CAJ | 학술논문

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I103A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I1iA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174A略有升高,为118.6％,L110A下降幅度最大,为5.2％,I103A仅次于L1 10A,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.
목적 학정신성역병독(NDV)혈응소신경안산매(HN)당단백촉세포융합구역내적보수안기산공능,탐토HN당단백적촉세포융합궤제.방법 채용PCR정점돌변여체내동원중조상결합적방법장HN당단백촉세포융합구역내6개보수안기산돌변위병안산(A),재BHK21세포내표체후,류식세포의정량분석단백적표체효솔,병분별검측기촉세포융합활성、혈세포흡부능력(야칭위수체식별활성)화신경안산매활성.결과 L74A단백표체효솔강저위72.7％,각돌변주단백표체효솔여야독주상비차별무통계학의의(P＜0.05).각돌변주단백촉세포융합활성도유불동정도적하강,기중I103A하강폭도최대,위야독주적9.1％,각돌변주단백혈세포흡부능력야도출현불동정도적강저,기중I1iA적하강최명현,위28.2％,각돌변주단백적신경안산매활성변화정도불동,기중174A략유승고,위118.6％,L110A하강폭도최대,위5.2％,I103A부차우L1 10A,위5.7％.결론 신성역병독HN당단백촉세포융합구역내적보수안기산재세포융합중발휘착중요작용,제103위이량안산(Ⅰ)시차구역내적관건안기산.
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.Methods 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 vacciniaT7 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 1103A decreased to 14.2％ in head.Hemadsorption activity of mutant proteins were reduced in different extent,the maximum reduction of which was also 1103A,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.