| Abstract:Fowlfox(FP) is an infective disease often occurring among domestic fowls and other birds all over the world. When domestic fowls are infected with Fowlfox virus (FPV), growth retardation, respiratory signs, abnormalities in egg production and a rise in mortality are observed. FP infection results in economic losses in the poultry industry. However, serious problems have not been reported since live vaccines have been employed in most farms and FP infection has been well controlled. But its prevention is still very important. The FPV genome, containing 260 to 309 kb of double-stranded DNA, consists of a central coding region bounded by identical 9.5 kb inverted terminal repeats and contains 260 open reading frames, of which 101 exhibit similarity to genes of known function. The 4b core protein gene is 1 971 nucleotides long, encoding a 75 200 protein. It is one of the major polypeptides, accounting for approximately 11% of viral protein and is conserved among the members of avipoxvirus. In order to establish a more rapid ,sensitive and special method for detection of fowlpox virus (FPV), two pairs of primers(p1,2 , g1,2) were designed and synthesized on the basis of published DNA sequence of the 4b core protein gene of the FPV genome(AF198100) in this study. The PCR method was developed for detection of FPV infection. The results revealed the expected 549 bp or 1 361 bp fragment could be amplified respectively by two sets of primers from extracted DNA or virus suspensions from recombinant FPV, FPV vaccine strains or FPV field isolates. There was no amplification in control samples e.g., Marek?s disease virus (MDV), avian infectious laryngotracheitis virus (AILTV), goatpox virus, DNA from chicken embryo fibroblast cell (CEF) using any of the primers. Two pairs of primers of this method could detect at least 10-2 fmol(g1,2 ), 10-3 fmol(p1,2 ) FPV DNA and the virus of 100.5(g1,2 ), 10-0.5 (p1,2 ) TCID50 ,respectively. And then, 10 feces samples of the poultry inoculated with rFPV vaccines were collected and the concentrations of rFPV were measured by PCR. It was showed that all samples were negative, and suggested that rFPV vaccines was safe and could not cause environmental pollution. The results showed the PCR technique had the specificity and high sensitivity, and would be applied for diagnosis of fowlpox virus infection. A recombinant plasmid was constructed by inserting the amplified 4b core protein gene 1 361 bp fragment into pMD18-T vector and identify by PCR and digestion with restriction enzyme. The positive plasmid (pMD18-T-4b) was selected and sequenced. Blast analysis showed the nucleotide homogeneity between the clones sequence and the published DNA sequence of 4b gene sequence of PFV (AF198100) was 99.5%.A total of 7 nucleotide differences were found between them, the nucleic acid C,T,G,T,T,C,A at sites 215,386,388,1 014,1 034,1 137,1 143 were substituted by A,A,A,G,G,T,G. The 4b core protein gene is very conserved according to the result of comparison, so it could be used to study the nucleotide probe, and so on. A 360 bp fragment was obtained by EcoR â… digestion of pMD18-T-4b...
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