CN106906141B - 一种嗜水气单胞菌活菌疫苗菌株的筛选方法 - Google Patents
一种嗜水气单胞菌活菌疫苗菌株的筛选方法 Download PDFInfo
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Abstract
本发明公开了一种嗜水气单胞菌活菌疫苗菌株的筛选方法，属于水产养殖疾病防控技术领域。本发明的技术方案要点为：一种嗜水气单胞菌活菌疫苗菌株的筛选方法，从养殖环境中分离出嗜水气单胞菌，并检测其毒力基因表达情况，通过细菌攻毒和免疫保护率动物实验，筛选出用于活菌疫苗的嗜水气单胞菌。本发明通过所列的系列引物，检测嗜水气单胞菌的毒力与毒力相关基因，筛选具有广阔应用前景的活菌疫苗，可以较好的保护养殖鱼类抵抗嗜水气单胞菌的感染。应用此种方法制备的疫苗，比灭活疫苗和减毒疫苗制备程序更简单，免疫保护效果更优，并且所分离的细菌为养殖环境中的天然细菌，不用担心复壮、返祖的风险，具有更高的安全性。
Description
技术领域
本发明属于水产养殖疾病防控技术领域，具体涉及一种嗜水气单胞菌活菌疫苗菌株的筛选方法。
背景技术
嗜水气单胞菌在河流、湖泊、池塘、食物、动物及人体等自然环境中广泛存在。嗜水气单胞菌属于条件性致病菌，可引起多种水生动物和陆生动物患病，是一种典型的人-畜-鱼共患病病原菌，当环境条件恶劣，机体抵抗力下降可导致鱼类、两栖类、爬行类等冷血动物和哺乳动物发病，引发水生动物细菌性出血病、体表溃疡症以及哺乳动物腹泻等，是鱼类、爬行类和人类的一种致命传染源。
目前，水产养殖中用于治疗细菌感染性疾病的药物主要为抗菌药物，但抗菌药物的滥用导致水环境污染、药物残留和细菌耐药性产生的问题，特别是多重耐药菌迅速增加，使其不能有效控制感染，成为细菌性疾病治疗的难题，也加重了水产养殖的成本。细菌疫苗从机体免疫出发，能提高水产动物对病原菌的抵抗力，降低病原菌感染的发生率，有利于感染性疾病的控制。所以，开发相关细菌疫苗一直是水产养殖领域的研究热点。
根据制备方式的不同，细菌疫苗可分为灭活疫苗和活菌疫苗。灭活疫苗是用免疫原性强的病原微生物制成的疫苗；活菌疫苗是用弱毒、但免疫原性强的病原微生物，经繁殖后制成的疫苗，也称减毒活疫苗。与灭活疫苗相比，活菌疫苗在免疫保护方面具有较大的优势，如(1)接种途径多样化，不仅可以注射，还可口服、浸泡等；(2)接种数量少，对动物品质影响较小；(3)产生免疫保护的时间短，一般一周内即可产生免疫保护力，而灭活疫苗需要二周以上；(4)免疫效果强且持久，一般只需要接种一次。然而，活菌疫苗有可能出现毒力返强的现象，使其具有一定的风险性。因此解决活菌疫苗毒力问题，是水产动物活菌疫苗发展至关重要的一步。本发明通过检测养殖水环境中的嗜水气单胞菌，选择特定基因型的无毒菌株，进一步通过动物实验证实其免疫保护率，发现免疫原性高的基因型特征，对嗜水气单胞菌活菌疫苗的研制具有重要的指导意义。
发明内容
本发明的目的是提供了一种嗜水气单胞菌活菌疫苗菌株的筛选方法，从鱼体及养殖水环境中筛选无毒、免疫原性高的嗜水气单胞菌菌株，用于活菌疫苗的制备，可达到高效、经济和安全的目的。
本发明为实现上述目的采用如下技术方案，一种嗜水气单胞菌活菌疫苗菌株的筛选方法，其特征在于：从养殖环境中分离出嗜水气单胞菌，并检测其毒力基因表达情况，通过细菌攻毒和免疫保护率动物实验，筛选出用于活菌疫苗的嗜水气单胞菌。
