摘要
目的 找到副粘病毒融合蛋白 (F)分子上与血凝素 神经氨酸酶 (HN)相互作用的特异性区域 ,弄清F融合细胞的分子机理。方法 采用基因定点突变法 ,创造一个酶切位点 ,得到突变株 ,然后用基因重组的方法得到各种重组株 ,并于真核细胞内进行表达 ,Gimsa染色和指示基因法检测细胞融合功能 ,荧光强度分析 (FACS)检测表达效率情况。结果 在将各有关F基因片段进行交换之前 ,创造一个酶切位点时所得突变株的细胞融合功能与野毒株相同 ;将新城疫病毒 (NDV)F和副流感病毒 (PIV)F在膜穿入部分和躯干部分交接处交换时 ,不影响细胞融合功能 ;进一步将F2进行交换时 ,也不影响细胞融合功能 ;而将F的头部进行交换时 ,则检测不到细胞融合作用 ,FACS分析表明 ,F没有达到细胞表面。结论 F分子上与HN相互作用的特异性区域位于膜穿入部分和躯干部分交接处与F1和F2交接处之间 ,即F1除去膜穿入部分和尾部的剩余部分。
Objective To identify an active domain that interacts with homologous hemagglutinin-neuraminidase(HN) on fusion protein (F) of paramyxoviruses and to find the molecular mechanism of cell fusion. Methods Site-directed mutagenesis was used to get mutants by creating a new enzyme site and gene recombination was used to get chimeric F proteins. All the wild type, mutant and chimeric F proteins were expressed in eukaryocytes and their fusion functions were assayed by Gimsa staining and fluorescence-activated cell sorter (FACS) analysis. Results All the mutants with a new enzyme site have the same functions as wild type. When the two F genes were recombined at the site between transmembrane and stalk parts, the fusion functions of the chimeric proteins were not affected. Also, this function was not affected when the F′s were exchanged with F2. But fusion activity disappeared when the F′s were exchanged with head parts and F molecules did not reach cell surface as shown by FACS analysis. Conclusion The specific interaction domain with HN on F protein is in the whole stalk and part of head, i. e. in F1 except transmembrane and tail parts.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2000年第4期289-292,共4页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金!资助项目 (39570 0 32 )
