CAJ | 학술논문

哈维氏弧菌(V.harveyi)的VHH溶血素是对海水养殖鱼类的潜在致病因子.哈维氏弧菌的VHH溶血素基因与副溶血弧菌(V.parahaemolyticus)的TLH热不稳定性溶血素基因具有高度相似性,其氨基酸序列的相似性达到85.6%.根据哈维氏弧菌vhhA溶血素基因序列,合成一个地高辛标记的VHH基因探针,利用其进行Southern Blot,检测VHH溶血素基因在57株弧菌(包括26株国际标准菌株,20株哈维氏弧菌,11株副溶血弧菌)中的分布情况.结果显示,VHH基因探针与13株弧菌标准菌株有强杂交信号,包括2株溶藻胶弧菌(V.alginolyticus),2株哈维氏弧菌以及1株霍氏格里蒙菌(Grimontia hollisae),坎贝氏弧菌(V.campbellii),辛辛那提弧菌(V.cincinatiensis),费氏弧菌(V.fischeri),拟态弧菌(V.mimicus),飘浮弧菌(V.natriegens),副溶血弧菌,解蛋白弧菌(V.proteolyticus)和火神弧菌(V.logei).与6株弧菌标准菌株有弱杂交信号,包括鳗弧菌(V.anguillarum),河口弧菌(V.aestuarianus),美人鱼发光杆菌(Photobacterium damselae subsp.damselae),河弧菌(V.fluvialis),弗尼斯弧菌(V.furnissii)和创伤弧菌(V.vulnificus),而另外7株弧菌标准菌株中无杂交信号.所有的哈维氏弧菌菌株至少含有一条杂交带,其中菌株VIB 645,VIB 648和SF-1分别含有2条杂交带.11株副溶血弧菌中均含有一条杂交带.上述数据表明,vhh/tlh溶血素基因广泛分布于弧菌中,尤其是哈维氏弧菌相关菌株和费氏弧菌相关菌株中.另外对鳗弧菌VIB 72,坎贝氏弧菌VIB 285,飘浮弧菌VIB 299和哈维氏弧菌VIB 647的vhh/tlh溶血素基因进行克隆并测序,其氨基酸序列与VHH溶血素和TLH溶血素氨基酸序列的同源性分别为67%～99%和69%～91%.对vhh/tlh溶血素基因在弧菌中的分布研究,将有助于进一步确定这类溶血素基因在病原弧菌致病性中的作用.
합유씨호균(V.harveyi)적VHH용혈소시대해수양식어류적잠재치병인자.합유씨호균적VHH용혈소기인여부용혈호균(V.parahaemolyticus)적TLH열불은정성용혈소기인구유고도상사성,기안기산서렬적상사성체도85.6%.근거합유씨호균vhhA용혈소기인서렬,합성일개지고신표기적VHH기인탐침,이용기진행Southern Blot,검측VHH용혈소기인재57주호균(포괄26주국제표준균주,20주합유씨호균,11주부용혈호균)중적분포정황.결과현시,VHH기인탐침여13주호균표준균주유강잡교신호,포괄2주용조효호균(V.alginolyticus),2주합유씨호균이급1주곽씨격리몽균(Grimontia hollisae),감패씨호균(V.campbellii),신신나제호균(V.cincinatiensis),비씨호균(V.fischeri),의태호균(V.mimicus),표부호균(V.natriegens),부용혈호균,해단백호균(V.proteolyticus)화화신호균(V.logei).여6주호균표준균주유약잡교신호,포괄만호균(V.anguillarum),하구호균(V.aestuarianus),미인어발광간균(Photobacterium damselae subsp.damselae),하호균(V.fluvialis),불니사호균(V.furnissii)화창상호균(V.vulnificus),이령외7주호균표준균주중무잡교신호.소유적합유씨호균균주지소함유일조잡교대,기중균주VIB 645,VIB 648화SF-1분별함유2조잡교대.11주부용혈호균중균함유일조잡교대.상술수거표명,vhh/tlh용혈소기인엄범분포우호균중,우기시합유씨호균상관균주화비씨호균상관균주중.령외대만호균VIB 72,감패씨호균VIB 285,표부호균VIB 299화합유씨호균VIB 647적vhh/tlh용혈소기인진행극륭병측서,기안기산서렬여VHH용혈소화TLH용혈소안기산서렬적동원성분별위67%～99%화69%～91%.대vhh/tlh용혈소기인재호균중적분포연구,장유조우진일보학정저류용혈소기인재병원호균치병성중적작용.
V. harveyi VHH haemolysin, which shows high homology to the TLH haemolysin ( the identities of their deduced amino acid sequences are up to 85.6%), is a putative virulence factor to marine cultured fish. A VHH probe, which is specific to V. harveyi vhhA haemolysin gene, was used to screen EcoR Ⅰ digests of total DNA from 57 vibrio strains, including 26 vibrio type strains, 20 V. harveyi isolates and 11 V. parahaemolyticus isolates. As a result, 1 strong hybridisation band was detected in 13 type strains, including 2 of Vibrio alginolyticus , 2 of V. harveyi , and 1 strain each of Grimontia hollisae , V. campbellii , V. cincinnatiensis , V. fischeri , V. mimicus , V.natriegens, V. parahaemolyticus, V. proteolyticus and V. logei. Also, 1 weak band was detected in 6 type strains, including V.anguillarum, V. aestuarianus, Photobacterium damselae subsp. damselae, V. fluvialis, V. furnissii and V. vulnificus. There was not any hybridization signal in other type strains. Also, vhh/tlh was present in all isolates of V. harveyi and V. parahaemolyticus. Moreover, 3 isolates of V. harveyi, i.e. VIB 645, VIB 648 and SF1, had duplicated vhh genes. The data indicates that vhh/tlh is widespread in vibrios,especially in V. harveyi related species and V. fischeri related species. To support this conclusion, the vhh/tlh homologue genes in V.anguillarum VIB 72, V. campbellii VIB 285, V. natriegens VIB 299 and V. harveyi VIB 647 were cloned and sequenced, and the deduced amino acid sequences showed high degree of identities to VHH (67% ～ 99% ) and TLH haemolysin (69% ～ 91% ). This study will help us to identify the role of vhh/tlh haemolysin gene in the pathogenicity of vibrios.