Cloning Analysis,Prokaryotic Expression And Dentify Of Major Gene Of Newcastle Disease Virus HN09-69Strain

Newcastle disease virus(NDV) is a member of the family Paramyxoviridae,and has been assigned to the genus Avulavirus in the subfamily Paramyxovirinae.It causes a highly contagious respiratory, neurological,or enteric disease in chickens.The genome was also observed to be typical of Baltimore group V-single stranded RNA of negative sense. its genome encode six proteins NP、P、M、 F、HN and L.Two additional proteins V and W are expressed by mRNAs which are derived from the P gene via RNA editing.From1926years since its discovery,NDV spread to the countries and regions in the world and the economic impact of ND and its effect on trade in commercial poultry was found to be more serious.ln2009,we collect epidemic materials from a poulty farm of Zhong Mou and demonstrate pathogeny is virulent NDV by virus isolation,identification and experimental infection and named HN09-69strain.In this study,the HN09-69strain of the study of major gene cloning,prokaryotic expression and identification in order to explore its recombinant proteins immunogenicity to provide a new method for the prevention of ND.Five pairs of primers were designed according to the sequence of the complete genome of NDV from Genebank,The ORF of NP、P、M、F、HN gene were succeedly amplified by RT-PCR from the HN09-69strain.The gene of HN09-69strain shared a lower homology in nucleotide acid sequence with the traditional vaccines La Sota、 Clone-30、VG/GA、F48E8、Mukteswar,however shared a high homology with waterfowl FP1-02、SD09、SF02、SDWF02、ZJ1and in the same branch with SD09、 SDWF02which by means of NP、P、M、F、HN nucleotide homology comparison,sequence and evolutionary analysis. HN09-69strain belong to genotype Ⅶd from genotyping.Meanwhile,in correlative analysis tools online,a series of bioinformatics amalysis were perfomed on the structure including prediction of domain,signal peptide and transmembrane domain.The results indicated that F and HN protein had signal peptides and transmembrane domain.With primers which containing BamH I and Xho I restriction site that NP、P and Vgene ORF and M(1-798bp), F(1119-1506bp), HN(675-1116bp) gene fragment were subcloned into the prokaryotic expression vector pET28a(+) use cloning vector as a template,and successfully constructed recombinant prokaryotic expression plasmids.the correct recombinant prokaryotic expression vector were transformed into BL21(DE3) with inducement of IPTG the results measured by SDS-PAGE manifested that it could be highly expressed.the expressed of NP、P existed in the form of solubility however the M、F、HN、V existed in the form of inclusion body.then Optimization each protein expression conditions and the expressed proteins were identified by Western blot.NP、P proteins were purified by NI affinity chromatography.The purified NP and P proteins emulsify with adjuvants to make vaccine for immune42days of age SPF chicken.7days post-second vaccine began to detect sera every7days.21days post-boosting,all chickens were challenged with F48E8virus.The results of the protection of the La Sota、NP protein、control、P protein immunization group was100%、0、0、20%respectively.In conclusion,in this study the HN09-69strain major gene were cloned successfully,the recombinant plasmids were constructed and made it get high expression in the prokaryotic expression system.NP and P fusion protein were purified by NI affinity chromatography and immune SPF chickens for challenged experiments that for lay the foundation further study of the immunogenicity of each protein subunit vaccine.