The Expression Of Recombinant Protein Of Fowl Adenovirus Serotype 4 And Establishment Of Indirect ELISA Antibody Detection Method

Fowl adenovirus serotype 4(FAdV-4)is the causative agent of hydropericardium hepatitis syndrome(HHS).Since 2015,the disease has broken out in chicken flocks in China,mainly infected 3-6 weeks old broiler chickens and the fatality rate is high,causing huge economic losses to the poultry industry.In this study,the non-structural proteins and structural proteins of FAdV-4 were expressed in e.coli,and the purified recombinant proteins were used as diagnostic antigen to establish indirect ELISA antibody detection methods for FAdV-4.According to the gene sequences of the non-structural proteins(100K,22K)and the structural proteins(Fiber-1,Fiber-2,Penton)of FAdV-4 in the Gen Bank,five pairs of specific primers were designed,the PCR amplified products were connected to the p ET-32 a expression vector,and five recombinant plasmids of p ET-32a-100 K,p ET-32a-22 K,p ET-32a-Fiber-1,p ET-32a-Fiber-2 and p ET-32a-Penton were constructed successfully.Then transferred into BL(21)DE3 cells for expression,the five recombinant proteins have expressed well by IPTG induction.The recombinant proteins of p ET-32a-Fiber-1,p ET-32a-Fiber-2,and p ET-32a-100 K were expressed as inclusion bodies.The recombinant proteins of p ET-32a-Penton and p ET-32a-22 K were soluble.After purification by nickel column,SDS-PAGE analysis showed that the five recombinant proteins were pure.The results of Western-blot showed that all the 5recombinant proteins could react with FAdV-4 positive serum and have good antigenicity,which provided effective antigens for FAdV-4 ELISA assay.The two indirect ELISA methods of 100K-ELISA and 22K-ELISA were established using the recombinant proteins of p ET-32a-100 K and p ET-32a-22 K as coated antigens,and were used to detect antibodies against FAdV-4 infection.The results showed that the optimal antigen-coated concentration were 6 μg/m L and 16 μg/m L,repectively.The optimal dilution of primary antibody serum and labeled secondary antibody were 1:100 and 1:5000,repectively.The optimal incubation time were both 30 min.The two methods have not cross-react with the positive serum of other avian diseases,and suggesting have good specificity.The positive serum of FAdV-4 640-fold dilution can still be detected by100K-ELISA and 22K-ELISA,and suggesting have good sensitivity.The maximum coefficient of variation was 4.7% and 4.9%,both less than 5 %,and suggesting that two methods have reproducibility.The 50 serum samples of FAdV-4 artificially infected SPF chickens were positive by the detection of two methods,50 serum samples from chickens which were vaccinated by FAdV-4inactivated vaccine tested negative,indicating that the two methods could identify antibodies between FAdV-4 immunized chicken and naturally infected chicken.The recombinant proteins of p ET-32a-Fiber-1,p ET-32a-Fiber-2 and p ET-32a-Penton were used as coated antigens,ELISA reaction conditions were optimized and three indirect ELISA methods were established for detecting FAdV-4 antibodies: Fiber-1-ELISA,Fiber-2-ELISA and Penton-ELISA.The results showed that the optimal antigen coated concentrations of the three methods were 4 μg/m L,2.5 μg/m L and 2 μg/m L,repectively.The optimal dilution of primary antibody serum were 1:200,1:400 and 1:400,repectively.The optimal dilution of labeled secondary antibody were 1:10000.None of the three methods cross-reacted with positive sera from other avian diseases and showed good specificity.The positive serum of FAdV-4 at 1:1600 dilution can still be detected by three methods,showed good sensitivity.The maximum coefficient of variation within and between batches of the three methods were all less than 5% and suggesting have good reproducibility.Three methods were used to detect SPF chickens serum samples of FAdV-4 artificially infected and inactivated vaccine immunized,all of which were positive.The 100K-ELISA,22K-ELISA,Fiber-1-ELISA,Fiber-2-ELISA and Penton-ELISA methods were used to detect 96 serum samples from FAdV-4inactivated vaccine immunized chickens,the positive rates were 3.1%,5.2%,93.8%,100.0% and 94.8%,respectively.The positive rates of 217 non-immune chicken serum samples were 30.9%,27.6%,35.5%,39.1% and 42.3%respectively.The results showed that 100K-ELISA and 22K-ELISA could distinguish FAdV-4 antibody from inactivated vaccine and natural infection.Fiber-1-ELISA,Fiber-2-ELISA and Penton-ELISA methods can detect antibody from FAdV-4 inactivated vaccine and natural infection,can be used to evaluate antibody level.In this study,five recombinant proteins of FAdV-4(p ET-32a-100 K,p ET-32a-22 K,p ET-32a-Fiber-1,p ET-32a-Fiber-2 and p ET-32a-Penton)were expressed by prokaryotic expression,and can be used as diagnostic antigen.Using recombinant proteins p ET-32a-100 K and p ET-32a-22 K as coated antigens,two indirect ELISA methods were developed to distinguish antibody produced by inactivated FAdV-4 vaccine and natural infection,provide technical support for the investigation and purification of FAdV-4.Three ELISA methods for detection of FAdV-4 antibody were established,using recombinant proteins of p ET-32a-Fiber-1,p ET-32a-Fiber-2 and p ET-32a-pendon as coated antigens,provide three effective methods for the evaluation of FAdV-4 antibody level.