Immune Efficacy Of Fiber2 Of Fowl Adenovirus Serotype 4 Expressed By P.pastoris And E.coli

Fowl adenoviruses are divided into groups Ⅰ,Ⅱ and Ⅲ,among them,Group Ⅰ and Ⅲviruses can infect chickens.Group Ⅰ fowl adenovirus has 12 serotypes,which can be divided into 5 genotypes such as A,B,C,D,E.In recent years,the incidence of hydropericardium syndrome(HPS)in chickens caused by the fowl adenovirus serotype 4(FAdV-4)has been rising,causing great losses to the poultry industry in China.In order to prevent and control the prevalence of HPS in China,the inactivated oil emulsion vaccine of FAdV-4 was introduced.However,the production of the inactivated vaccine requires the use of leghorn male hepatocellular(LMH)to culture viruses.This caused not only the higher cost of the vaccine,but exist a certain tumorigenic risk.In order to solve these problems,in this study,the main neutralization antigen,fiber2(Fiber2)of FAdV-4 was expressed in eukaryotic and prokaryotic systems,respectively,and the immune efficacy of the expressed products was evaluated.1.Expression of the Fiber2 protein of fowl adenovirus serotype 4The FAdV-4 GX2013 strain was used as a template and design primers for the Fiber2 gene.The Fiber2 gene was amplified by PCR and cloned into the yeast expression vector pPICZaA obtained the recombinant expression plasmid pPICZaA-Fiber2.This recombinant plasmid was transformed into GS115,and yeast recombinant protein(Y-Fiber2)was induced with methanol.SDS-PAGE and mass spectrometry identification showed that Y-Fiber2 protein was secretory expression with a size of about 60 KDa.The ammonium sulfate precipitation method combine the activated carbon powder were used for the initial purification of fermentation liquid and the adsorption of the pigment and odor,and then 30 KDa ultrafiltration tube was used for further concentration.Y-Fiber2 protein was well purified and concentrated identified by Western blot,and the content could reach 6.3 mg/mL.In the current study,the E.coli expression system also used to express the Fiber2 protein.The recombinant expression plasmid pET32a(+)-Fiber2 was constructed and transformed into BL21(DE3)to express induced by IPTG.The results of SDS-PAGE identification showed that the recombinant Fiber2 protein(E-Fiber2)expressed in E.coli was soluble and with a size of 80 KDa approximately.The E-Fiber2 protein concentration was 1.67 mg/mL after purified by using Ni-NTA affinity tomography.2.Development of subunit vaccines for fowl adenovirus serotype 4The recombinant Fiber2 protein(Y-Fiber2,E-Fiber2)expressed by Pichia pastoris and E.coli and sterilized Tween-80 were mixed respectively in a 95:5 ratio to prepare into a water phase,then mixed with the sterilized oil phase 1:1,fully emulsified,prepared into subunit vaccine,and evaluated its immune effect.The 40 4-day-old SPF chicks were randomly divided into four groups,namely Y-Fiber2 immunization group,E-Fiber2 immunization group,FAdV-4 whole virus oil emulsion inactivated vaccine immunization group and non-immune challenge control group.At 7 days of age,Y-Fiber2 and E-Fiber2 subunit vaccine were immunized by subcutaneous injection in the neck,respectively,and each chick was vaccinated with 10 μg recombinant Fiber2 protein;the other two groups were inoculated with a 0.25 mL commercial virus oil emulsion inactivated vaccine or PBS respectively.All test chicks were challenged FAdV-4 GX2013 with a dose of 105.5TCID50/0.2mL by intramuscular injection at 28 days of age.At this time,the mean agar expansion titers of serum antibodies in the Y-Fiber2 immunization group were 1:16(4Log2).The mean value of the agar expansion titers of E-Fiber2 immunization group and FAdV-4 whole virus oil emulsion inactivated vaccine immunization group was 1:32(5Log2).All chicks in the control group died within 2 days after the challenge,and the chicks in each immunized group performed normal.The results showed that the three immunization groups can all achieve 100%clinical protection efficacy on FAdV-4 GX2013 strain.The viral load showed that the quantitative of the respiratory and digestive tracts on the 3rd and 7th days after challenge in the E-Fiber2 immunization group was lower than that in the Y-Fiber2 immunization group and FAdV-4 whole virus oil emulsion vaccine immunization group test by fluorescence quantitative PCR.In summary,the E-Fiber2 subunit vaccine has a better immune protection effect.