新城疫病毒HN糖蛋白促细胞融合区域内保守氨基酸基因突变分析  

Mutational analysis of conserved amino acids in the fusion-promoting domain of Newcastle disease virus hemagghtinin-neuraminidase protein

作　　者：褚福禄[1] 温红玲[1] 林彬[2] 孙成玺[1] 李振梅[1] 宋艳艳[1] 许洪芝[1] 王志玉[1] 

机构地区：[1]山东大学公共卫生学院病毒学研究室,济南250012 [2]山东省疾病预防控制中心艾滋病防治所

出　　处：《中华微生物学和免疫学杂志》2012年第4期351-357,共7页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目（30970142）;山东大学创新团队资助项目

摘　　要：目的确定新城疫病毒（NDV）血凝素神经氨酸酶（HN）糖蛋白促细胞融合区域内的保守氨基酸功能，探讨HN糖蛋白的促细胞融合机制。方法采用PCR定点突变与体内同源重组相结合的方法将HN糖蛋白促细胞融合区域内6个保守氨基酸突变为丙氨酸（A），在BHK21细胞内表达后，流式细胞仪定量分析蛋白的表达效率，并分别检测其促细胞融合活性、血细胞吸附能力（也称为受体识别活性）和神经氨酸酶活性。结果L74A蛋白表达效率降低为72．7％，各突变株蛋白表达效率与野毒株相比差别无统计学意义（P〈0．05）。各突变株蛋白促细胞融合活性都有不同程度的下降，其中1103A下降幅度最大，为野毒株的9．1％，各突变株蛋白血细胞吸附能力也都出现不同程度的降低，其中1103A的下降最明显，为28．2％，各突变株蛋白的神经氨酸酶活性变化程度不同，其中L74A略有升高，为118．6％，L110A下降幅度最大，为5．2％，1103A仅次于L110A，为5．7％。结论新城疫病毒HN糖蛋白促细胞融合区域内的保守氨基酸在细胞融合中发挥着重要作用，第103位异亮氨酸（Ⅰ）是此区域内的关键氨基酸。Objective To determine the function of conserved amino acids in the fusion-promoting domain of Newcastle disease virus （NDV） hemagglutinin-neuraminidase （HN） protein, clearly understanding mechanism of cell fusion. Meth.ods Using a PCR-based site-directed mutagenesis method and the method of homology recombination occurred in vivo to change six conservative amino acids into alanine respectively. Wild type （WT） and all mutant HN proteins were exepressed in BHK-21 cells by the vaccinia- T7 RNA polymerase expression system. The amount of each HN protein at the cell surface was determined by fluorescence-activated cell sorter （FACS）. Cell fusion efficiency, hemadsorption activity （or receptor binding activity） and neuraminidase activity were determined. Results There was no statistic difference of cell surface expression among WT and each mutant HN protein （ P〈0.05 ）. Cell fusion efficiency of each mutant protein decreased to some extent, especially I103A decreased to 14.2% in head. Hemadsorption activity of mutant proteins were reduced in different extent, the maximum reduction of which was also I103A, 28.2% of wt NDV HN. There was different neuraminidase activity among each mutant HN protein. L74A increased slightly to 118.6%. L110A decreased most to 5.2%. I103A decreased second most to 5.7%. Conclusion Conserved amino acids in fusion-promoting domain of NDV HN played an important role in cell fusion. I103 was identified as a key amino acid in this domain.

关 键 词：新城疫病毒 细胞融合 血细胞吸附 血凝素神经氨酸酶 

分 类 号：R3[医药卫生—基础医学]