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首页> 外文期刊>Journal of bacteriology >Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp.
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Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp.

机译：编码致病性钩端螺旋体的跨膜外膜蛋白OmpL1的基因的分子克隆和序列分析。

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摘要

Pathogenic Leptospira spp. are spirochetes that have a low transmembrane outer membrane protein content relative to that of enteric gram-negative bacteria. In a previous study we identified a 31-kDa surface protein that was present in strains of Leptospira alstoni in amounts which correlated with the outer membrane particle density observed by freeze fracture electron microscopy (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 1991). The N-terminal amino acid sequence was used to design a pair of oligonucleotides which were utilized to screen a lambda ZAP II library containing EcoRI fragments of L. alstoni DNA. A 2.5-kb DNA fragment which contained the entire structural ompL1 gene was identified. The structural gene deduced from the sequence of this DNA fragment would encode a 320-amino-acid polypeptide with a 24-amino-acid leader peptide and a leader peptidase I cleavage site. Processing of OmpL1 results in a mature protein with a predicted molecular mass of 31,113 Da. Secondary-structure prediction identified repeated stretches of amphipathic beta-sheets typical of outer membrane protein membrane-spanning sequences. A topological model of OmpL1 containing 10 transmembrane segments is suggested. A recombinant OmpL1 fusion protein was expressed in Escherichia coli in order to immunize rabbits with the purified protein. Upon Triton X-114 extraction of L. alstoni and phase separation, anti-OmpL1 antiserum recognized a single band on immunoblots of the hydrophobic detergent fraction which was not present in the hydrophilic aqueous fraction. Immunoelectron microscopy with anti-OmpL1 antiserum demonstrates binding to the surface of intact L. alstoni. DNA hybridization studies indicate that the ompL1 gene is present in a single copy in all pathogenic Leptospira species that have been tested and is absent in nonpathogenic Leptospira species. OmpL1 may be the first spirochetal transmembrane outer membrane protein for which the structural gene has been cloned and sequenced.
机译：致病性钩端螺旋体螺旋体的肠膜革兰氏阴性菌跨膜外膜蛋白含量低。在先前的研究中，我们鉴定出31 kDa表面蛋白存在于阿尔斯通体钩端螺旋体菌株中，其数量与通过冷冻断裂电子显微镜观察到的外膜颗粒密度相关（DA Haake，EM Walker，DR Blanco，CA Bolin，JN Miller，和MA Lovett，Infect.Immun.59：1131-1140，1991）。 N末端氨基酸序列用于设计一对寡核苷酸，所述寡核苷酸用于筛选包含阿尔斯通乳杆菌DNA的EcoRI片段的λZAP II文库。鉴定出包含整个结构ompL1基因的2.5kb DNA片段。从该DNA片段的序列推导的结构基因将编码具有24个氨基酸的前导肽和前导肽酶I切割位点的320个氨基酸的多肽。 OmpL1的加工产生了成熟的蛋白质，预测的分子量为31,113 Da。二级结构预测确定了典型的跨膜蛋白跨膜序列的两亲性β-折叠的重复延伸。建议的OmpL1拓扑模型包含10个跨膜段。重组OmpL1融合蛋白在大肠杆菌中表达，以用纯化的蛋白免疫兔。在Triton X-114提取阿尔氏乳杆菌并进行相分离后，抗OmpL1抗血清在疏水性洗涤剂级分的免疫印迹上识别出一条单条带，而该条带并不存在于亲水性水性部分中。带有抗OmpL1抗血清的免疫电子显微镜显示与完整的阿尔斯通乳杆菌表面结合。 DNA杂交研究表明，ompL1基因以单拷贝形式存在于所有经过测试的致病性钩端螺旋体物种中，而在非致病性钩端螺旋体物种中则不存在。 OmpL1可能是第一个已经克隆并测序了结构基因的螺旋形跨膜外膜蛋白。

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