Isolation, Identification And Differential Diagnosis Of Fowl Adenovirus Group Ⅰ By Recombinant Protein Elisa Method

Fowl adenovirus(FAV) belongs to Adenovirus family Aviadenovirus. It contains three groups according to diverse antigenicity. Fowl adenovirus group I(FAVI) has congenerous antigen and twelve serotypes, and exists in chickens, ducks and geese, being infected in recessive manner and working together with other pathogens in fowl.259 samples in live poultry market of nanning city were collected from year 2008 to 2010, and 92 isolations are positive by chick embryos proliferation and PCR, positive rate is 35.5%(92/259). Eight isolations are chosen from different years of postive samples to be evaluated through hemagglutination assay, immune agar diffusion assay, morphology, cell cytopathic effect and embryos pathogensis test. The results indicate that eight strains do not agglutinate chicken red blood cell, but react with positive serum of FAVI. Typical characteristics of adenovirus are seen in electron microscope, chick embryo liver cells become round, diacaustic rate is enhanced and affect growth of chicken embryo. Eight hexon genes are cloned and sequence alignment analysis indicated that similarity is highest with FAVI. Phygenetic analysis suggests genetic relationship is closest with FAVI. It suggests that eight strains were belongs to FAVI.The primers are designed according to hexon, penton,100K and 33K genes sequences of FAVI in GenBank, PCR and linked in procaryon express vetor pGEX-4T-1 and transformed into DH5a competence cells. With IPTG induction, four recombinant proteins expressed well. With solubility analysis indicate that the products of hexon and penton are existed as inclution bodies, but 100K and 33K are solubility. The result of purification suggests that purity and concentration are much better. Their reactionogenicitys are demonstrated well by western-blot analysis suggests they can react with positive serum of FAVI, so could to be ELISA diagnosis antigen.As both structure proteins, the hexon and penton are coated as antigens respectively, indirect hexon-ELISA and penton-ELISA methods for detecting FAVI antibodies are established. The results show that the optimal concentrations of hexon and penton are 10.0ug/mL and 15.0ug/mL, respectively, the dilution of serum is 1:100, and the optimal dilution of enzyme-labeled antibody is 1:2000, the optimal effect time of the serum and enzyme-labeled antibody are 75min and 45min, respecitively. The two methods both have good specificity, sensitivity and repeatability. The two proteins of hexon and penton are mingled then coated with the different proportion to establish the hexon-penton-ELISA method. Three ELISA methods had detected the FAVI infected and inactivated serum each 30 copies, and the results show that the two kinds of serum both has reacted, hexon-penton-ELISA sensitivity is higher. These three ELISA methods detected 100 portions of the clinical samples, and the percent of positive samples are 45%,41%,48%, respectively. The establishment of three ELISA methods have provided easy, fast serodiagnostic methods for the antibody detection and epidemiological survey of FAVI.As both nonstructural protein, the 100K and 33K are coated'as antigens respectively, indirect 100K-ELISA and 33K-ELISA methods for detecting FAVI antibodies are established. The results show that the optimal concentrations of antigens are 10.8ug/mL and 12.0ug/mL, respectively, the dilution of serum sample is 1:100, and the optimal dilution of enzyme-labeled antibody is 1:3000, the optimal effect time of the serum and enzyme-labeled antibody are 65min and 45min, respectively. The two methods both have good specificity, sensitivity and repeatability. The two proteins of 100K and 33K are mingled then coated with the different proportion to establish the indirect 100K-33K-ELISA method. Three ELISA methods have detected the infected and inactivated serum each 30 copies, and 100K-33K-ELISA sensitivity is higher. The three ELISA methods detected 100 portions of the clinical samples, and the positive percent are 40%, 35%,42%, respectively. The three ELISA methods can detect the infected serum, but no respond to inactivated serum, and provide technical support for diagnosis and purification of FAVI.