Molecular Epidemiology Of Newcastle Disease Virus Isolated In Partial Regions Of Henan Province And The Application On Detection Of NDV Antibody Of Main Antigen Region Of NDV HN Protein

Newcastle disease(ND) is a highly contagious disease, the causative agent of ND isNewcastle disease virus (NDV), which cusing severe economic losses in poultry farmingaround the world. With the widely application of NDV vaccines, outbreaks of ND were mildand sporadic, which show some new epidemical characterizations such as atypical ND,emergence of new genotypes and enlargenment of host range. The HN protein is one of themain structural glycoproteins and protective antigens, which locates on NDV envelopesurface, it plays an important role on the antigenicity. In this study, the main antigenic regionof NDV xx08strain HN glycoprotein of xinxiang area was obtained as a recombinant antigenby using prokaryotic expression system on the basis of analyzing NDV molecularepidemiology in the parts of Henan province. Using recombinant HN protein as antigen, anELISA for the detection of antibodies against NDV was developed.Allantoic fluid samples with different isolates were harvested by specific-pathogen-free(SPF) eggs and were applied to identify the NDV isolates by haemagglutination(HA) andhaemagglutination inhibition (HI) tests. The F gene fragment which codes the main functionalregion of the F protein was obtained by RT-PCR and sequenced. The phylogenetic tree wasconstructed using the MegAlign program in the Lasergene package. The results showed thatsix strains Newcastle disease virus (NDV) were isolated from outbreaks in chicken in partialregions in Henan province, phylogenetic analysis revealed that all of NDV strains belonged toClass II (genotypes I and VII), which had the velogenic motif112RRQKRF117at the F proteincleavage site. Which has the typical characteristics of Virulent strain. The main functional region homology of the F protein mino acids sequence was82.5%-97.6%. It was thepredominant strain in chickens in some areas of Henan province.On the basis of above epidemiology，the gene of main antigen domain of NDV xx08strainHN was amplified by RT-PCR. The target gene fragment was sub-cloned into pMD18-Tvector to construct recombinant cloning vector pMD-rHN, which were identified by PCR,restriction enzymes and sequencing. The target gene was recycled and subcloned intoprokaryotie expression vector pET-28a(+) to obtain the recombinant prokaryotic expressionvector pET28a-rHN. The recombinant expression vectors which were identified by PCR andrestriction enzymes were induced in BL21(DE3). SDS-PAGE and Western-blot were used toanalyse expression status and expression form of recombination protein and identifyantigen-specific of recombination protein. The purified products of recombination proteinwere obtained by Ni-NTA column affinity. The results showed that target protein was highlyexpressed with the condition of0.2mm IPTG and induced for4h in37℃, with molecularweight is about28kDa. The analysis showed that recombinant protein in the form ofinclusion bodies. Western-blotting identification results showed that the recombinant proteinwas recognized by NDV chicken hyperimmune serum. Recombinant proteins was given aconcentration of0.8mg/mL.The purified recombinant HN protein was used as the coating antigen to thedevelopment of a enzyme-linked immunosorbent assay (ELISA) to measure the specificantibody in sera of chickens against NDV. By optimizing the detection conditions, theoptimal coating concentration of antigen was5μg/mL and the serum sample for testing wasdiluted to1:80for detection at37°C for40min. And the working condition for HRP-labeled rabbit anti-chicken IgG was1:1000at37°C for30min. A S/P ratio of X+3SD=0.220was setas a negative-positive cut-off. The diagnostic sensitivity, specificity and accuracy of theELISA for the detection of anti-NDV antibodies in comparison to the HI was92.85％,91.2％and91.95％. The kappa value was0.979, indicating excellent agreement between the rELISAand the hemagglutinin inhibition tests. It was confirmed that there was no cross reactionbetween the antibodies of ND poultry viruses,such as IBV, INDV, AIV and MDV, it laid thefoundation of detection, monitoring and epidemiological investigation of NDV antibodies.