Cloning of a sapB homologue (sapB2) encoding a putative 112-kDa Campylobacter fetus S-layer protein and its use for identification and molecular genotyping
Cloning of a sapB homologue (sapB2) encoding a putative 112-kDa Campylobacter fetus S-layer protein and its use for identification and molecular genotyping
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DOI：
10.1016/s0928-8244(98)00081-9
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发表时间：
1998-08-01
影响因子：
--
通讯作者：
Guesdon, JL
中科院分区：
文献类型：
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作者：
Casadémont, I;Chevrier, D;Guesdon, JL
从一株弯曲杆菌中分离到一个编码表面层蛋白的汁液基因。胎儿CIP 53.96T宇宙文库。该基因与sapB保守区具有显著的同源性，命名为sapB2。完整的ORF包含3339个核苷酸，编码一个1112个氨基酸的多肽，计算分子质量为112 kDa。与sapB基因高度同源的区域从ORF前67 bp开始，到ORF后546 bp。同样，在起始密码子后540 bp开始的2038-bp区域，与sapA2基因的同源性达到98%。在本研究中，我们发现这个sapB2基因有两个主要有趣的特征：在每个C.胎儿菌株中都发现了与sapA2同源区域高度同源的区域5‘端，并且包含与sapB保守区域和sapA2同源区域5’端同源区域的片段（IG01），当用作探针时，可以揭示C.胎儿菌株之间的基因组多态性。我们利用这些特点，建立了C -胎儿特异性PCR检测方法，并建立了C -胎儿分型方法。发现该PCR检测具有物种特异性。利用从sapA2同源区5′端提取的寡核苷酸引物，对101株弯曲菌胎儿、18株非弯曲菌胎儿和7株非弯曲菌基因组DNA进行聚合酶链反应试验。只有当C.胎儿DNA作为靶标时，才能扩增出220 bp的片段。在Southern blot分析中，IG01探针只与从C，胎儿菌株中提取的DNA杂交。此外，IG01与几个hindiii消化的DNA片段杂交，为每个菌株提供了特定的模式。(C) 1998年欧洲微生物学会联合会。Elsevier Science B.V.版权所有。
A sap gene encoding a surface layer protein was isolated from a Campylobacter fetus ssp. fetus CIP 53.96T cosmid library. This sap gene, which shows significant homology with the sapB conserved region, was named sapB2. The complete ORF of 3339 nucleotides encodes a 1112-amino acid polypeptide with a calculated molecular mass of 112 kDa. High homology with the sapB gene was found in a region beginning 67 bp before the ORF and proceeding 546 bp into the ORF. Similarly, 98% homology with the sapA2 gene was observed in a 2038-bp region beginning 540 bp after the initiation codon. In the present study, we show that this sapB2 gene has two main interesting features: the 5' end of the region which presents high homology with the sapA2 homologue was found to be present in every C. fetus strain, and the fragment (IG01) comprising the region which presents homology with the sapB conserved region and the 5' end of the sapA2 homologue region, when used as a probe, can reveal genomic polymorphism among C. fetus strains. We exploited these features to develop a PCR assay for the specific detection of C, fetus and to set up a method for typing C. fetus isolates. The PCR assay was found to be species-specific. Oligonucleotide primers derived from the 5' end of sapA2 homologue region were used in a polymerase chain reaction test on genomic DNA extracted from 101 Campylobacter fetus, 18 Campylobacter non-fetus and seven non-Campylobacter strains. A 220-bp fragment was amplified only when C. fetus DNA was used as a target. In Southern blot analysis, the IG01 probe was found to hybridize only with DNA extracted from C, fetus strains. Moreover, IG01 hybridized with several fragments of HindIII-digested DNA, giving a specific pattern for each strain. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.