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从哈维氏弧菌(Vibrio harveyi)、溶藻弧菌(V.alginolyticus)、副溶血弧菌(V.parahaemolyticus)克隆、测定了共19株海水鱼类致病性弧菌外膜蛋白OmpK基因序列,探讨其作为海水鱼类致病性弧菌共同抗原的分子基础。根据已知的弧菌外膜蛋白OmpK序列设计1对简并引物,利用聚合酶链式反应(PCR)方法从19株弧菌总DNA中分别扩增得到约800 bp外膜蛋白OmpK的基因片段,将其克隆到pDM18-T Vector载体筛选阳性重组子进行序列测定。结果显示,OmpK基因分别含有786 bp~849 bp的开放读码框,编码261~282个氨基酸,其核苷酸序列之间的相似性在72%~100%,推测氨基酸序列的相似性为71%~100%,且种内OmpK氨基酸序列的相似性比种间高。序列分析还表明,每一种弧菌OmpK基因都有一段特异性序列,可用于设计核酸探针或特异性引物来诊断、检测哈维氏弧菌等海水鱼致病性弧菌。本研究不仅从基因水平上证实了外膜蛋白OmpK广泛存在于海水鱼致病性弧菌中,而且证明了它们之间具有较高的相似性。由结果推测外膜蛋白OmpK是哈维氏弧菌、溶藻弧菌、副溶血弧菌等致病性弧菌的一种共同抗原,是较好的亚单位疫苗候选成分,为进一步研制广谱的海水鱼类致病性弧菌外膜蛋白基因工程亚单位疫苗提供了理论基础。
A total of 19 strains of marine pathogenic Vibrio outer membrane protein OmpK genes were determined from Vibrio harveyi, V. alginolyticus and V. parahaemolyticus clones Sequence, to explore its molecular basis for common antigens of marine fish pathogenic Vibrio. One pair of degenerate primers was designed according to the known OmpK sequence of Vibrio outer membrane protein, and the gene fragment of OmpK of about 800 bp OMPK was amplified from 19 strains of Vibrio spp. By polymerase chain reaction (PCR) , Cloned into pDM18-T Vector Vector Screening positive recombinant sequence determination. The results showed that OmpK gene contained open reading frame of 786 bp to 849 bp, encoding 261 to 282 amino acids, respectively. The similarity of nucleotide sequence of OmpK gene ranged from 72% to 100%, and the deduced amino acid sequence similarity was 71 % ~ 100%, and the intraspecific OmpK amino acid sequence similarity higher than the species. Sequence analysis also showed that each Vibrio OmpK gene has a specific sequence can be used to design nucleic acid probes or specific primers to diagnose and detect Vibrio harveyi and other marine fish pathogenic Vibrio. This study not only confirmed from the gene level that OmpK, an outer membrane protein, is widely found in marine fish pathogens, but also shows a high similarity between them. The results suggest that the outer membrane protein OmpK is Vibrio harveyi, Vibrio alginolyticus, Vibrio parahaemolyticus and other pathogenic Vibrio common antigen, is a good subunit vaccine candidate components, to further develop a broad spectrum The marine fish pathogenic Vibrio outer membrane protein gene engineering subunit vaccine provides a theoretical basis.