, two free doses of 80μg/only. Ten days after the second immuniz-ation and blood tests carried out in Serum neutralization titers. The results showed that successfully cloned PEDVS534 (534bp) and COE (530bp) gene, PEDVS534 protein and protein size COE prokaryotic expression were 37.5kDa and 37.0kDa, consistent with the theoretical value, the expression of the protein and immunogenic. Eukaryotic protein expression COE size of 27kDa, larger than the theoretical value, the protein may be due to the glycosylation reaction occurs, the neutralizing antibody titers in mice immunized with the protein produced by expression reaches 1:36. PEDVS534 prokaryotic expression of neutralization titer PEDV COE proteins and whole virus as an immunogen prepared yolk antibodies were 1:12,1:25 and 1:120. The recombinant protein yolk antibody anti-COE clinical trials showed diarrhea yolk antibody group was 5.56%, while the mortality rate reached 61.11% in the control group,The piglets diarrhea rate of experimental group and control group was not significant. In this study, Pichia expression system of PEDV COE, protein expression and can induce neutralizing antibodies in mice; successfully prepared three yolk antibodies, and three egg yolk antibody showed some ability to neutralize the virus; The resistance to COE recombinant protein yolk antibody has significant effect on control of diarrhea piglets mortality, for further study and use it as a feed additive to provide a theoretical basis.