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目的探讨牙龈卟啉单胞菌血凝素2(Porphyromonas gingivalis hemagglutinin-2,Pg HA-2)的氯化血红素结合位点多肽对牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)摄取氯化血红素生长的影响。方法合成多肽DHYAVMISK(肽1),DEYAVMISK(肽2,肽1中第2位氨基酸突变为谷氨酸),ALHPDHYLI(肽3,HA-2结合位点不相关多肽)。将肽1、肽2、肽3分别与氯化血红素琼脂糖珠预孵育,加入Pg重组血凝素2(Porphyromonas gingivalis recombinant HA-2,Pg rHA-2),收集与氯化血红素结合的Pg rHA-2,SDSPAGE电泳,分析多肽对Pg rHA-2与氯化血红素结合的抑制作用。肽1、肽2、肽3与氯化血红素预孵育后,加入到CDC液体培养基中培养Pg,测定菌液A600值,分析多肽对Pg生长的抑制作用。结果肽1浓度依赖性抑制Pg rHA-2与氯化血红素结合,而肽2和肽3对Pg rHA-2与氯化血红素的结合无抑制作用。在24、48和72 h时间点,肽1组的A600值较肽2、肽3和PBS组明显降低(P<0.05)。结论本研究表明Pg HA-2氯化血红素结合位点多肽DHYAVMISK与Pg竞争结合氯化血红素,抑制Pg的生长,为开发新的牙周病防治方法奠定基础。
Objective To investigate the effect of hemin binding sites of Porphyromonas gingivalis hemagglutinin-2 (Pg HA-2) on the uptake of Porphyromonas gingivalis (Pg) The impact of prime growth. Methods DHYAVMISK (peptide 1), DEYAVMISK (peptide 2, mutation of amino acid 2 in peptide 1 to glutamic acid) and ALHPDHYLI (peptide 3, unrelated polypeptide of HA-2 binding site) were synthesized. Peptide 1, peptide 2 and peptide 3 were respectively preincubated with hemin agarose beads, and PgrHA-2 (Pg rHA-2) was added to collect hematin binding Pg rHA-2, SDSPAGE electrophoresis, the inhibitory effect of the polypeptide on the binding of Pg rHA-2 to hemin. After preincubation, Peptide 1, Peptide 2, Peptide 3 and hemin, were added into CDC liquid medium to culture Pg. The A600 value of the bacteria liquid was determined, and the inhibitory effect of the polypeptide on Pg growth was analyzed. As a result, peptide 1 inhibited Pg rHA-2 binding to hemin in a concentration-dependent manner, while peptide 2 and peptide 3 did not inhibit the binding of Pg rHA-2 to hemin. At the time points of 24, 48 and 72 h, the A600 values in peptide 1 group were significantly lower than those in peptide 2, peptide 3 and PBS groups (P <0.05). Conclusion This study shows that Pg HA-2 heme-binding site polypeptide DHYAVMISK competes with Pg for the binding of hemin and inhibits the growth of Pg, which lays the foundation for the development of a new periodontal disease prevention and treatment method.