副粘病毒F1蛋白胞外非保守区对其特异性膜融合的影响

为了解融合蛋白F1分子的胞外非保守区在融合蛋白(F)与血凝素．神经氨酸酶(HN)的特异性膜融合中的作用，采用基因定点突变方法，在新城疫病毒(NDV)F1与人副流感病毒(hPIV)F1基因的胞外非保守区进行定点突变，创造酶切位点，得到分别含3个相同酶切位点的突变株NDV-M和hPIV-M。经检测，突变体的细胞融合功能与野毒株相同。然后用3个限制性内切酶分别从NDV-M与hPIV-M中切出两个片段NDVF-1、F-2及hPIVF-1、F-2。NDV-M和hPIV-M相互交换对应的F-1片段后进行基因重组，得到2个嵌合体(Chimera)，即NDV-C1和hPIV-C1；同样方法交换F-2片段后又得到2个嵌合体NDV-C2和hPIV-C2。将各种嵌合体DNA与同源及异源HN基因共转染BHK21细胞后，在真核细胞中表达。Giemsa染色和指示基因法检测细胞融合功能，荧光强度分析(FACS)检测F蛋白的表达效率。结果表明，突变体：NDV-M和hPIV-M的细胞融合功能与野毒株相同，可用于构建嵌合体。NDV-1C和NDV—C2分别与NDV HN共表达后，融合功能达到野毒株的76．34％和96．2％，与hPIV HN共表达后均无细胞融合发生；hPIV-C1和hPIV—C2分别与hPIV HN共表达后，融合功能达到野毒株的65．82％和93．78％，与NDV HN共表达后无细胞融合发生。FACS分析表明，突变体及所有嵌合体蛋白F的表达效率与野毒株相比均没有明显变化。实验结果说明在F1蛋白的胞外非保守区中，NDV F-1和hPIV F-1这两个片段对于NDV和hPIV的特异性膜融合具有重要作用；而NDV F-2和hPIV F-2这两个片段对于NDV和hPIV的膜融合来讲，则特异性较低。

Effects of Ectodomain Non-conservative Sequences of F1 Protein of Paramyxoviruses on the Specific Membrane Fusion

To understand the effects of ectodomain non-conservative sequences of F1 protein on the specific interaction with its homologous hemagglutinin-neuraminidase (HN), site-directed mutagenesis was used to obtain mutants containing new enzymatic sites and the other four non-conservative segments (NDV F-1, hPIV F-1, NDV F-2 and hPIV F-2) were obtained by cutting mutant F genes with these new enzymes. Gene recombination was used to get chimeric F protein NDV-C1 and hPIV-C1 by exchanging NDV F-1 and hPIV F-1 each other, and NDV-C2 and hPIV-C2 were obtained by the same way. All the mutants and chimeric F proteins were co-expressed in eukaryocytes with their homologous HN DNA or heterogenous HN DNA. The fusion functions were assayed by Giemsa staining and reporter gene method, the expression of F protein was assayed by fluorescence-activated cell sorter (FACS). Results of experiment were that the mutants of paramyxoviruses fusion proteins had the same functions as their relevant wild types. Chimeric F proteins NDV-C1 and hPIV-C1 had 76.34% and 65.82% of fusion activities, and chimeric F proteins NDV-C2 and hPIV-C2 had 96.25% and 93.78% of fusion activities, respectively, as compared with their relevant wild types. The analysis of FACS indicated that all the mutants and chimeric F proteins had almost the same expression efficienciy as their relevant wild types. The results suggested that NDV F-1 and hPIV F-1 were important for their specific membrane fusion, but NDV F-2 and hPIV F-2 were not.