The Preparation And Protection Effect Of Chicken Egg Yolk Immunoglobulin (IgY) Against White Spot Syndrome Virus (WSSV)

White spot syndrome(WSS),is one of the most virulent diseases,causing high mortality and large economic losses in cultured shrimp worldwide.Up to date no effective measuer is available to pervent the spreading of WSSV.Therefore,it is urgent to find an effective method to control this pathogen.Chicken egg yolk immunoglobulin(IgY) provides an inexpensive,effective and stable source of antibodies for the passive immunization of animals suffering from different kinds of diseases.To complicated water environment and invertebrates lack a true adaptive immune response system,the passive immunization of specific IgY is a promising immunity preparation for the treatment and prevention of aquatic diseases.In this study,WSSV were amplified,separated,identified.The major antigen region gene VP28,VP19,VP15 of WSSV were amplified by PCR,and then linked together by overlap extension PCR with the designed linkers(GGDAGD and GNSPNA) as overlap regions.The single gene and fusion gene was cloned into the eukaryotic expression plasmid pCI,and then pCI-28,pCI-19,pCI-15,pCI-28-19-15 DNA vaccine was sucessfuly constructed.The E.coli BL21 cells and HeLa cells were transfected by DNA vaccine and the expression of goal gene was detected by Western Blotting and indirect immunofluorescence assay(IFA).The results showed genes that could express and recognise antibody,and these DNA vaccines had good immunogenicitySubsequently,hens were immunized with recombinant plasmids as antigens which used CpG oligodeoxynucleotides(CpG ODNs) as adjuvant,compared with inactivated WSSV vaccine.And the activity of IgY was detected by indirect enzyme-linked immunosorbent assay(ELISA).The results demonstrated that the CpG ODNs enhanced the immune response of DNA vaccine significantly(P＜0.05).The DNA vaccine and inactivated WSSV showed significant difference compared with negative control(P＜0.05).Anti-WSSV IgY still maintained high antibody activity on day 110.But There was no significant difference between single DNA groups.The single DNA group showed no significant difference compared with fusion gene DNA group and the mixture group.IgY was purified from the water-soluble fraction by a combination of several purification techniques including salt precipitation,ultrafiltration and freeze-drying.The yield of IgY is above 120mg/egg,and the contents of specific IgY in yolk varied at the same trend to that of IgY tite.The average contents of total IgY from different antigens are consistent.But the contents of specific IgY from DNA vaccines are more than negative control,and less than inactivated WSSV.The protective effects of IgY powder and egg yolk powder against WSSV in the experiment ainimals were evaluated.In viral neutralization experiment by intramuscular injection,specific IgY against WSSV could inhibit WSSV to some extent.In oral administration experiment,those fed a diet containing 1%IgY power of inactivated WSSV and 10%egg yolk powder showed mortalities of 53.3%and 66.7%,respectively.The inactivated WSSV immersion group showed a mortality of 33.3%,and the DNA immersion group was 80%.These results have significantly differences compared to the positive control group which showed a mortality of 100%(P＜0.05).The results suggested that passive immunization of specific IgY antibodies through oral administration and immersion were effective to protect crayfish against WSSV.In conclusion,the specific IgY preparation in this study can provide a novel approach to the prevention and treatment of WSSV infection,which will have a industrial prospect.