固相合成法合成葡萄球菌RNAⅢ抑制肽的鉴定

Synthesis and identification of RNA Ⅲ inhibiting peptide of Staphylococcus using solid phase method

背景:葡萄球菌感染和生物被膜形成受群体感应机制调控。葡萄球菌RNAⅢ抑制肽可以干预葡萄球菌群体感应系统,阻断葡萄球菌细胞间的信号传导通路,进而抑制葡萄球菌的毒素分泌和生物被膜形成,防治葡萄球菌感染。目的:探讨葡萄球菌RNAⅢ抑制肽(RIP)的人工合成方法。设计、时间及地点:单一样本观察。于2006-08/11在北京宣武医院中心实验室完成。材料:Fmoc-AA、Pipredine、TFA、HMP购自美国MERCK公司;DCC、HOBT、DMAP购自美国ABI公司。方法:采用固相合成法合成RNAⅢ抑制肽,HPLC法纯化,进行质谱分析法、酸水解法、红外光谱分析法和内酰胺酶活性测定法检测。主要观察指标:①合成和纯化结果。②分子量测定结果。③组成成分分析结果。④氨基酸序列分析结果。⑤结构分析结果。⑥生物活性鉴定结果。结果:合成的RNAⅢ抑制肽纯度为97.42%,相对分子质量为914.37,与理论相对分子质量基本一致;与天然RNAⅢ抑制肽具有相同的氨基酸组分、氨基酸序列以及空间结构;生物活性检测证明合成RNAⅢ抑制肽具有和天然RNAⅢ抑制肽相同的功效,可以有效抑制细菌产生RNAⅢ。结论:采用固相合成法成功合成RNAⅢ抑制肽,鉴定分析证实合成的多肽和天然RNAⅢ抑制肽蛋白具有相同的结构和功能。

BACKGROUND： Staphylococcal infections and its biofilm formation are regulated by quorum sensing. RNAⅢ inhibiting peptide of Staphylococcus （RIP） can intervine staphylococcal quorum-sensing system by blocking the signal transduction among staphylococcal cells, and inhibiting staphylococcal toxin secretion and biofilm formation to prevent staphylococcal infections. OBJECTIVE： To investigate the synthesis method of RNAⅢ-inhibiting peptide of Staphylococcus. DESIGN, TIME AND SETTING： Single sample observation was performed at the Central Laboratory of Beijing Xuanwu Hospital from August to November 2006. MATERIALS： Fmoc-AA, Pipredine, TFA, and HMP were purchased from MERCK, USA; DCC, HOBT, and DMAP were purchased from ABI, USA. METHODS： RIP was synthesized with solid phase chemistry and purified by HPLC. The peptide＇s composition and structure were identified with mass spectroscopy, amino acid analysis and Fourier transform infrared spectroscopy. MAIN OUTCOME MEASURES： Results of synthesis and purification; molecular weight; composition; amino acid sequencing structure analysis; bioactivity results. RESULTS： The result of mass spectroscopy revealed the polypeptide purity was 97.42% and molecular weight was 914.37, consistent with theoretic molecular mass; the synthesized RIP had identical amino acid composition and sequence to natural RIP. Results of activity verification reveal the peptide had high biological activity as natural RIP. CONCLUSION： RIP can be synthesized well by the way of solid phase chemistry. The synthesized RIP has the same structure and function with the natural RIP.