| Four primers were designed according to the sequences of nucleoprotein (NP) genes of all subtype avian influenza virus(AIV) and hemagglutinin(HA) genes of H5,H6,H9subtypeAIV. A multi-reverse transcription polymerase chain reaction (RT-PCR) was developed for detection of the RNAs from these three subtype AIVs chorioallamoic fluid. Aditional comparing research on RNA extraction from H6 subtype AIV was also carried on. The results suggested that two amplific bands appeared for detection the three subtype AIVs, which including the NP and corresponding HA bands. And three bands appeared when amplifying the samples of mixture virus with two subtypes. And the diagnosis were negative for newcastle disease virus(NDV), infectious bronchitis virus(IBV), infectious bursal disease viru(IBDV) .Also the 64 times diluted samples of the three subtype AIV could be detected. The results of RNA extraction methods suggested that high sensitivity but more time were the characters for TRIzol with centrifugal.The assay costs of SDS with heating were little but the sensitivity was also low. The method of TRIzol with microcolumn had the same advantages but having expensive costs. The last method of TRIzol with nylon membrane had the characters of rapid, low cost and high sensitivity.HA gene of H6 subtype AIV was cloned and sequence was also analyzed before the DNA vaccine was constructed. The green fluorescent protein gene (GFP) was chose as reporting gene. The H6A antigene and GFP gene were fused and cloned to the pAVX! Vector. The recombinant plasmid pAVX!-H6A-GFP was identified by restricted enzyme digestion with EcoRâ… or Xhoâ… . The positive plasmid pAVX!-H6A-GFP were transfected to BHK cell with liposome. And the fluorescent was observed. The results showed that the purpose gene (H6A-GFP) was successfully cloned to the vaccine vector. And the faint fluorescent and strong fluorescent were observed on the 24 hours and 48 hours after lipofection. |
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