19株海水鱼致病性弧菌OmpK基因序列及其抗原性分析

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800bp外膜蛋白OmpK的基因片段,将其克隆到pDM18-Tvector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786bp～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.

Sequence analysis on OmpK genes and antigenicity of pathogentic Vibrios isolated from marine fishes

Outer membrane protein OmpK was a kind of receptor for broad-host-range vibriophage. To study whether the OmpK was the common antigen of different Pathogenic Vibrios in marine fish at molecular level, the OmpK genes of V. harveyi, V. alginolyticus and V. parahaernolyticus were cloned and sequenced. 19 strains of Pathogenic Vibrios were isolated from different marine fish in Guangdong, Hainan and Fujian province. A pair of degenerate primers were designed and synthesized according to the nucleotide sequence of the OmpK gene of Vibrio reported. Using the total genomes of 19 stains Vibrio as templates, OmpK genes fragments were obtained by PCR amplification and cloned into pMD18-T Vector. Recombinants were confirmed by digesting with restriction enzyme, EcoR I and Sal, and then the inserts were sequenced. The result showed that OmpK genes contained an open reading frame of 786 bp- 849 bp that coded 261-282 amino acids in 19 strains of Vibrios. The similarities of the nucleotide sequences of OmpK genes were 72%-100% and the deduced amino acid sequences were 71%-100% among 19 strains of Vibrios. As a result, the OmpK genes not only were distributed widely in all tested Vibrios, but also shared quite high similarity in nucleotide sequences and deduced amino acid sequences. So OmpK could be a kind of effective protective common antigen and an effective potential vaccine candidate against different Vibrios. It suggested that the OmpK would be a candidate for subunit vaccine to prevent the disease caused by different Vibrios in marine fish. Among the deduced amino acid sequences of three species of Vibrios, each had a special sequences of 6-9 amino acids which were different from each other. The special sequences would lead to further studies of a rapid and sensitive method to diagnose the pathogenic V. harveyi, V. alginolyticus or V. parahaemolyticus.