副粘病毒融合蛋白活性位点中亮氨酸基因突变分析

为了确定副粘病毒融合蛋白（Ｆ）分子上活性位点中亮氨酸在Ｆ的细胞融合作用中的作用，弄清Ｆ融合细胞的分子机理，采用基因定点突变法创造一个酶切位点，用酶切反应初步筛选突变株，然后用ＤＮＡ序列分析进一步确定，并在真核细胞内进行表达，Ｇｉｅｍｓａ染色和指示基因法检测细胞融合功能，荧光强度分析（ＦＡＣＳ）检测表达效率。结果表明，ｈＰＩＶ３等４６０位亮氨酸（Ｌ）和第４７４位异亮氨酸（Ｉ）分别突变成丙氨酸（Ａ）（

Mutation Analysis of Leucines in the Active Domain of Paramyxovirus Fusion Protein

To make sure the effect of leucines in the active domain of fusion protein (F)which interacts with homologous hemagglutinin neuraminidase(HN) of paramyxoviruses and to know the molecular mechanism of cell fusion, site directed mutagenesis was used to obtain mutants by creating a new enzyme site.The mutants were sequenced, and expressed in eukaryocytes. Their fusion activities were assayed by Giemsa staining, reporter gene assay and expression efficiency by fluorescence activated cell sorter(FACS) analysis. The results showed that hPIV3 F mutants of L460A and I474A had 51.24% and 48.73% of fusion activities as wild type, respectively, and L467A and L481A had the same as wild type; NDV F mutants of L481A and L488A had 24.78% and 19.96% of functions as wild type, respectively. Double mutants of L460A L467A had 20.87% of functions as wild type, while I474A L481A kept the same as I474A. Double mutant of NDV F L481A L488A had only 10.13% of activities as wild type. The expression efficiency of each mutant was the same by FACS analysis. These data proved that the leucines in the specific domain of hPIV3 F or NDV F for fusion play an important role in the cell fusion process. If they are mutated, the fusion activity of F will decrease for most of them, although some keep the same as wild type. Some double mutants of adjacent leucines will further decrease F fusion activity.