副血链球菌fap1-orf4基因座位编码的ORF3调控Fap1糖基化与成熟的研究

Study on Fap1 glycosylation and maturation regulating by ORF3 coded by fap1-orf4 gene locus of Streptococcus parasanguis

目的研究副血链球菌fap1-orf4基因座位编码的ORF3是否调控Fap1的糖基化与成熟，并探讨其对副血链球菌黏附功能的影响。方法采用基因置换技术构建副血链球菌arf3等位基因置换突变株，利用互补分析和Western blot检测副血链球菌Fap1的表达水平，并采用全唾液包被的羟磷灰石黏附试验检测副血链球菌的黏附能力。结果（1）副血链球菌orf3基因置换突变株VT1774未发生极化；（2）Western blot检测菌株VT1774显示成熟的Fap1（M，约220×10^3）被不成熟的Fap1（M，约470×10^3）所取代，互补分析显示VT1774的互补株VT1775能恢复表达成熟的Fap1；（3）菌株VT1774黏附能力显著下降。结论副血链球菌fap1—orf4基因座位编码的ORF3是Fap1糖基化与成熟所必需的，orf3基因置换导致Fap1成熟障碍与菌株黏附力显著下降。

Objective To study whether ORF3 coded byfap1-orf4 gene locus of Streptococcus parusanguis is involved in the regulation of Fap1 glycosylation and maturation and to investigate whether ORF3 influences Streptococcus parasanguis adhesion. Methods A gene replacement strategy was adapted to constrnet orf3 alleic replace mutant of Streptococcus parasanguis. Complementation assay and Western blot were used to test Fap1 expression levels. Whole saliva-coated hydroxyapatite （SHA） adhesion assay was adapted to determine Streptococcus parasanguis adhension. Results （ 1 ） Non-polar was found in strain VT1774, the orf3 alleie replace mutant of Streptococcus parasanguis. （2） Western blot showed that mature Fap1 （Mr about 220×10^3） disappeared and were substituted with high molecular weight Fap1 （Mr about 470×10^3） in strain VT1774, furthermore, eomplementation assay showed VT1775, the complementation strain of VT1774, restored mature Fap1 expression. （3） The binding ability reduced significantly in strain VT1774. Conclusion ORF3 coded byfapl-off4 gene locus was required for Fap1 glycosylation and maturation in Streptococcus parasanguis, orf3 alleic replacment resulted in Fap1 glycosylation and mature disorder and decreasing of adhension ability of Streptococcus parasanguis.