Development Of Detection Method For Pathogen And Antibody Of Fowl Adenovirus Group ? And It's Application In Serological Investigation

Fowl adenovirus(FAdV)'s DNA is linear and double strand.The virus particles are nonenveloped.FAdV belongs to the genus adenovirus.FAdV Group I is one of three subpopulations of FAdV.The Group I FAdV mainly infected chickens,which caused the inclusion body hepatitis and pericardial effusion syndrome.The virus is one of the most serious diseases in poultry infectious diseases because of its immune suppression.Since June 2015 the outbreak of the disease in our country caused heavy losses to the poultry industry in China,but also to the disease prevention and control in China has brought enormous pressure.Currently there is no effective treatment for the disease,but also the lack of a formal and effective vaccine for the prevention and control of the disease.At this stage what we can do is to do a good job of disease monitoring,timely detection and to take effective measures timely to reduce losses.The hexon protein of FAdV is located on the surface of virus particles,which is the most important capsid protein and the highest structural protein of virus particles,accounting for about 60%of the total molecular weight.The antigen epitopes on the hexon protein have the characteristics of type,group and subgroup,which are the criteria for the diagnosis of serotypes.The hexon protein has a large number of FAdV protective antigen epitopes.In this study,we selected FAdV-I PZ strain which was isolated by our laboratory as the research object,in order to develop method of the traditional PCR and LAMP assay to detect it's pathogen and hexon protein indirected ELISA to detect it's antibody.This study includes the following four parts.Test I Development of PCR method and Development and optimization of LAMP method for FAdV group I.In order to develop the detection method of fowl adenovirus group I.According to the conserved sequence of hexon gene 2 PCR primers were designed by the help of Oligo software,After that we developed PCR method for detecting pathogen.At the same time we were succeed using Primer explorer V4 to design 4 LAMP primers and establishing LAMP method to detect pathgen.After that the dNTP concentration,Mg2+ concentration and betaine concentration were optimized,so we determined the optimum conditions of LAMP.The results of PCR method showed that the PCR method has good specificity.The PCR method could detect dilutions of DNA extracted at 10-7 concentration.The results of LAMP method showed that the sensitivity of the LAMP detection method is 10 times higher than the traditional PCR method.Moreover,LAMP detection method also has very good specificity.From these we can concluse that the LAMP detection method provides a simpler method for detecting fowl adenovirus.Test ? Prokaryotic expression of FAdV hexon protein and preparation of chicken anti-hexon serum.In order to obtain the prokaryotic expression of hexon protein of fowl adenovirus.We seeked the fowl adenovirus hexon gene sequence in Gen Bank.By the help of Oligo we designed a pair of primers in order to clone the hexon protein gene of fowl adenovirus.The size of hexon gene is 1227 bp.After that we cloned it into the prokaryotic expression vector PET-32a.and constructed expression plasmid PET-32a-hexon successfully.Then we transformed PET-3 2a-hexon into Escherichia coli BL21 and the hexon protein was expressed after induced by IPTG.The results of SDS-PAGE of recombinant protein showed that the expression of hexon protein existed in inclusion bodies.Then we used the refolded hexon protein as antigen to immune SPF chickens to obtain high titer of chicken anti hexon serum.The result of western blot showed distinct brown bands can be expected in the target protein position.The serum of chicken anti hexon protein prepared with this antigen laid the foundation for the subsequent development of ELISA method.Test ? Development and optimization of hexon indirected ELISA methodIn order to develop the hexon indirect ELISA antibody detection method.In this study the prokaryotic expressied and purificatied hexon protein was used as a coated antigen The reaction conditions include The concentration of antigen,the optimum dilution of serum,the condition of inclusion,the sealing condition,the blocking liquid,the resistance and the two reaction time of the ELISA method were optimized.The results showed that:the best package concentration of antigen is 1.5 ?g/mL,the optimal dilution of serum is 1:800,the optimal coating conditions is 4 degrees overnight,the best sealing condition is 37 ? for 1 h,using 1%BSA,the optimal reaction time of the serum to is 30 min,the optimal reaction time of enzyme labeled antibody is 45 min.The cut-off value was determined by testing the OD450 values of 40 negative serum.The development of indirect ELISA method provides a simple and rapid serological detection method for antibody detection and epidemiological investigation of fowl adenovirusTest ? Investigation on antibody level of chicken adenovirus in broiler farms in Pizhou.In order to investigate the antibody level of avian adenovirus group I in Pizhou in 2015 and also exam the practicability of the hexon protein indirect ELISA method edeveloped in Test III.We used the hexon protein indirected ELISA method to detect the antibody level of the serum samples which were collected in Pizhou in the most prevalent period of this disease.The results showed that there were 25 positive samples,the positive rate was 15.6%.Among them,the highest positive rate of serum in August was 30%,and the positive rate of serum 9?10 decreased obviously.The results of antibody levels were consistent with the prevalence of the disease in Pizhou.Which suggested that the indirect ELISA we established for detecting antibody is of good clinical relevance.