进一步优选，所述嗜水气单胞菌活菌疫苗菌株的筛选方法的具体过程为：从池塘水样、泥土及发病的黄河鲤和淇河鲫中分离获得9株不同批次的嗜水气单胞菌，将分离出的嗜水气单胞菌直接划线在普通营养琼脂平板上，培养单菌落，分别挑取单菌落于液体LB培养基中培养16h，按照试剂盒说明书提取细菌基因组DNA，与嗜水气单胞菌毒力相关的基因为鞭毛系统fla、粘附素aha、外膜蛋白omp、气溶素aerA、溶血素hlyA、β-溶血素β-hly、细胞兴奋性肠毒素altA、细胞毒性肠毒素act、胞外丝氨酸蛋白酶ahpA、弹性蛋白酶ahpB、脂酶酶lip、III型分泌系统aopB和VI型分泌系统vasH，根据嗜水气单胞菌毒力相关基因的基因序列，利用Primer premier 5.0软件设计引物，通过PCR检测嗜水气单胞菌的毒力相关基因和免疫保护率动物实验，筛选出毒力较弱的至少具有β-hly-/act-/fla-基因型的嗜水气单胞菌活菌疫苗菌株和免疫原性较强的至少具有ahpB+/omp+/apoB+基因型的嗜水气单胞菌活菌疫苗菌株。
进一步优选，所述用于活菌疫苗的嗜水气单胞菌为毒力相对较弱且免疫原性相对较强的具有β-hly-/act-/fla-/ahpB+/omp+/apoB+基因型的嗜水气单胞菌活菌疫苗菌株。
进一步优选，所述的毒力较弱的至少具有β-hly-/act-/fla-基因型的嗜水气单胞菌活菌疫苗菌株、免疫原性较强的至少具有ahpB+/omp+/apoB+基因型的嗜水气单胞菌活菌疫苗菌株及毒力相对较弱且免疫原性相对较强的具有β-hly-/act-/fla-/ahpB+/omp+/apoB+基因型的嗜水气单胞菌活菌疫苗菌株在指导活菌疫苗生产中的应用。
本发明与现有技术相比具有以下有益效果：通过本发明所列的系列引物，检测嗜水气单胞菌的毒力相关基因筛选具有广阔应用前景的活菌疫苗，可以较好的保护养殖鱼类抵抗嗜水气单胞菌的感染。应用此种方法制备的疫苗，比灭活疫苗和减毒疫苗制备程序更简单，免疫保护保护效果更优，并且所分离的细菌为养殖环境中的天然细菌，不用担心复壮、返祖的风险，具有更高的安全性。
附图说明
图1是9株不同来源的嗜水气单胞菌基因组DNA电泳图；
图2是9株细菌16S rRNA和13个毒力相关基因的PCR扩增结果图；
图3是4种疫苗的免疫保护效果图。
具体实施方式
以下通过实施例对本发明的上述内容做进一步详细说明，但不应该将此理解为本发明上述主题的范围仅限于以下的实施例，凡基于本发明上述内容实现的技术均属于本发明的范围。
以下实施例中所用的材料及试剂来源
菌株：本发明中所用的菌株来源于池塘水、泥土及发病的黄河鲤和淇河鲫，共分离获得9株不同批次的嗜水气单胞菌。
PCR相关试剂及生化试剂：PCR相关试剂和细菌基因组DNA提取试剂盒均购自宝生物工程(大连)有限公司，引物由生工生物工程(上海)股份有限公司合成，培养基所需试剂均购自生工生物工程(上海)股份有限公司。所述实施例中的未注明具体条件的实验方法，按照常规方法进行。
实施例1
1、9株细菌基因组DNA的提取
表1 9株不同批次的嗜水气单胞菌来源
将采集到的细菌直接划线培养在普通营养琼脂平板上，培养单菌落，分别挑取单菌落于液体LB培养基中培养16h。按试剂盒说明书提取细菌基因组DNA，结果如图1。
2、9类嗜水气单胞菌毒力相关基因及其引物设计
与嗜水气单胞菌毒力相关的基因主要有粘附因子、外毒素及其分泌系统，因此本发明中主要选择如下9类共13种基因：
(1)鞭毛系统：fla
(2)粘附素：aha
(3)外膜蛋白：omp
