Kostrzynska M, Betts J D, Austin J W, Trust T J
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
J Bacteriol. 1991 Feb;173(3):937-46. doi: 10.1128/jb.173.3.937-946.1991.
Flagellar filaments were isolated from Helicobacter pylori by shearing, and flagellar proteins were further purified by a variety of techniques, including CsCl density gradient ultracentrifugation, pH 2.0 acid disassociation-neutral pH reassociation, and differential ultracentrifugation followed by molecular sieving with a Sephacryl S-500 column or Mono Q anion-exchange column, and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to an Immobilon membrane. Two flagellin species of pI 5.2 and with apparent subunit molecular weights (Mrs) of 57,000 and 56,000 were obtained. N-terminal amino acid analysis showed that the two H. pylori flagellin species were related to each other and shared sequence similarity with the N-terminal amino acid sequence of Campylobacter coli, Bacillus, Salmonella, and Caulobacter flagellins. Analysis of the amino acid composition of the predominant 56,000-Mr flagellin species isolated from two strains showed that it was comparable to the flagellins of other species. The minor 57,000-Mr flagellin species contained a higher content of proline. Immunoelectron microscopic studies with polyclonal monospecific H. pylori antiflagellin antiserum and monoclonal antibody (MAb) 72c showed that the two different-Mr flagellin species were located in different regions of the assembled flagellar filament. The minor 57,000-Mr species was located proximal to the hook, and the major 56,000-Mr flagellin composed the remainder of the filament. Western immunoblot analysis with polyclonal rabbit antisera raised against H. pylori or Campylobacter jejuni flagellins and MAb 72c showed that the 56,000-Mr flagellin carried sequences antigenetically cross-reactive with the 57,000-Mr H. pylori flagellin and the flagellins of Campylobacter species. This antigenic cross-reactivity did not extend to the flagellins of other gram-negative bacteria. The 56,000-Mr flagellin also carried H. pylori-specific sequences recognized by two additional MAbs. The epitopes for these MAbs were not surface exposed on the assembled inner flagellar filament of H. pylori but were readily detected by immunodot blot assay of sodium dodecyl sulfate-lysed cells of H. pylori, suggesting that this serological test could be a useful addition to those currently employed in the rapid identification of this important pathogen.
通过剪切从幽门螺杆菌中分离出鞭毛丝，并通过多种技术进一步纯化鞭毛蛋白，包括氯化铯密度梯度超速离心、pH 2.0酸解离-中性pH重结合，以及差速超速离心，随后用Sephacryl S-500柱或Mono Q阴离子交换柱进行分子筛分离，并通过制备性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳纯化至同质，然后转移到Immobilon膜上。获得了两种等电点为5.2、表观亚基分子量（Mr）分别为57,000和56,000的鞭毛蛋白。N端氨基酸分析表明，这两种幽门螺杆菌鞭毛蛋白彼此相关，并且与空肠弯曲菌、芽孢杆菌、沙门氏菌和柄杆菌鞭毛蛋白的N端氨基酸序列具有序列相似性。对从两株菌株中分离出的主要56,000-Mr鞭毛蛋白的氨基酸组成分析表明，它与其他物种的鞭毛蛋白相当。较小的57,000-Mr鞭毛蛋白含有较高含量的脯氨酸。用多克隆单特异性幽门螺杆菌抗鞭毛蛋白抗血清和单克隆抗体（MAb）72c进行的免疫电子显微镜研究表明，两种不同Mr的鞭毛蛋白位于组装好的鞭毛丝的不同区域。较小的57,000-Mr物种位于钩的近端，而主要的56,000-Mr鞭毛蛋白构成了鞭毛丝的其余部分。用针对幽门螺杆菌或空肠弯曲菌鞭毛蛋白产生的多克隆兔抗血清和MAb 72c进行的蛋白质免疫印迹分析表明，56,000-Mr鞭毛蛋白携带与57,000-Mr幽门螺杆菌鞭毛蛋白和弯曲菌属鞭毛蛋白抗原交叉反应的序列。这种抗原交叉反应并不延伸到其他革兰氏阴性菌的鞭毛蛋白。56,000-Mr鞭毛蛋白还携带另外两种MAb识别的幽门螺杆菌特异性序列。这些MAb的表位在组装好的幽门螺杆菌内鞭毛丝表面未暴露，但通过对幽门螺杆菌十二烷基硫酸钠裂解细胞的免疫斑点印迹分析很容易检测到，这表明这种血清学检测可能是目前用于快速鉴定这种重要病原体的检测方法的有益补充。
