红斑丹毒丝菌免疫原性蛋白的鉴定及其编码基因的克隆和测序

Identifying immunogenic proteins of Erysipelothrix rhusiopathiae C43065 by MALDI-TOF mass spectrometry and cloning their encoding genes

摘要:

[目的] 为深入研究红斑丹毒丝菌的免疫保护性抗原及其致病机制,采用免疫蛋白组学技术鉴定红斑丹毒丝菌的免疫原性蛋白。[方法] 通过SDS-PAGE电泳分离红斑丹毒丝菌C43065株的NaOH提取抗原,用兔抗NaOH提取抗原抗血清经Western blot检测免疫原性蛋白,通过MALDI-TOF质谱技术鉴定蛋白种类,并对部分免疫原性蛋白的编码基因进行克隆和测序。[结果] 通过MALDI-TOF质谱技术从C43065株NaOH提取抗原中鉴定出9个免疫原性蛋白,分别为SpaA、伴侣蛋白GroEL、烯醇化酶、ATP结合盒转运蛋白、丙酮酸脱氢酶复合物E1、甘油醛-3-磷酸脱氢酶、果糖二磷酸醛缩酶、50S核糖体蛋白L1、30S核糖体蛋白S4。其中烯醇化酶、ATP结合盒转运蛋白、甘油醛-3-磷酸脱氢酶和果糖二磷酸醛缩酶已被证实与链球菌、牙龈卟啉单胞菌、脑膜炎奈瑟菌和结核分枝杆菌的致病性相关。C43065株伴侣蛋白GroEL、烯醇化酶、ATP结合盒转运蛋白、丙酮酸脱氢酶复合物E1、甘油醛-3-磷酸脱氢酶和果糖二磷酸醛缩酶编码基因大小分别为1614、1296、1260、1005和867 bp,与已公布的红斑丹毒丝菌Fujisawa株相应基因的相似度高达98%。[结论] 本文所鉴定的9个免疫原性蛋白,为进一步开展红斑丹毒丝菌保护性抗原及其致病机制研究奠定基础。

Abstract:

[Objective] To identify immunogenic proteins of Erysipelothrix rhusiopathiae C43065. [Methods] Antigens were extracted from E. rhusiopathiae C43065 by the alkaline extraction method. Proteins in the NaOH-extracted antigen were separated by SDS-PAGE and transferred to nitrocellulose membranes, and then Western blotting was performed with rabbit antiserum against the NaOH-extracted antigens. The immunogenic protein bands were identified by MALDI-TOF mass spectrometry. The genes encoding 5 major immunogenic proteins was amplified by PCR from the genomic DNA of E. rhusiopathiae C43065, and inserted into the pMD18-T vector and then sequenced. [Results] A total of 9 immunogenic surface proteins in the NaOH-extracted antigen from E. rhusiopathiae C43065 were successfully identified by MALDI-TOF mass spectrometry. Four of the proteins were putative virulence-associated proteins: enolase, ATP-binding cassette transporter, glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase class-II. The genes encoding the chaperone protein GroEL, enolase, ATP-binding cassette transporter, glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase class-II were 1614, 1296, 1260, 1005 and 867 bp in length, and the nucleotide sequences homologies of the genes between the C43065 strain and the previously reported E. rhusiopathiae Fujisawa strain was more than 98%. [Conclusion] Several putative virulence-associated proteins in the NaOH-extracted antigen of E. rhusiopathiae C43065 will be useful for elucidating the roles of these proteins in the pathogenesis of the organism.