Clone And Expression Of VP1Gene Of Avian Encephalomyelitis Virus Shaanxi Strain And Establishment Of Indirect ELISA Method For Detecting Antibody

Avian encephalomyelitis (avian encephalomyelitis, AE), caused by avianencephalomyelitis virus (avian encephalomyelitis virus, AEV), is primarily affecting thecentral nervous system of young chickens. AE was first discovered in United States in1930and spread to our country in the early1980s, subsequently caused epidemic in Guangdong,Henan, Hebei and Inner Mongolia. The epidemic of AE has been an upward trend in recentyears in Shaanxi province since been reported in1997. AE is characterized by ataxia andtremors, especially of the head and neck, and it’s fatality rate is10%to70%.Laying hens hasa sudden drop in egg production, a drop of16%to43%after infection, which results in greatharm to the commercial poultry industry. The AEV antibody test kit of IDEXX company onthe market is expensive and not suitable for the detection of large quantities of the grass-rootslevel in our country, and there is no commercial kit on our domestic market. So method ofspecific, high sensitive, simple and fast, suitable for a large number of samples testing isimperative. In this study, an AEV of YL strain was isolated from clinically suspected avianencephalomyelitis chickens，and the VP1gene of AEV Shaanxi isolates SX and YL strainwere cloned and sequenced，VP1protein of SX strain was expressed, the preliminary theELISA method for detecting AEV antibody was established used the purified protein asantigen. Three different parts of work have been finished as follows:1.An AEV of YL strain was isolated from clinically suspected avian encephalomyelitischickens by Agar Gel precipitin test、experimental infection in Shaanxi Yangling, and a pair ofAEV special primers was designed according to the nucleotide sequences of AEV VP1geneof1134strain published in GenBank, the VP1gene of AEV SX and YL strain were amplifiedby RT-PCR, then cloned into pMD18-T vector and sequenced. The sequences of VP1genewere both compared with those of the known strains, and analyzed by DNAStar. The resultsshowed that the full length sequence of VP1gene both of SX and YL strain consisted of810nucleotides, coding270amino acid residues. The isolates shared88.5%~99.8%and89.3%~99.4%homology with the reference strains based on nucleotides and deduced acid,respectively; Mutation could be found in VP1gene of the two isolates,but on insertion, anddeletion. Evolution analysis indicated that SX and YL strain were close related to1143andL2Z strains, and distantly related to VR、HB、SD and NH937strains.2. The VP1gene of AEV SX strain was digested with EcoR Ⅰa ndSalⅠ, and thenconnected with pET-32a(+)prokaryotic expression vector. The recombinant plasmid pET32a-VP1was transformed into competent cells E. Coli. BL21（DE3）whose expressioninduced by IPTG., then optimized of the expression conditions and purified through HisTrapHP affinity column. The results of restriction enzyme digestion identification and genesequencing both proved that the pET32a-VP1vector were successfully constructed; targetfusion protein was successfully expressed in E.coli, the IPTG induction concentration of0.1mmol/L,37℃inducted5h. The SDS-PAGE electrophresis showed that the fusion proteinwas about50ku in molecular weigh. Western blot showed it existed specific reaction betweenthe fusion protein and AEV positive serum, the protein has a good reactogenicity.380μg/mLtarget protein was obtained by purificating of HisTrap HP affinity column.3. The purified recombinant protein was used for establishment of an indirect ELISA fordetecting of anti-AE antibody, then optimized of the reaction conditions.The results showedthat through optimization to determine the optimal ELISA working conditions for coatingantigen at4℃overnight after37℃2h at a concentration of3.8μg/mL of the recombinantprotein,1%BSA as blocking agent at37℃2h, serum samples（1:50）at37℃for30min,HRP labeled goat anti-chicken （1:4000）at37℃for30min, the substrate TMB for ELISAbeing incubated at37℃for5min; the threshold of positive and negative sera was0.360bystatistical analysis. The ELISA assay was confirmed to have a good repeatability、ensitivityand specificity. The coincidence rate of the two assays was up to91.9%compared with thebird AE-Ab ELISA kit of IDEXX by detecting180clinical sera. The method can be used forthe detection of large quantities of clinical samples.To sum up, the results showed that VP1gene of AEV SX and YL strain were both closerelated to1143and L2Z strains, and distantly related to other known AEV strains, theysuggested that there was a certain degree of difference in evolution with AEV known strains;VP1gene of AEV SX strain was highly expressed in prokaryotic system; the product was asoluble fusion protein of50ku, and has a good reactogenicity; the ELISA assay wasestablished using purified protein as antigen, and has a good repeatability, sensitivity andspecificity. It can be used for detecting large quantities of clinical samples.