摘要
新城疫是由新城疫病毒(Newcastle disease virus,NDV)引起禽的一种急性、热性、败血性和高度接触性传染病,具有很高的发病率和病死率,对养禽业危害极大。为了探讨新城疫病毒融合蛋白(Fusion protein,F)胞内尾部(Cytoplasmic tail,CT)后段的氨基酸(Amino acid,aa)在细胞融合中的作用,定位该区域影响F蛋白功能的关键氨基酸,本研究利用基因定点突变和体内同源重组技术构建7个突变体,经BHK-21细胞表达后,分别用间接免疫荧光法和流式细胞术定性和定量检测各突变体蛋白的表达效率,采用R18染料转移实验、指示基因法、吉姆萨染色对各突变体蛋白促细胞融合的三个阶段分别进行分析,并利用Western Blot检测突变体功能的改变是否与F蛋白的裂解活化有关。结果显示,与野生型F蛋白相比,突变体N542A、L544A、M547A的半融合活性均发生了显著变化,分别为野生型的189.8%、58.5%和17.3%,而突变体G540A、N541A、G545A、Q546A的半融合活性处于野生型的80.1%~117.5%之间(P均大于0.05)。突变体N542A、L544A、M547A的内容物混合能力分别为野生型的248.5%、62.2%和12.6%,其它突变体的内容物混合能力处于野生型F蛋白的95.8%~119.9%之间(P均大于0.05)。突变体L544A和M547A形成合胞体的大小与数目均有下降,其中M547A几乎丧失了形成合胞体的能力,突变体N542A形成的合胞体的能力则大幅上升。除了M547A的表达水平有所降低之外(为野生型的66.3%),其它各突变体F蛋白在细胞表面的表达水平均处于野生型F蛋白的91.3%~111.4%之间(P均大于0.05)。由此可见,NDV F蛋白胞内尾部N542、L544和M547为关键氨基酸,对F蛋白的融合活性起到了重要作用。
Newcastle disease is an avian infectious disease that can cause serious economic losses to the poultry industry. To determine the function of amino acids(aa)in the cytoplasmic tail(CT)of Newcastle disease virus fusion protein(F),and clarify the role of CT in mediating cell membrane fusion,seven mutants were constructed with site-directed mutagenesis and in vivo homologous recombination method. Indirect immunofluorescence assay(IFA)and flow cytometry(FCM) were used to qualitatively and quantitatively detect the efficiencies of wild type(wt)and mutant F proteins expressed in BHK-21 cells. Semi-fusion,as well as content mixing and cell fusion activity were detected with dye transfer assay,content mixing assay and syncytium formation experiment. Western Blot assay was used to detect whether or not functional changes of mutants are associated with cleavage activation of the F protein. The semi-fusion activities of the mutants N542 A,L544 A and M547 A were significantly changed,which were 189.8%,58.5% and 17.3% compared with that of the wt fusion protein,respectively,while those of mutants G540 A,N541 A,G545 A,Q546 A were between 80.1% and 117.5% compared with that of the wt fusion protein(P > 0.05). The content mixing ability of mutants N542 A,L544 A and M547 A were 248.5%,62.2% and 12.6% of wt F protein,respectively.Other four mutants were between 95.8% and 119.9% compared with that of the wt fusion protein(P > 0.05).The area of syncytia formed by mutants L544 A and M547 A decreased,and M547 A almost lost the ability to form syncytia,but the ability of mutant N542 A to form syncytia increased significantly. In addition to the decreased expression level of M547 A(66.3% that of the wt),the expression level of other mutant F proteins on the cell surface were between 91.3% and 111.4% that of the wt fusion protein(P > 0.05). These data suggest that N542,L544 and M547 of the NDV F protein play important roles in the membrane fusion activity of the virus.
引文
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