牙龈卟啉单胞菌血凝素2与氯化血红素结合多肽的序列分析

Sequence analysis of hemin-binding peptide derived from recombinant hemagglutinin-2 of Porphyromonas gingivalis

目的明确牙龈卟啉单胞菌血凝素2（Porphyromonas gingivalis hemagglutinin-2，PgHA-2）与氯化血红素结合的多肽片段氨基酸序列，为牙周病保护性抗体的制备奠定基础。方法提取Pg ATCC33277基因组DNA，原核克隆重组表达HA-2，质粒测序，蛋白质印迹法及质谱分析法鉴定重组HA-2（recombinant HA-2，rHA-2）。利用氯化血红素琼脂糖珠验证rHA-2的氯化血红素结合功能。利用多肽内切酶水解与氯化血红素结合的rHA-2，获得与氯化血红素结合的多肽片段，质谱分析多肽片段的氨基酸序列。结果PCR鉴定、质粒测序、蛋白质印迹法及质谱分析结果均证实原核表达蛋白为Pg的HA-2。氯化血红素结合实验明确了rHA-2具有与氯化血红素结合的功能。质谱分析与氯化血红素结合的多肽片段的氨基酸序列为DHYAVMISKTGTNAG。结论成功基因克隆重组与原核表达了具有与氯化血红素结合功能的PgHA-2，明确了与氯化血红素结合的多肽片段的氨基酸序列，为针对Pg的牙周病免疫预防和治疗药物的开发奠定了基础。

Objective To determine the sequence of the active peptide derived from recombinant hemagglutinin （rHA-2） of Porphyromonas gingivalis （Pg）. Methods The HA-2 gene from PgATCC33277 was cloned, expressed in Escherichia coli（Ec） BL21 （DE3） , and purified. The purified recombinant protein was evaluated for its ability to bind hemin-linked agarose. The active peptide was subjected to endoproteinase-mediated sequence analysis. Results The protein expressed in Ee BL21 （DE3） was identified as PgHA-2 by plasmid sequence analysis, Western blotting, and mass spectrometry. The recombinant protein was confirmed functional by its ability to bind hemin. The sequence of the active peptide of rHA-2 was determined to be DHYAVMISKTGTNAG. Conclusions The availability of sequence of the active peptide of rHA-2 provides a foundation for the development of immunoprophylactic and therapeutic agents against this human pathogen.