Study On Preparation And Purification Technology Of IgY Against TGE Virus And PED Virus

Porcine epidemic diarrhea and Porcine transmissible gastroenteritis are causedby virus, bring digestive infection disease, close contact get strong infection.They arecharacterized by vomiting, severe diarrhea, dehydration, high mortality of pigs undertwo weeks. According to the data of Veterinary Epidemic Prevention Department, inthe all kinds of swine contagious diseases, incidence rate and mortality caused byviral diarrhea are highest, and become the main diseases of swine. Now there are noeffective drugs to cure. The trial aims to develop egg yolk immunoglobulin of TGEand PED, and its extract purification technology, expects to offer foundation andtechnique support for disease prevention and IgY scale manufacture.In trail, PED-TGE duplex inactivated vaccines was as immunogen,settingdifferent dosees groups and control group, according to certain plan to immunize hens,stimulating organism produce egg yolk immunoglobulin against TGE and PED. ThenTGE ELISA kit and PED ELISA kit were used to monitor the serum antibody andIgY, building and adopting a indirect ELISA method to monitor the combinationperformance of two antibodies:the method adopted checkerboard means to definitethe best work concentration of coating and Rabbit-anti-Chicken secondary antibody,the coating is PED-TGE duplex inactivated vaccines;To its extract technology,makethe water solution-octanoic acid, ultrafiltration, ammonium sulfate salt fractionationunion means apply to scale manufacture IgY, also determine the technologicalparameters, results are as follow:In trial, PED-TGE duplex inactivated vaccines was as immunogen, immunizedhens,collected and dealt with eggs, finally obtained the growth and decline patterns ofPED/TGE IgY titers, IgY concentration and combination performance of twoantibodies.IgY apperared in one week, antibody titers were low undulate,both OD450of two virus could reach2.900, high titers could keep over4weeks after strenghenimmune;IgY concentration was also low undulate, high concentration could be9.50mg/mL; The antibody combination performance in serum could be5500.There are onsignificant different between dosees (P>0.05), significant differet exists beween testgroups and control group(P<0.01).The immune program is as follows: chest inject,the basic immune dose is1.0mL, interval every two weeks are routineimmunization,the first is1.0mL, second is1.5mL, third is1.5mL, then interval fourweeks is strengthen immunization,immunize1.0mL, control group inject the same dose physiological saline. To the selfbuilt indirect ELISA method, the best workconcentration of coating and HPR-antibody are400times and7000times dilution,through sensitivity test, specificity test and repeatability test determine the ELISAmethod is feasibility.The parameters of definitive union method and the ways to purify is: put9times4℃distilled water,1%octanoic acid in one egg yolk,then fully stir and standing12hin4℃, hypothermia high speed centrifugation to get supernate,then adopt its pH to7.5, temperature25℃,use50kDa membrane carry out hyperfiltration under0.025MPa,then adopt the concentrated solution pH to7.0,add same volume PBS and the finalconcentration50%、33%saturated ammonium sulfate in it, then PBS is used todissolve sediment. Ultraviolet spectrophotometer detects the highest IgYconcentration is9.89mg/mL,extraction yield rate is about90%,SDS-PAGE decectsthe purity,the more steps, the higher purity,finally purity is about95%, ELISA kitsdecect antibody activity,test shows its titer is about2.700.Through these steps, theIgY achieved are high content,purity and strong specificity, the methods are fit forlarge scale produce.