| Newcastle disease virus(NDV) belongs to Paramyxoviridae whose genome is a non-segmented single-strand negative-sense RNA. It causes ND in avian. Through years of integrated control, the epidemicity of this disease has been fundamentally controlled in our country. In recent years, some new characteristics appeared, For example, non-typical ND cases are increasing day by day and chicken flocks with highly antibody could also be infected. Some scholars considered that it is possibly related with the variation of NDV strain. So it was quite important to carry out the etiologic study on NDV and develop new vaccine. In order to study the molecular difference between NDV isolates and vaccinal strains and explore the causes of immunity failure of ND vaccine, genetic variation of F and HN genes of 6 isolates were studied in this research. The F protein structure of NDV isolates were compared with the reference strains to explain the biological characteristics variation of NDV isolates at nucleotide and amino acid level. NDV-F gene and IL-2 gene were cloned and nucleic acid vaccine were constructed which provide theoretical basis and technical support for the assisted immune.1. The main functional region of F gene of 6 NDV isolates (ZD,JZ,XB,CA,ZZ,CC) were amplified by RT-PCR. PCR products were cloned and sequenced , the sequences of nucleotide and deduced amino acid were analyzed compared with NDV reference strain published in GenBank. The results showed that the sequence homologous rates of nucleotide and amino acids of the 6 isolates were 99.6%-99.9% and 99.1%-99.8% respectively. Compared with reference strains of NDV, the sequence homologous rates of nucleotide and amino acids of the isolates were 83.2%-87.3% and 90.2%-93.8% respectively. The F protein cleavage sites of those isolates were 112R-R-Q-K-R-F117, and the molecular characteristics of F protein showed that those 6 isolates all belonged to genotypeⅦof NDV. The result indicated that: those 6 isolates might derived from the same strain which variated in its propagation process and had low homology with vaccine strains. Those NDV isolates have a distant relationship with vaccine strains, this may be one of the important reasons why clinical incidence appeared in immunity chicken. 2. HN gene of those 6 isolates were amplified by RT-PCR, PCR products were cloned and sequenced, then the sequences were analyzed compared with HN genes of NDV reference strains published in GenBank. The results showed that the sequence homologous rates of nucleotide and amino acids of those 6 isolates were 98.7%-99.8% and 98.1%-99.7% respectively. Compared with reference strains of NDV , the sequence homologous rates of nucleotide and amino acids of the isolates were 78.22%-85.3% and 82.6%-90.0% respectively. And those 6 isolates belonged to subtype HN571 of NDV. Those 6 isolates had low homology with vaccine strains. Analysis of the molecular characterizations of HN protein indicated that 5 of 6 isolates lacked the potential glycosylation site at 538~540aa.3. The F gene of ZD isolate was sequenced. Compared with reference strains of NDV published in Genbank, the structure of F0 protein was analyzed. The open reading frame of F gene of ZD coded 553 amino acids. The analysis of the secondary structure indicated that the dispositions ofα-helices centre,β-strands, angle and irregular crimp of F gene of ZD was similar to those of JS-2-06. Compared with LaSota, Clone-30, B1 and V4, theα-helices centre at 124-167aa was cleavage into two sections, aβ-strands region was missed at 377-386aa. The analysis of the B cell epitopes of protein and transmembrane protein showed that the disposition of common region of 4 parameters of B cell epitopes of ZD was similar to that of F48E9, Mukteswar and JS-2-06. An additional common region is introduced at 110-117aa compared with La Sota, Clone-30, B1 and V4. The three dimensional structure of F0 protein of ZD isolate was predicted by biological software. The research was meaningful to explain the biological characteristic variation of NDV isolates at the nucleotide and amino acide level.4. NDV nucleic acid vaccine pcDNA4-F was constructed by successfully inserting F gene into pcDNA4/HisMax, and NDV nucleic acid vaccine pcDNA4-F+IL-2 was constructed by inserting the connection of F gene and IL-2 gene into pcDNA4/HisMax, those two plasmids were used to immune chicken and challenge of NDV isolate, the result showed that NDV nucleic acid vaccines were effective against NDV infection. The research provide useful reference for development of nucleic acid vaccine. |
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