(4)气溶素：aerA
(5)溶血素：溶血素(hlyA)、β-溶血素(β-hly)
(6)肠毒素：细胞兴奋性肠毒素(altA)、细胞毒性肠毒素(act)
(7)蛋白酶：胞外丝氨酸蛋白酶(ahpA)、弹性蛋白酶(ahpB)
(8)脂酶酶：lip
(9)分泌系统：III型分泌系统(aopB)，VI型分泌系统(vasH)
根据嗜水气单胞菌毒力相关基因的基因序列(NCBI登陆号)，利用Primer premier5.0软件设计引物，如表2。所有引物由生工生物工程(上海)股份有限公司合成。
表2毒力相关基因所用引物
3、PCR检测嗜水气单胞菌的毒力相关基因
以9株细菌的基因组DNA为模板，得用上述引物对毒力相关基因进行扩增，其退火温度及目标片段长度见表2，具体操作方法及步骤如下：
(1)PCR反应体系为：
(2)PCR反应条件设置为：
95℃3min；
94℃30s；50-60℃30s或1min(见表1)；72℃30s；30个循环；
72℃5min；
16℃5min；
最后对PCR产物进行1.0％琼脂糖凝胶电泳，检测PCR扩增效果，如图2。
4、毒力基因检测与其致病力的相关性分析
分离菌株的LD50检测：以无菌生理盐水洗涤培养好的细菌3次，并配成菌悬液，以分光光度计将菌悬液的细菌浓度调整到OD600为0.6，约为5×108cfu/mL。按5倍稀释法制备5个不同浓度的细菌悬液。挑取大小规格一致的健康淇河鲫，随机分为对照组和5个实验组，每组30尾。对照组注射0.1mL生理盐水，实验组注射0.1mL不同浓度菌液，胸鳍基部腹腔注射。注射后的鱼置于25℃水箱中，连续观察7d，每天记录受试鱼的发病和死亡情况，最后统计累积死亡率并计算其LD50。
研究表明，细菌所携带的毒力基因对嗜水气单胞菌的致病性有直接的影响。根据图2对毒力相关基因检测结果发现，所有9株细菌具有不同的基因型。二株嗜水气单胞菌Ah-05和Ah-06基本无致病性。而Ah-01、Ah-02和Ah-03菌株的致病力最强，1×108cfu/mL菌量致死率为100％。以往研究发现毒力基因AerA+/hlyA+基因型的菌株最为普遍(Heuzenroeder,et al.,FEMS Microbiology Letters,1999,174:131-136等)，在本发明分离的具有致病力的菌株中，除Ah-07和Ah-08缺失hlyA基因，其它均具有AerA+/hlyA+基因型。缺失hlyA基因的Ah-07和Ah-08菌株其毒力相对较弱，说明本发明的结果与以往研究结果相一致。非常重要的是，β-hly/act/fla基因对嗜水气单胞菌的致病性是非常重要的，β-hly-/act-/fla-基因型的Ah-06对淇河鲫基本无致病能力。
表3 13个毒力基因型与其致病力的相关性
注：“+”表示基因检测呈阳性，“-”表示基因检测呈阴性
实施例2
菌苗制备及免疫：挑取Ah-02、Ah-03、Ah-05和Ah-06单菌落于新鲜LB培养基中。待细菌生长至OD 1.0时，收集菌液，4000g离心15min，生理盐水洗涤菌体3次并悬浮。Ah-02、Ah-03用0.5wt％福尔马林于30℃灭活24h。灭活或活菌用无菌生理盐水调OD600至0.2，菌数约1×108cfu/mL。180尾淇河鲫随机分为6组，每组30尾，对照组注射0.1mL生理盐水，实验组注射0.1mL不同免疫状态的菌株。免疫14d后，每尾注射0.1mL1×108cfu/mL Ah-01菌液进行攻毒，注射后的鱼置于25℃水箱中，连续观察7d，每天记录受试鱼的发病和死亡情况，最后统计累积死亡率。免疫保护率＝[1–(免疫组死亡率/对照组死亡率)]×100％。
