H5N1亚型禽流感病毒HA糖基化位点缺失突变株的构建及生物学特性  

Construction and Biological Characteristics of H5N1 Avian Influenza Viruses with Different Patterns of the Glycosylation Sites in HA Protein

作　　者：张晓剑[1] 李彦芳[1] 熊丽平[1] 陈素娟[1] 彭大新[1] 刘秀梵[1] 

机构地区：[1]扬州大学兽医学院农业部畜禽传染病学重点开放实验室禽类预防医学教育部重点实验室,扬州225009

出　　处：《病毒学报》2013年第5期495-499,共5页Chinese Journal of Virology

基　　金：国家科技重大专项(2012ZX10004214-001-002);973计划(2011CB505003);江苏省自然科学研究重大项目(10KJA230055);江苏省研究生科研创新计划(CXZZ12_0915);江苏省青蓝工程项目(苏教师[201027号];教育部创新团队项目(IRT0978);江苏省高校优势学科建设工程资助项目;国家自然科学基金项目(31372450)

摘　　要：不同H5亚型禽流感(AIV)HA上糖基化位点的分布是不同的,然而不同糖基化位点对病毒的作用仍不十分清楚。本研究利用定点突变和反向遗传操作构建了3个不同糖基化位点单缺失突变病毒,并对其增殖特性和毒力进行了测定。结果表明,通过HA基因序列测定和免疫印迹分析证实已成功去除了特定糖基化位点。158位糖基化位点缺失突变株rS△158在鸡胚中的EID50和MDCK细胞上TCID50比野生型rS略低,空斑直径显著减小;而单独去除169位和290位糖基化位点的rS△169和rS△290的EID50和TCID50略高于野生型rS,空斑直径相近,但空斑数要比后者高出10倍以上。所有突变株及野生型病毒在CEF上增殖速率相同,对SPF鸡均是高致病性的。因此,糖基化位点可影响病毒的体外增殖,但结果因细胞而异。The distribution of glycosylation sites in HA proteins was various among H5 subtype avian influ- enza viruses （AIVs）, however, the role of glycosylation sites to the virus is still unclear. In this study, avian influenza H5N1 viruses with deletion of the glycosylation sites in HA were constructed and rescued by site direct mutation and reverse genetic method, and their biological characteristics and virulence were determined. The result showed that the mutants were confirmed to be corrected by HA gene sequencing and Western blot analysis. The EID50 and TCID50 tested in SPF chick embryo and MDCK ceils of a mutant rSf158 with deletion of glyeosylation site at position 158 were slight lower than that of wild type rescued virus rS, and the plaque diameter of rSA158 was significant smaller than that of rS. The EIDs0 and TCIDs0 of mutants rSA169 and rSf290 with deletion of glycosylation sites at position 169 and 290, respectively, were slight higher than that of wild type rescued virus rS, the plaque diameters of rS△69 and rS△290 were similar as that of rS, but the plaque numbers of rSf169 and rSf290 were 10-fold higher than that to rS. On the other hand, the rS△158, rS△169 and rS△290 showed similar growth rate in chicken embryo fibroblast as rS. All viruses remained high pathogenicity to SPF chickens. Therefore, the growth of AIV can be affected by changes of glycosylation sites in HA protein, by which the effect is variable in different cells.

关 键 词：H5N1亚型禽流感 HA 糖基化位点 突变 

分 类 号：S855.3[农业科学—临床兽医学]