CHARACTERIZATION, CLONING, AND BINDING-PROPERTIES OF THE MAJOR 53-KILODALTON TREPONEMA-DENTICOLA SURFACE-ANTIGEN
CHARACTERIZATION, CLONING, AND BINDING-PROPERTIES OF THE MAJOR 53-KILODALTON TREPONEMA-DENTICOLA SURFACE-ANTIGEN
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DOI：
10.1128/iai.60.5.2058-2065.1992
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发表时间：
1992-05-01
影响因子：
3.1
通讯作者：
MCBRIDE, BC
中科院分区：
文献类型：
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作者：
HAAPASALO, M;MULLER, KH;MCBRIDE, BC
对齿密螺旋体表面蛋白的生化和生物学特性进行了研究。全细胞洗涤剂提取液经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法(SDS-PAGE)分析，主要蛋白为53 kDa，少量次要蛋白。针对齿纹夜蛾ATCC 35405全细胞制备的抗血清可与53 kDa蛋白和72 kDa蛋白反应，而不与其他蛋白反应。抗53-kDa蛋白抗体免疫电子显微镜显示，53-kDa蛋白定位于细胞表面。未加热样品的SDS-PAGE分析表明，53 kDa的蛋白质是低聚物的主要成分，分子量在130~300 kDa之间。Western印迹(免疫印迹)分析表明，大分子寡聚体可与全细胞抗血清和抗53 kDa蛋白抗体反应。在加热到70摄氏度后，聚集体解离成它们的亚单位。等电聚焦和SDS-PAGE分析表明，该53 kDa蛋白可分为几种形式，表观等电点在8.0~5.5之间。低聚物高度抵抗胰酶和蛋白酶K的蛋白降解，而单体蛋白易被消化。从齿状毛滴虫ATCC 35405的ZAP II基因文库中分离到一个在大肠杆菌中表达53 kDa抗原的克隆。重组蛋白与主要的53 kDa齿状螺旋体表面蛋白的分子质量完全相同，并与抗该蛋白的抗血清发生反应。用改良的Western印迹结合法研究了齿状毛滴虫ATCC 35405表面蛋白与层粘连蛋白、纤维连接蛋白、明胶、纤维蛋白原和牛血清白蛋白结合的作用。纤维连接蛋白、层粘连蛋白和纤维蛋白原附着在齿状毛滴虫53 kDa的表面蛋白和72 kDa的蛋白上，而没有观察到与明胶或BSA的附着。纤维蛋白原可抑制贴壁，明胶或BSA则不能。我们的结果表明，齿纹夜蛾53 kDa的主要表面蛋白可能在与宿主蛋白的结合中发挥作用，因此可能是该物种的一个重要毒力决定因素。
Treponema denticola surface proteins were studied for their biochemical and biological characteristics. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of detergent extracts of whole cells revealed a major protein of 53 kDa and a number of minor proteins. Antiserum raised against whole cells of T. denticola ATCC 35405 reacted with the 53-kDa protein and a 72-kDa protein but not with the other proteins. Immunoelectron microscopy with anti-53-kDa-protein antibodies showed that the 53-kDa protein is located on the surface of the cell. SDS-PAGE analysis of unheated samples indicated that the 53-kDa protein is the major component of oligomers with molecular masses ranging from 130 to 300 kDa. Western blot (immunoblot) analysis showed that the high-molecular-mass oligomers reacted with whole-cell antiserum and anti-53-kDa-protein antibody. The aggregates dissociated into their subunits after heating to 70-degrees-C. Isoelectric focusing followed by SDS-PAGE indicated that the 53-kDa protein was separated into several forms with apparent pI values ranging from 8.0 to 5.5. The oligomeric forms were highly resistant to proteolysis by trypsin and proteinase K, whereas the monomeric proteins were readily digested. A clone expressing a 53-kDa antigen in Escherichia coli was isolated from a lambda ZAP II DNA library of T. denticola ATCC 35405. The recombinant protein had exactly the same molecular mass as the major 53-kDa T. denticola surface protein and reacted with antisera raised against this protein. The role of T. denticola ATCC 35405 surface proteins in attachment to laminin, fibronectin, gelatin, fibrinogen, and bovine serum albumin (BSA) was studied by a modified Western blot binding assay. Fibronectin, laminin, and fibrinogen attached to the 53-kDa surface protein of T. denticola as well as to a 72-kDa protein, whereas no attachment to gelatin or BSA was observed. Attachment could be inhibited by pretreating the blots with fibrinogen but not with gelatin or BSA. Our results suggest that the 53-kDa major surface protein of T. denticola may play a role in the attachment to host proteins and may thus be an important virulence determinant of this species.