H1N1亚型猪流感病毒广东分离株全基因克隆及其遗传演化分析

Cloning and Phylogenetic Analysis of the Entire Gene of an H1N1 Subtype Swine Influenza Virus Isolated from Guangdong Province

为了研究H1N1亚型SIV遗传演化与变异的特性,采用RT-PCR技术分别扩增A/Swine/Guangdong/LM/2004(H1N1)的8个基因片段,分别将其克隆到pMD18-T载体,进行全基因组序列测定。核苷酸序列测定结果显示:LM株SIV各基因片段均未发现核苷酸插入或缺失现象。HA切割位点处的氨基酸序列序列为IPSIQSR↓G,与高致病性SIV的H1N1亚型毒株的分子特征不符合。HA基因含有6个潜在的N-糖基化位点,4个在HA1的第11、23、87、和276位,增加2个分别在HA2的154和213位点;NA基因不仅在58、63、68、88和146位含有高度保守的N-糖基化位点,而且在44和235位增加2个潜在的N-糖基化位点,这可能是近期H1N1亚型SIV的一个分子特征。核苷酸同源性结果:HA基因与类人谱系的流感病毒分离株有很高的同源性(99%),而其他基因均与古典猪谱系的流感病毒分离株同源性较高(87%～98%)。从绘制的各个片段进化树和核苷酸同源性分析结果,可以推测该毒株HA基因可能来源于类人谱系的流感病毒;而其他基因来源于古典猪谱系的流感病毒。

To study the genetic variation and evolutionary characteristics of H1N1 swine influenza virus, all the eight genes of LM were amplified by RT- PCR, cloned into pMD18-T vector and sequenced respectively. The results showed that neither insertion nor deletion was observed in nucleotides of LM. The amino acids sequence of cleavage site of HA is IPSIQSR ↓ G, suggesting that LM did not have the molecular characteristics of high pathogen. HA had highly conservative N-glycosylation site at position 11, 23, 87 and 276 sites of HA1, and two more at position 154 and 213 sites of HA2. NA had highly＇conservative N-glycosylation site at position 58, 63, 68, 88, 146, and two more at position 44 and 235 sites, which might be one molecular characteristics of H1N1 subtype of SIV. The results of Bast showed HA gene had high homology to the strain of human-like SIV（99%）, while others had high homology to the ＂classical＂ SIV. So it is inferred that HA of LM might originate from human-like linage swine influenza virus, while others might originate from ＂classical＂ swine influenza virus.