细菌攻毒结果显示，Ah-02灭活菌、Ah-03灭活菌、Ah-05活菌和Ah-06活菌的累积死亡率分别为36.66％、56.66％、43.33％和13.33％，远低于对照组的90％，这说明这4种疫苗均具有一定的免疫保护效果，如表4和图3。从以上结果可以看出，Ah-06活菌疫苗的保护效果最优，这说明该菌株具有非常好的免疫原性，是一个较好的候选活菌疫苗。相比Ah-05活菌疫苗，其毒力相关基因相对完整，保留有很多可能与细菌免疫原性相关的分子，如altA、Aha、Omp(LeClaire RD et al.,Infect.Immun,2002,70:2278-2281等)，即细菌具有altA+/aha+/omp+基因型可能对其保持免疫原性的保持至关重要。
综合以上结果，通过本发明所列的系列引物，检测嗜水气单胞菌的毒力相关基因筛选具有广阔应用前景的活菌疫苗，可以较好的保护养殖鱼类抵抗嗜水气单胞菌的感染。应用此种方法制备的疫苗，比灭活疫苗和减毒疫苗制备程序更简单，免疫保护保护效果更优，并且所分离的细菌为养殖环境中的天然细菌，不用担心复壮、返祖的风险，具有更高的安全性。
表4活菌及灭活菌免疫后HNAh01菌株对淇河鲫的人工感染结果
以上实施例描述了本发明的基本原理、主要特征及优点，本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明原理的范围下，本发明还会有各种变化和改进，这些变化和改进均落入本发明保护的范围内。
SEQUENCE LISTING
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<120> 一种嗜水气单胞菌活菌疫苗菌株的筛选方法
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Claims (1)
1.一种结合免疫原性和致病力特性筛选嗜水气单胞菌活菌疫苗的方法，其特征是包含如下步骤：
（1）将从黄河鲤或淇河鲤分离出的嗜水气单胞菌直接划线在普通营养琼脂平板上，培养单菌落，分别挑取单菌落于液体LB培养基中培养16h，按照试剂盒说明书提取细菌基因组DNA；
（2）与嗜水气单胞菌毒力相关基因可以分为9大类，检测这9大类共13个基因的分布情况，这些基因包括鞭毛系统fla、粘附素aha、外膜蛋白omp、气溶素aerA、溶血素hlyA、β-溶血素β-hly、细胞兴奋性肠毒素altA、细胞毒性肠毒素act、胞外丝氨酸蛋白酶ahpA、弹性蛋白酶ahpB、脂酶lip、III型分泌系统aopB和VI型分泌系统vasH，根据上述基因的基因序列，利用Primer premier 5.0软件设计引物，并进行PCR扩增；
（3）细菌攻毒实验，统计鱼体的累积死亡率并计算其LD50，分析毒力基因的分布与其致病力的相关性；
（4）筛选候选菌株制备活菌疫苗，并以含有完整毒力基因型的菌株制备灭活疫苗作为对照，验证上述活菌疫苗的免疫保护性；
筛选用于活菌疫苗制备的嗜水气单胞菌至少具有β-hly - /act - /fla - 基因型，这样的菌株致病性较弱或无致病性；同时具有ahpB+/omp+/apoB+基因型，这样保证其免疫原性较强。
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