Dubreuil J D, Kostrzynska M, Logan S M, Harris L A, Austin J W, Trust T J
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
J Clin Microbiol. 1990 Jun;28(6):1321-8. doi: 10.1128/jcm.28.6.1321-1328.1990.
A protein antigen with an apparent molecular weight (Mr) of 31,000 was isolated from 0.2 M glycine hydrochloride (pH 2.2) extracts of a typical human fecal isolate, Campylobacter jejuni VC74. The protein was purified to homogeneity on a preparative scale by immunoaffinity chromatography followed by molecular sieving with a Superose 12 column. Isoelectric focusing under nondenaturing conditions indicated a pI of 9.3, and amino acid composition analysis showed that the protein was unusually rich in lysine, containing 14.9 mol% of this basic amino acid. Cysteine and tryptophan were absent. The protein also contained approximately 35% hydrophobic amino acid residues, and N-terminal amino acid analysis showed that 17 of the first 38 residues were hydrophobic. This amino-terminal sequence to residue 22 was virtually identical to that of an antigenically cross-reactive 31,000-Mr protein isolated from another C. jejuni strain belonging to a different heat-labile serogroup. Western blotting (immunoblotting) of glycine extracts of other C. jejuni, Campylobacter coli, and Campylobacter laridis strains belonging to different thermolabile and thermostable serotypes, as well as Campylobacter fetus, with a rabbit polyclonal antiserum raised against the purified C. jejuni VC74 protein showed that all C. jejuni, C. coli, and C. laridis strains tested contained a 31,000-Mr protein with epitopes which were antigenically cross-reactive with the C. jejuni VC74 protein. The antigenically cross-reactive epitopes of this protein were also readily detected by immunodot blot assay of glycine extracts of C. jejuni, C. coli, and C. laridis with monospecific polyclonal antisera to the 31,000-Mr protein, suggesting that this serological test could be a useful addition to those currently employed in the rapid identification of these important pathogens. Slide agglutination reactions, immunofluorescence assay, and immunogold electron microscopy with antisera to purified 31,000-Mr protein and trypsin treatment of whole cells indicated that the cross-reactive epitopes of the 31,000-Mr protein were not exposed on the cell surface. Cell fractionation analysis and immunogold electron microscopy located the protein on the outer surface of the cytoplasmic membrane. This finding suggests that the 31,000-Mr protein is not a good candidate for inclusion in a monovalent subunit Campylobacter vaccine.
从典型的人粪便分离株空肠弯曲菌VC74的0.2M甘氨酸盐酸盐（pH 2.2）提取物中分离出一种表观分子量（Mr）为31,000的蛋白质抗原。通过免疫亲和层析，然后用Superose 12柱进行分子筛，将该蛋白质大规模纯化至同质。非变性条件下的等电聚焦表明其pI为9.3，氨基酸组成分析表明该蛋白质富含赖氨酸，这种碱性氨基酸含量为14.9mol%。不含半胱氨酸和色氨酸。该蛋白质还含有约35%的疏水氨基酸残基，N端氨基酸分析表明前38个残基中有17个是疏水的。该蛋白质第22位残基之前的N端序列与从属于不同热不稳定血清群的另一株空肠弯曲菌中分离出的具有抗原交叉反应性的31,000-Mr蛋白质的序列几乎相同。用针对纯化的空肠弯曲菌VC74蛋白质产生的兔多克隆抗血清对属于不同热不稳定和热稳定血清型的其他空肠弯曲菌、大肠弯曲菌和Laridis弯曲菌菌株以及胎儿弯曲菌的甘氨酸提取物进行蛋白质印迹（免疫印迹）分析，结果表明所有测试的空肠弯曲菌、大肠弯曲菌和Laridis弯曲菌菌株都含有一种31,000-Mr蛋白质，其表位与空肠弯曲菌VC74蛋白质具有抗原交叉反应性。用针对31,000-Mr蛋白质的单特异性多克隆抗血清对空肠弯曲菌、大肠弯曲菌和Laridis弯曲菌的甘氨酸提取物进行免疫斑点印迹分析，也很容易检测到该蛋白质的抗原交叉反应性表位，这表明这种血清学检测可能是目前用于快速鉴定这些重要病原体的检测方法的有益补充。用针对纯化的31,000-Mr蛋白质的抗血清进行玻片凝集反应、免疫荧光测定和免疫金电子显微镜检查以及对全细胞进行胰蛋白酶处理表明，3l,000-Mr蛋白质的交叉反应性表位未暴露在细胞表面。细胞分级分离分析和免疫金电子显微镜检查将该蛋白质定位在细胞质膜的外表面。这一发现表明，31,000-Mr蛋白质不是单价亚单位弯曲菌疫苗的理想候选物。
