【摘要】 采用RT_PCR技术,以A/Goose/Guangdong/3/96(H5N1)RNA为模板,扩增了1.73kb 的HA 全基因cDNA。将HAcDNA克隆后进行了序列测定,测序结果表明所扩增的1728 个核苷酸片段包含了完整的HA基因的开放阅读框架和上下游引物序列、蛋白质合成的起始密码子和终止密码子。核苷酸序列比较分析结果表明:A/Goose/Guangdong/3/96(H5N1) 与A/Goose/Guangdong/1/96(H5N1)有11 个核苷酸差异,同源率99.4% ;与A/HongKong/156/97(H5N1) 有25 个核苷酸差异,同源率98.6 % ;与A/Chicken/HongKong/258/97 (H5N1) 有30 个核苷酸差异,同源率98.3% ;它们的氨基酸序列同源率依次分别为99 .2 % 、98.6% 和98.1% 。受体结合位点的氨基酸序列完全一致;HA裂解位点氨基酸序列也完全一致,各有5 个碱性氨基酸插入。这说明上述4 个流感病毒分离株可能来自同一个祖先,具有相同的毒力和相似的生物学特性。更多还原

【Abstract】 In this study, full_length cDNA of HA gene of A/Goose/Guangdong/3/96(H5N1)(GD/3) was amplified using RT_PCR method. The cDNA was cloned into Sma I site of pUC18 plasmid and then sequenced. The result of sequencing shows that the cDNA contains whole open reading frame of HA gene, initiation codon and termination codon. The results of comparative sequence analysis indicated that the sequence of the cDNA had 11 nucleotide differences and 99.4% of homology with A/Goose/Guangdong/1/96(H5N1)(GD/1),25 nucleotide differences and 98.6% of homology with A/HongKong/156/97(H5N1)(HK/156),and 30 nucleotide differences and 98.3% of homology with A/Chicken/HongKong/258/97(H5N1)(HK/258). Furthermore, the HA of GD/3 had the homology of 99.2% with GD/1,98.6% with HK/156, and 98.1% with HK/258 at amino acid level. They shared the same basic amino acid insert at the cleavage site, which indicates that all 4 influenza virus isolates are probably evolved from same ancestor and have similar virulence and biological properties. In this study, full_length cDNA of HA gene of A/Goose/Guangdong/3/96(H5N1)(GD/3) was amplified using RT_PCR method. The cDNA was cloned into Sma I site of pUC18 plasmid and then sequenced. The result of sequencing shows that the cDNA contains whole open reading frame of HA gene, initiation codon and termination codon. The results of comparative sequence analysis indicated that the sequence of the cDNA had 11 nucleotide differences and 99.4% of homology with A/Goose/Guangdong/1/96(H5N1)(GD/1),25 nucleotide differences and 98.6% of homology with A/HongKong/156/97(H5N1)(HK/156),and 30 nucleotide differences and 98.3% of homology with A/Chicken/HongKong/258/97(H5N1)(HK/258). Furthermore, the HA of GD/3 had the homology of 99.2% with GD/1,98.6% with HK/156, and 98.1% with HK/258 at amino acid level. They shared the same basic amino acid insert at the cleavage site, which indicates that all 4 influenza virus isolates are probably evolved from same ancestor and have similar virulence and biological properties.更多还原