医学检验信息网 检验医学 2007-2-26 16:16:50

【摘要】 目的 了解近几年H1N1亚型毒株在深圳地区人群中活动加强及“O”相特性出现的分子生物学基础及其基因演变的特性。方法 病毒粒RNA经逆转录合成cDNA，用PCR扩增，产物纯化，采用双脱氧链末端终止法进行核苷酸序列测定并推导出其所编码的氨基酸序列。进化树分析用DNA-STAR公司出品的序列分析软件，MegAlign(1.03版)的Editseq(3.69版)。结果 根据病毒粒HA1基因特性，至少1995年以来深圳地区人群中同时流行着基因特性不同的三系毒株；1995～1997年H1N1毒株与A/新加坡/6/86(H1N1)病毒相比较，其HA1蛋白分子上第54和155位上分别插入和缺失一个糖基化点，同时氨基酸序列发生了替换。结论 近年来深圳地区人群中同时流行着HA1基因不同的三系H1N1亚型毒株。由于HA1蛋白分子上氨基酸序列发生了替换，尤其糖基化位点插入和缺失，造成1995年以来H1N1毒株活动加强，这些可能与毒株“O”相特性再现密切相关。 Characterization of HA1 genes of influenza A (H1N1 ) viruses isolated in Shenzhen city

CHENG Xiaowen, LI Liangcheng, HE Jianfan.

Municipal Hygiene and Anti-epidemic Station of Shenzhen, Shenzhen 580020

【Abstract】 Objective To understand the molecular bases of intension of influenza virus activity and emergence of “O” phase of influenza A(H1N1) strains in human population in Shenzhen in recenty years ,and also the evolutionary characterization of influenza A(H1N1) HA1 gene.Methods Virion RNA was transcribed into cDNA by reverse transcriptase, cDNA was amplified by PCR, the products of PCR were purified. Afterward, RNA sequence analsis was performed by the dideoxynucleotide chain termination method using synthetic oligodeoxynucleotide primers. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign(version 1.03) and Editseq (version 3.69) softwares.Results Since 1995, there were three different genetic lineages of influenza A(H1N1) virus HA1 gene cocirculating in men in Shenzhen city.Adding one and deleting one of potential glycosylation sites at 54 and 155 positions of amino acid sequences on HA1 protein domain of H1N1 viruses isolated recently was found as compared with those of A/Singapore/6/86(H1N1) virus. Meanwhile, there were some differences of amino acid sequences on HA1 protein molecules among H1N1 viruses tested and A/Singapore/6/86(H1N1) virus.Conclusion The intension of influenza A(H1N1) virus activity occurred since 1995 was due to emergence of substitution in amino acid sequences, especially the appearance of one addition and one deletion of potential glycosylation sites on their HA1 protein domains, and also was due to occurrence of influenza A(H1N1) virus with feature of “O” phase.

【Key words】 Influenza A(H1N1) virus Gene

甲1(H1N1)亚型流感病毒在人群中消失20年后，于1977年5月在我国北方突然重现，同年秋天蔓延至深圳地区。从此而后，深圳地区出现了人流感流行史上罕见的、同时流行着两个亚型(H3N2和H1N1)毒株的局面。1990年以来，H1N1与H3N2亚型和乙型流感病毒相比较，它在深圳地区一直处于劣势。然而，从1995年开始，它的活动有明显加强，同时重现了“O”相特性。故有必要了解H1N1亚型毒株近几年活动加强和“O”相特性出现的分子生物学基础，及其HA1基因演变的特点，为进一步搞好流感监测提供科学依据，也为深圳地区流感病毒基因库建立提供参考依据。

1 材料和方法

1.1 病毒(H1N1)亚型 A/深圳/227/95，A/深圳/30/96，A/深圳/223/97和A/深圳/240/97毒株，于1995～1997年流感监测中从流感患者中分离出，经鸡胚尿囊腔常规传代，收获的新鲜尿囊液用于RNA提取。A/新加坡/6/86(H1N1)病毒为1986～1996年国际性代表株。A/北京/262/95(H1N1)病毒为1998～1999年国际性代表株。它们的HA1基因核苷酸序列及其HA1区蛋白分子的氨基酸序列由国家流感中心提供。新加坡、北京和深圳分别用Sing, Bei和SZ代表。

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