Establishment And Application Of ELISA Detection Methods Based On Hexon And Fiber-2 About Serotype 4 Fowl Adenovirus

Since 2015,Hydropericardium syndrome(HPS)and Inclusion body hepatitis(IBH)caused by group I fowl Adenovirus(FAdV-I)have caused huge losses in the poultry breeding industry in China.Among them,Serotype 4 of fowl-adenovirus is the main epidemic strain in China.It caused a lot of losses in poultry because of its short course,high mortality and urgency.Hexon and fiber-2 are the two main structural proteins of serotype 4 avian adenovirus.Hexon has different epitopes of specific antigens,and there are parts of the neutralizing antibodies epitopes.Fiber-2 contains epitopes associated with viral virulence and cell-surface receptors.In order to promptly monitor the infection status of chickens and reduce economic losses,in this study,a group I serotype 4 adenovirus isolated and preserved in the laboratory was used as the research object,the indirect ELISA method based on Hexon and fiber-2 was established,In order to be used for clinical diagnosis monitoring and prevention of the disease.1.Epitope identification of the Hexon monoclonal antibody and the establishment of detection methodsBased on a monoclonal antibody 1B4 that has been prepared in our laboratory for Hexon truncated body(named Hexon879bp),in order to determine the epitope of the monoclonal antibody.In this study,the truncated body was analyzed to identify possible antigen site regions based on hydrophobic and hydrophilic regions,Primers were designed separately and each small fragment was amplified,Subclones were incorporated into prokaryotic expression vector,induced expression was detected by SDS-PAGE and Western blot.The results showed that the antigenic sites of monoclonal antibody 1B4 were between 210 and 219aa(between 630bp and 657bp corresponding to Hexon gene).In order to establish an ELISA detection method based on Hexon for enveloped antigens,in this study,we used two enveloped antigens to establish the detection method:1)the antigen epitope polypeptide was synthesized and coupled with keyhole blood blue protein(KLH).An ELISA method was established by using the coupled peptides as encapsulated antigens.The results showed that the best results were obtained when the concentration of polypeptide was 5 g/100 L;2)purified prokaryotic expression of Hexon879 antigen in inclusion bodies by protein,Matrix method was used to determine the optimal protein concentration of 0.1 g/100 L and the optimal dilution ratio of monoclonal antibody was 1:2000.The results showed that when the concentration of protein envelope was 0.1 g/100 L,the detection positive rate of chicken serum antibody against the inactivated avian adenovirus vaccine was 100%compared with the detection kit of avian adenovirus antibody.This method provides important reference for laboratory and clinical diagnosis of serum type 4 avian adenovirus.2.Establishment of serotype 4 avian adenovirus fiber-2 antibody detection methodThe high hydrophilic region of fiber-2 gene 5 'terminal 11 lbp based on fadv-4 AQ strain(login number:KY436520)affects the expression efficiency of this protein in pET32a(+)vector,in this study,a pair of specific primers were designed from the fiber-2 5 'end at 112bp,and the Fiber-2?N sequence of the 112-1440bp fragment was amplified by PCR.When coupled with pET32a(+)vector,the correct positive plasmid was sequenced and named pet-32a-Fiber-2?N,and the positive plasmid was transferred into the expressing bacterium BL21(DE3).The protein of Fiber-2?N was verified to be of correct size by SDS-PAGE,and the protein expressed in the inclusion body was purified.After purification,it was used to detect the antigen envelope,and an indirect ELISA method was established,and the experimental conditions were optimized.The results showed that the optimal concentration of Fiber-2?N protein was 0.1 g/100 L,The method has good specificity and repeatability.Compared with the AGP experiment,the detection rate of chicken serum antibody against inactivated vaccine of avian adenovirus type 4 was 100%.3.Comparison of serum antibody detection methods for inactivated avian adenovirus type 4 vaccineThe inactivated vaccine was prepared by inactivation and emulsification of FAdV-4-AQ virus prepared in our laboratory,the jugular vein blood were collected one week later in SPF chickens immunized at 7 days of age for 8 consecutive weeks.Respectively with Hexon879 ELISA assay,peptide ELISA assay and I avian adenovirus type detection kit to detect antibody levels in serum and compare several detection methods of testing effect.The results showed that both the Hexon879 ELISA assay and the polypeptide ELISA assay had better detection rate.The 10 positive serum samples collected by ELISA were tested by AGP experiment.The results showed,AGP test results and the experiment to establish the Hexon 879,peptide ELISA assay and ELISA assays of the I avian adenovirus type detection kits were completely accordance.This suggests that these methods provide a feasible method for clinical detection of avian adenovirus and evaluation of vaccine titer.