将新城疫病毒 (NDV)长春株和四平株 HN插入 p IRES1多克隆位点 (Eco R )中 ,构建成核酸表达疫苗 p IRc HN和 p IRs HN,然后切除 p IRc HN和 p IRs HN的新霉素基因 ,将长春株和四平株 F基因分别插入其中 ,构建成 p IRc HNF和 p IRs HNF,...将新城疫病毒 (NDV)长春株和四平株 HN插入 p IRES1多克隆位点 (Eco R )中 ,构建成核酸表达疫苗 p IRc HN和 p IRs HN,然后切除 p IRc HN和 p IRs HN的新霉素基因 ,将长春株和四平株 F基因分别插入其中 ,构建成 p IRc HNF和 p IRs HNF,而后分别转染 Hela细胞。经血凝效价测定、Western blot分析 ,弱毒株构建的核酸疫苗的血凝活性比强毒株高 1个数量级 ,Hela细胞表达的 HN蛋白量以 p IRc HN最高 ,p IRs HN次之 ,p IRc HNF和 p IRs HNF较低。将重组疫苗转染 Hela细胞 ,用兔抗鸡 Ig Y进行间接免疫荧光试验 ,结果 ,在细胞膜和细胞浆中观察到了特异性的黄绿色荧光 ,证明表达产物具有特异性。展开更多
Virion RNA was abstracted from purified type I Avian paramyxovirus strain YN-PA01(isolated from parrot)and used as a template. The fragment containing the fusion gene(F) and hemagglutinin-neuraminidase gene(HN) of the...Virion RNA was abstracted from purified type I Avian paramyxovirus strain YN-PA01(isolated from parrot)and used as a template. The fragment containing the fusion gene(F) and hemagglutinin-neuraminidase gene(HN) of the isolate was amplified by RT-PCR and cloned to the pMD18-T Vector. Using primer walking method the complete sequence of F-HN genes was obtained finally.And the respective amino acid sequence was deduced. Through relative software the phylogenetic trees on F gene and HN gene were constructed between strain YN-PA01 and reference strains. The results showed that strain YN-PA01 comparing with reference strains displays 98.7%-83.2% nucleotide homology and 98.1%-86.2% amino acid homology on F gene; 97.4%-79.1% nucleotide homology and 97.2%-83.2% amino acid homology on HN gene. Additional 6 amino acids are encoded by the HN gene ORF of strain YN-PA01 comparing with national reference strains.The studied strain YN-PA01 exhibits highest identities with strain JS/5[O1/Go either analyzed on F gene or HN gene.展开更多
文摘Virion RNA was abstracted from purified type I Avian paramyxovirus strain YN-PA01(isolated from parrot)and used as a template. The fragment containing the fusion gene(F) and hemagglutinin-neuraminidase gene(HN) of the isolate was amplified by RT-PCR and cloned to the pMD18-T Vector. Using primer walking method the complete sequence of F-HN genes was obtained finally.And the respective amino acid sequence was deduced. Through relative software the phylogenetic trees on F gene and HN gene were constructed between strain YN-PA01 and reference strains. The results showed that strain YN-PA01 comparing with reference strains displays 98.7%-83.2% nucleotide homology and 98.1%-86.2% amino acid homology on F gene; 97.4%-79.1% nucleotide homology and 97.2%-83.2% amino acid homology on HN gene. Additional 6 amino acids are encoded by the HN gene ORF of strain YN-PA01 comparing with national reference strains.The studied strain YN-PA01 exhibits highest identities with strain JS/5[O1/Go either analyzed on F gene or HN gene.
