Cloning Of Fusion Gene From Newcastle Disease Virus B₉₅ Strain And Construction Of Nucleotide Vaccine

Newcastle Disease virus (NDV) strain 695, a thermostable nature avirulent strain, were replicated in embryonated chicken eggsand its RNA was extracted from allantoic fluid. Referred to the reported sequence of F gene, a pair of primers were designed and synthesized. F gene of NDV B95 strain was amplified by RT-PCR, The PCR products were checked by agrose gel electrophoresis and purified by agrose gel fracion method. The purified products were cloned into PGEM-T-easy vector successfully, then the cloned plasmids were transformed into E.coli JM109. The specific recombinant plasmid was identified by molecular weight, PCR and restriction endonuclease analysis. The results indicated that the resuctant construct contained the gene of interest F. The positive cloned was sequenced by Sanger's dideoxy sequencing method. The results demonstrated that the F gene included an open reading frame which was 1662bp in length and encoded a protein of 553 amino acid. The cDNA of F gene from B95 strain shares a high identity between 88.3%-96.4% with that of reported F fene. The predicted amino acid residues share an identity between 92.1%-96.4% with that of other strains. Major difference of F gene lies in No.1-No.32 amino acid residues on N-terminal, which is the signal sequence of F protein and cut off short after the new peptide of F protein is combined with the membrane of vesicle. The sequence is not the composition of the F protein and the evolutronary force it bears is very small.Furthermore, the fragment encoding F gene was excised from the positive clone PGEM-F with Not I enzyme and purified by agrose gel fracion method. Then the fragment was linked to the PcDNA3.1 eukaryotic expressing vector digested by Not I enzyme and dephosphated and transformed into E.coli JM109 cell. The recombinant plasmids were identified by PCR and restriction enzyme analysis. The results indicated that the fragment was conformed to a open reading frame. Then the nucleotide vaccine was constructed. The immune experiment was carried out with the combination of the established nucleotide vaccine of ND and vector gene positive liposome. The expression of vaccine in chicks was detected by ELISA and Lymphocyte transformation test. The special response reaction was produced when using expressing products as antigen stimulates the body, which causes the special cell and humoral immunity. The products protect chicks against attack of virulent NDV to a certain extent and lay a foundation for studying the recombinant vaccine.