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首页> 美国卫生研究院文献>Infection and Immunity >Cloning nucleotide sequence and expression of the Brucella melitensis omp31 gene coding for an immunogenic major outer membrane protein.
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Cloning nucleotide sequence and expression of the Brucella melitensis omp31 gene coding for an immunogenic major outer membrane protein.

机译：布鲁氏布鲁氏菌omp31基因的克隆核苷酸序列和表达编码免疫原性主要外膜蛋白。

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摘要

The gene coding for the major outer membrane protein (OMP) of 31 to 34 kDa, now designated Omp31, of Brucella melitensis 16M was cloned and sequenced. A B. melitensis 16M genomic library was constructed in lambda GEM-12 XhoI half-site arms, and recombinant phages expressing omp31 were identified by using the anti-Omp31 monoclonal antibody (MAb) A59/10F09/G10. Subcloning of insert DNA from a positive phage into pGEM-7Zf allowed the selection of a plasmid bearing a 4.4-kb EcoRI fragment that seemed to contain the entire omp31 gene under control of its own promoter. omp31 was localized within a region of the EcoRI insert of approximately 1.1 kb. Sequencing of this region revealed an open reading frame of 720 bp encoding a protein of 240 amino acids and a predicted molecular mass of 25,307 Da. Cleavage of the first 19 amino acids, showing typical features of signal peptides for protein export, leaves a mature protein of 221 amino acids with a predicted molecular mass of 23,412 Da. The predicted amino acid sequence of B. melitensis 16M Omp31 showed 35.2% identity with the RopB OMP of Rhizobium leguminosarum bv. viciae 248 and 34.3% identity with Omp25 of B. abortus 544. As in Brucella spp., Omp31 was located in the outer membrane of recombinant Escherichia coli, but its reported peptidoglycan association in Brucella cells was not detected in E. coli. The ability of Omp31 to form oligomers resistant to sodium dodecyl sulfate denaturation at low temperatures, a characteristic described for several bacterial porins, was observed in both B. melitensis and recombinant E. coli. The epitope recognized by the anti-Omp31 MAb A59/10F09/G10, for which a protective activity has been suggested, has been delimited to a region of 36 amino acids of Omp31 covering the most hydrophilic part of the protein. The availability of recombinant Omp31 and the identification of the antigenic determinant recognized by MAb A59/10F09/G10 will allow the evaluation of their potential protective activity and their potential for the development of subcellular vaccines against brucellosis.
机译：克隆并测序了编码布鲁氏菌16M的31至34kDa的主要外膜蛋白（OMP）的基因，现在命名为Omp31。在λGEM-12 XhoI半位臂中构建了苜蓿芽孢杆菌16M基因组文库，并使用抗Omp31单克隆抗体（MAb）A59 / 10F09 / G10鉴定了表达omp31的重组噬菌体。将来自阳性噬菌体的插入DNA亚克隆到pGEM-7Zf中，可以选择带有4.4 kb EcoRI片段的质粒，该质粒似乎在其自身启动子的控制下包含整个omp31基因。 omp31位于EcoRI插入片段的大约1.1 kb区域内。该区域的测序揭示了一个720 bp的开放阅读框，编码240个氨基酸的蛋白质，预测分子量为25,307 Da。前19个氨基酸的切割显示出蛋白质输出信号肽的典型特征，留下了221个氨基酸的成熟蛋白，预测分子量为23,412 Da。棉铃虫16M Omp31的预测氨基酸序列与豆科根瘤菌Bop的RopB OMP具有35.2％的同一性。蚕豆248和与流产芽孢杆菌544的Omp25具有34.3％的同一性。如在布鲁氏菌属中一样，Omp31位于重组大肠杆菌的外膜中，但是在大肠杆菌中未检测到其报道的布鲁氏菌细胞中的肽聚糖结合。在双歧杆菌和重组大肠杆菌中均观察到了Omp31在低温下形成的耐十二烷基硫酸钠变性的低聚物的能力，这是几种细菌孔蛋白所描述的特征。由抗Omp31 MAb A59 / 10F09 / G10识别的表位已被建议具有保护活性，该表位被限定为Omp31的36个氨基酸区域，覆盖了蛋白质的最亲水部分。重组Omp31的可用性以及MAb A59 / 10F09 / G10识别的抗原决定簇的鉴定将有助于评估其潜在的保护活性以及开发针对布鲁氏菌病的亚细胞疫苗的潜力。

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