Development Of Quail Egg Yolk Antibody Against Transmissible Gastroenteritis Virus Prevalent Strains

Transmissible gastroenteritis(TGE)is an acute and contact infectious disease caused by transmissible gastroenteritis virus(TGEV).TGEV is especially harmful to piglets,which leads to degeneration and necrosis of intestinal mucosa epithelial cells,disorder of water and electrolyte metabolism and dystrophic absorption,serious diarrhea,dehydration and nutrition loss,and high mortality rate of piglets.Vaccination is the main means to prevent tge.Through the immunization of pregnant sows,the primary piglets suck colostrum and get passive protection,but the actual effect in production is not very ideal;With the continuous variation of the virus,the vaccine immune antibody can not provide complete immune protection for piglets,so it is more difficult to prevent and control tge in production,which makes the disease outbreak in some areas of pig farms,resulting in a huge loss.In the practice of tge prevention and control,the effect of anti TGEV specific antibody was obtained by injection or administration.Among them,the yolk antibody(IgY)is an important antibody product.IgY has the advantages of convenient source,simple preparation,relatively low price and good effect,and has been widely used in animal epidemic control.In this study,the inactivated vaccine was prepared after the proliferation of TGEV epidemic strain.Egg producing quail was immunized and anti TGEV IgY was extracted.By studying the anti TGEV effect,specific titer and stability of the specific IgY,a quail derived yolk anti TGEV was obtained,which provided the basic material for further clinical application.The results of this study are as follows:1.The TGEV strain had the highest viral titer at 24 h in PK-15 cells.The proliferation curve of TGEV was measured within 96 h after the virus was inoculated with PK-15 cells.The results showed that the cytopathic effect(CPE)appeared 16 h after the virus was exposed,and reached the peak of virus titer(TCID₅₀=10^-7.3/ml)24 h after the virus was received,which could meet the needs of vaccine preparation.2.TGEV isolate is a popular strain.The N and S gene sequences of TGEV isolate were obtained and analyzed.The results showed that TGEV isolate belonged to purgue like group branch,and was the most popular strain with the strains SY-C and HE-1 in Ya'an and Harbin.3.The indirect ELISA method of TGEV antibody was established.The best concentration of TGEV S protein C anti-situ point protein is 1.5?g/m L.The best dilution of the sample was 1:200,the dilution of enzyme antibody was 1:2000,and the incubation time was 20 min.When OD₄₅₀?0.15,the result is positive.4.Prepare inactivated immunogen to inoculate quail to obtain high immunity egg.The inactivated seedling of TGEV oil in water was prepared.80 egg producing quails were inoculated and serum and high immunity eggs were collected.The results showed that the heavy chain of IgY was about 70 k Da and the light chain was 28 k Da.The specific anti TGEV strain IgY was isolated and purified.5.The anti TGEV epidemic strain,quail IgY,which has high purity and high potency,simple preparation and suitable for mass production,was obtained.The titer and specificity of TGEV IgY were detected.The results showed that the antibody level reached the peak value(OD₄₅₀was 2.219)1 week after the third immunization.The trend of antibody growth in the control group of commercial vaccine was consistent with that of the experimental group,and reached the peak at the seventh week after immunization(OD₄₅₀was 2.100).It is suggested that the inactivated vaccine of TGEV epidemic strain has good immunogenicity.The neutralization titer of IgY was 1:109,which was 5-7 days later than the peak value of IgG antibody(1:120).The maximum antibody dilution of Western blot positive bands was1:16000.IgY was stable at 70?and p H was 4.0-10.0;IgY had a certain resistance to pepsin within 6 hours.