Sequencing Of Fusion Gene Of Newcastle Disease Virus Isolates And Eukaryotic Expression Of Fusion Gene Of F₄₈E₉ And La Sota Strain

In this study, Fusion glycoprotein(F)gene of NDV Qinhai(QH3), Heilongjiang(HLJ) and (Gansu)A4 isolates were amplified by reverse transcriptase ploymerase chain reaction(RT-PCR) and cloned into pMD18-T vector. After recombinant plasmids were identified by restriction endonucleases digestion and PCR, The sequences of the cNDA were obtained and their amino acid sequences were deduced, analysed and compered. The sequences analysis indicated that complete open read frame of F gene of 3 isolates was 1662bp and encoded F protein of SS3 amino acids/There were 6 identically potential asparagine-linked glycosylation sites and the deduced amino acid sequence of the cleavage site region of F protein of the 3 NDV isolates that had the same amino acids pattern as velogenic strain was ¹¹²R-R-Q-K/R-R-F¹¹⁷, which was accordant with the results of mean death time of chicken embryos (MDT) and intracerebral pathogenicity index flCPI).On the basic of the first 374bp nucleotides initiated from the first ATG of F gene, a phylogenetic tree of 122 NDV strains including the 3 strains,the other strains isolated recently in China and the some strains abroad was created. The phylogenetic analysis showed that QH3,HLJ and A4 belonged to the genetype â…§,â…¨,â…¥ respectively. It indicated that not only NDV genetypes among epizootic strains isolated from different areas were evidently variable, but also special genetype DC which hadn't prevailed in abroad was present in China.The homology of the nucleotide sequences and genetic variation analysis of NDV strains suggested that mere were obvious divergence in gene lever among NDV vaccine strains and epizootic strains isolated from different areas and different ages.The divergence may have effect on amino acid and structure and function of protein in some extent and lead to difference of immunogenicity ,which was one of reason being difficult in preventing from ND. Therefore, to analyze difference of immunogenicity among NDV epizootic isolates and vaccine strains in details and to establish methods to check immune and epidemic situation,we expressed F protein of strain F₄₈E₉ and strain La Sota respectively.The specific primers of F gene were firstly designed and synthesized, which based on the sequence of the known fusion protein gene of NDV. F gene of F₄₈E₉ and La Sota strains were cloned into the transposion vector pFastBac^?HTa to construct two recombinant transposon vectors (pFastBacHT-F₄₈F and pFastBacHT-LaF) respectively. Positive recombinants were screened in LB/AMP* culture after transformation into E.coli DH5a and further identified with restriction endonucleases digestion and PCR and sequenced. To generate recombinant bacmids, the positive pFastBacHT-F₄₈F, pFastBacHT-LaF were transformed into E. coli DHlOBac.White bacterial colonies were selected as positive recombinant expression vectors after screening in GM,Ka,Tel,X-gal and IPTG selection LB agar plates. Then mediated by Cellfectin reagent, recombinant bacmid DNA (rBacmid-F₄₈F and rBacmid-LaF) were transfected into sf-9 cells to generate recombinant baculovirus that express the F proteins of NDV F₄₈E₉ and La Sota. The results of SDS-PAGE and Western-blot showed that the expressed F proteins were specific with a molecular weight 56KD and were partly glycosylated and provided with reactinogenicity.In addition, bacluovirus expression vector pFastHTa had 6×His tag which was beneficial to pure protein that help analysis and sift specific antibody. It provided a basis for analyzing difference of immunogenicity among NDV virulent and avirulent strains in details and establishing special diagnosis method of ELISA .Meanwhile it is import to further to research pathogenicity and immune foundation of NDV.