A single amino-acid change between the antigenically different extracellular serine proteases V2 and B2 from Dichelobacter nodosus
A single amino-acid change between the antigenically different extracellular serine proteases V2 and B2 from Dichelobacter nodosus
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DOI：
10.1016/0378-1119(95)00664-8
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发表时间：
1995-12-29
期刊：
影响因子：
3.5
通讯作者：
Stewart, DJ
中科院分区：
文献类型：
--
作者：
Riffkin, MC;Wang, LF;Stewart, DJ
结节双杆菌（Dn），羊足病的致病生物，分泌三种不同类型的细胞外丝氨酸蛋白酶，这与毒力有关。南方分析表明，蛋白酶由三个独立的基因编码，编码酸性蛋白酶V5和碱性蛋白酶的基因已经从毒力Dn菌株198中被鉴定出来。利用基于蛋白酶V2氨基酸（aa）序列的寡脱氧核糖核苷酸聚合酶链反应（PCR）片段，从菌株198的EcoRI-BamHI基因组DNA文库中分离到了酸性蛋白酶V2的编码基因。从RsaI文库中分离出一个克隆，完成该基因的5'区，得到一个长度为1803 bp的ORF，编码601 aa的蛋白前体。酸性蛋白酶V2基因aprV2与bprV和aprV5基因具有相同的前体结构，在核苷酸（nt）水平上的相似性分别为72%和69%，在aa水平上的相似性分别为73%和69%。由于单克隆抗体一致地区分了该蛋白酶的毒性(V)和良性(B)形式，我们利用PCR方法从良性Dn菌株305中分离出酸性蛋白酶B2的编码基因，并对其进行了特征分析，以探讨这种抗原性差异的分子基础。在单个密码子中发现了一个2 bp的替换，这似乎是导致表位改变的原因。
Dichelobacter nodosus (Dn), the causative organism of ovine footrot, secretes three distinct types of extracellular serine proteases which have been implicated in virulence. Southern analyses have shown that the proteases are encoded by three separate genes, and the genes encoding an acidic protease V5 and a basic protease have already been characterised from virulent Dn strain 198. The gene encoding the third protease type, as represented by acidic protease V2, was isolated from an EcoRI-BamHI library of strain 198 genomic DNA by probing with a polymerase chain reaction (PCR) fragment generated with oligodeoxyribonucleotides based on protease V2 amino acid (aa) sequences. A further clone from an RsaI library was isolated to complete the 5' region of the gene to yield an ORF of 1803 bp encoding a protein precursor of 601 aa. The acidic protease V2 gene, aprV2, shows the same precursor structure as the bprV and aprV5 genes with 72% and 69% similarity at the nucleotide (nt level and with 73% and 69% similarity at the aa level, respectively. As monoclonal antibodies consistently distinguish the virulent (V) and benign (B) forms of this protease, the gene encoding the acidic protease B2 from benign Dn strain 305 was isolated using the PCR and characterized to investigate the molecular basis for this difference in antigenicity. A 2-bp substitution in a single codon was identified which appeared to be responsible for a change of epitope.