一株海豚链球菌的分离鉴定及其亚单位疫苗研究
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摘要
海豚链球菌是一种革兰氏阳性菌,可感染多种海水和淡水养殖鱼类,是一种重要的水产养殖病原菌。本实验室从濒死的患病牙鲆体内分离到一株细菌SF1,初步的攻毒实验表明SF1可以感染牙鲆并且显示出较强的毒力。16S序列分析SF1为海豚链球菌;通过API 20 Strep检测和随机扩增多态性DNA标记分析,确认SF1为血清型I型海豚链球菌。通过本实验室构建的一个分泌序列的捕获系统pBU自SF1筛选到一个候选抗原Sip11。将Sip11用原核表达系统在BL21(DE3)进行诱导表达,进一步将其纯化,得到重组蛋白Sip11。用牙鲆为模型动物,腹腔注射Sip11,用SF1攻毒检测其保护效率,其相对保护率为69.7%。为了克服常规亚单位疫苗的缺点以及提高Sip11的免疫效应,进一步构建了一个可将Sip11分泌到胞外的菌株DH5α/pTAS11,以DH5α/pTAS11为活体疫苗免疫牙鲆,其免疫相对保护率为100%。用ELISA检测免疫之后5到8周的血清抗体效价,Sip11蛋白免疫组和DH5α/pTAS11活体疫苗免疫组都有特异性针对Sip11的抗体产生。这些结果表明,DH5α/pTAS11可作为一个有效的抵御SF1感染的候选疫苗。
Streptococcus iniae is a Gram-positive bacterium and a severe aquaculture pathogen that can infect a wide range of farmed fish species. A strain named SF1 was identified from a dying Janpanese flounder. SF1, which exhibited apparent virulence in a Japanese flounder infection model and conformed to the description of S. iniae by 16S rRNA sequence analysis and API 20 Strep test. Sip11 was by a previously established exported proteins system pBU from SF1. Recombinant Sip11 was purified from Escherichia coli BL21(DE3) and tested to be an immunoprotective protein agaist SF1 in Janpanese flounder. Sip11 exhibited highly RPS (Relative Percentage Survival)≈69.7% aganist SF1. To enhance the RPS, one antigen delivery vector expressed in DH5αwas constructed, which expresses and secrets recombinant Sip11 covalently linked to a carrier protein in the form of a chimera. Vaccination of Japanese flounder with live Sip11-secreting E. coli afforded complete protection upon the fish following lethal SF1 challenge. Specific antibody production was observed in fish immunized with both Sip11 and DH5α/pTAS11, which detected from 5th week to 8th week after vaccination. These results indicate that Sip11, especially when delivered by a live bacterial carrier, is an effective vaccine candidate against SF1 infection.
Streptococcus iniae is a Gram-positive bacterium and a severe aquaculture pathogen that can infect a wide range of farmed fish species. A strain named SF1 was identified from a dying Janpanese flounder. SF1, which exhibited apparent virulence in a Japanese flounder infection model and conformed to the description of S. iniae by 16S rRNA sequence analysis and API 20 Strep test. Sip11 was by a previously established exported proteins system pBU from SF1. Recombinant Sip11 was purified from Escherichia coli BL21(DE3) and tested to be an immunoprotective protein agaist SF1 in Janpanese flounder. Sip11 exhibited highly RPS (Relative Percentage Survival)≈69.7% aganist SF1. To enhance the RPS, one antigen delivery vector expressed in DH5αwas constructed, which expresses and secrets recombinant Sip11 covalently linked to a carrier protein in the form of a chimera. Vaccination of Japanese flounder with live Sip11-secreting E. coli afforded complete protection upon the fish following lethal SF1 challenge. Specific antibody production was observed in fish immunized with both Sip11 and DH5α/pTAS11, which detected from 5th week to 8th week after vaccination. These results indicate that Sip11, especially when delivered by a live bacterial carrier, is an effective vaccine candidate against SF1 infection.
引文
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[8]杜佳银.海水养殖鱼类链球菌病.渔业现代化,2001,5:28–29.
[9]杜佳银,吴晓慧.牙鲆链球菌病的防治.中国水产,1999,7:29–30.
[10] Klesius P, Evans J, Shoemaker C, Yeh H, Goodwin AE, Adams A, Thompson K. Rapid detection and identification of Streptococcus iniae using a monoclonal antibody based indirect fluorescent antibody technique. Aquaculture 2006; 258:180–6.
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[15] Shoemaker CA, Vandenberg GW, Désormeaux A, Klesius PH,Evans JJ. Efficacy of a Streptococcus iniae modified bacterin delivered using Oralject? technology in Nile tilapia (Oreochromis niloticus). Aquaculture 2006; 255:151–6.
[16] Buchanan JT, Stannard JA, Lauth X, Ostland VE, Powell HC, Westerman ME, Nizet V. Streptococcus iniae phosphoglucomutase is a virulence factor and a target for vaccine development. Infect Immun 2005; 73:6935–44
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[18]Zlotkin A, Hershko H, Eldar A. Possible transmission of Streptococcus iniae from wild fish to cultured marine fish. Appl Environ Microbiol 1998; 64:4065–7.
[19] Shoemaker CA, Klesius PH, Evans JJ. Prevalence of Streptococcus iniae in tilapia, hybrid striped bass, and channel catfish on commercial fish farms in the United States. Am J Vet Res 2001;62:174–7.
[20] Barnes AC, Young FM, Horne MT, Ellis AE. Streptococcus iniae: serological differences, presence of capsule and resistance to immune serum killing. Dis Aquat Org 2003; 53:241–7.
[21] Fuller J D, Camus A C, Duncan C L, et al. Identification of a StreptolysinS-Associated Gene Cluster and Its Role in the Pathogenesis of Streptococcus iniae Disease. Infect Immun, 2002, 70:5730–9
[22] Locke JB, Aziz RK, Vicknair MR, Nizet V, Buchanan JT. Streptococcus iniae M-like protein contributes to virulence in fish and is a target for live attenuated vaccine development. PLoS One, 2008, 3: 2824.
[23]林为民,李新苹,张孝恩等.鱼链球菌病的诊断和防制.中国兽医杂志,2000 26(3):51
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[26]Evans J J, Shoemaker C A, Klesius P H. Therapeutic and prophylactic immunisation against Streptococcus iniae infection in hybrid striped bass Morone chrysops x M saxatilis [J]. Aquaculture Research, 2006c; 37: 742–50.
[27]Eldar A, Bejerano Y, Bercovier H. Streptococcus shiloi and Streptococcus difficile: two new streptococcal species causing a meningoencephalitis in fish. Curr Microbiol 1994; 28:139–43.
[28] Klesius PH, Shoemaker CA, Evans JJ. Efficacy of a killed Streptococcus iniae vaccine in tilapia (Oreochromis niloticus). Bull Eur Assoc Fish Pathol 1999; 19:39–41.
[29] US6379677B1
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