CN111172119B - 具有宽裂解谱的新副溶血弧菌噬菌体、其特异性引物及其应用 - Google Patents
具有宽裂解谱的新副溶血弧菌噬菌体、其特异性引物及其应用 Download PDFInfo
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Abstract
本发明公开了一种具有宽裂解谱的新副溶血弧菌噬菌体及其扩增应用,以及其特异性扩增引物,上述噬菌体对副溶血弧菌具有强烈的裂解作用,可由宿主菌特异性扩增,且增殖时间短,发酵产物效价高,为工业化生产噬菌体及其应用提供了噬菌体来源。上述噬菌体可用于可制备抗副溶血弧菌的药物,为控制由副溶血弧菌感染引起的急性肝胰腺坏死综合征提供新型药物。所述噬菌体单次泼洒养殖水体后,能够显著降低水体中副溶血弧菌的数量;将噬菌体拌料后饲喂南美白对虾,或者将对虾浸浴噬菌体,能够有效预防和治疗由副溶血弧菌引起的感染,提高对虾成活率。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一株具有宽裂解谱的新副溶血弧菌噬菌体及其应用。
背景技术
对虾是全球性的水产养殖种类,也是全球范围内交易量最大的水产品。但据中国水产频道报道,GSMC报告显示:2016年中国对虾产量下降15万吨, 2017年仍然呈下降趋势。病毒和细菌性疾病是阻碍对虾可持续发展和农业市场波动的主要因素。急性肝胰腺坏死综合征(acute hepatopancreatic necrosis disease,AHPND)是对虾养殖场一种新型的爆发性的病害,即早期死亡综合征 (early mortality syndrome,EMS)。该病最早于2009年在中国海南首次报道,主要危害凡纳滨对虾和斑节对虾。AHPND致死率高、发病急、传染性强,该病害已经对全球对虾养殖业造成巨大的冲击,使得对虾产量急剧下降并造成了严重的经济损失。
目前,对该疾病的防治方法多停留在改良养殖环境、改善虾苗的抗病性、使用化学性药物和抗生素等。但是大量抗生素及化学药物的使用,严重污染了水体,降低了水产品的品质。近年来,人们对生物防治更加关注,包括使用蛭弧菌和噬菌体防治的方法。
噬菌体是一类能特异性感染细菌、真菌、放线菌或螺旋体的病毒,可以从污水、粪便以及土壤等自然环境中分离得到。噬菌体具有严格的宿主特异性,副溶血弧菌噬菌体只特异性感染副溶血弧菌,对人、动植物和其他微生物没有感染作用,且只能依附于相应的宿主菌存在,所以噬菌体具有安全性的特征,对环境没有副作用。噬菌体裂解细菌具有高效的特点,一个噬菌体颗粒感染宿主菌后,便呈指数级增长,能够持续感染其他宿主菌。噬菌体可通过多种方式进行给药,便于养殖人员操作,且作用效果持久。所以噬菌体能够作为一种具有杀菌作用的高效生物消毒剂和药物。
目前,仅有关于副溶血弧菌噬菌体应用于控制食品表面的副溶血弧菌,或给加工器具消毒的相关报道,但是副溶血弧菌噬菌体作为养殖环境水体消毒的使用方式,以及作为饲料添加剂饲喂对虾的噬菌体给药途径还未见报道。此外,公开的副溶血弧菌噬菌体都具有相同的缺点,即裂解谱都较窄。本身申请中的副溶血弧菌噬菌体针对123株副溶血弧菌,裂解率高达68.3%,具有宽裂解谱的特点。
发明内容
针对上述问题,本发明提供了一株具有宽裂解谱的新副溶血弧菌噬菌体及应用,对新副溶血弧菌具有广谱的杀菌能力,可被用于防治副溶血弧菌感染导致的疾病,也可作为水体消毒剂应用于海产养殖,旨在解决水产养殖场因副溶血弧菌感染而导致发病急、改水慢等困难。
本发明的技术方案如下:
本发明提供了一种具有宽裂解谱的新副溶血弧菌噬菌体,其拉丁名为Vibrioparahaemolyticus,被命名为PG07,保藏编号为CGMCC NO.16392,于 2018年9月26日被保藏于中国微生物菌种保藏管理委员会普通微生物中心。
本发明还提供所述新副溶血弧菌噬菌体在制备预防或治疗副溶血性弧菌引起的疾病的药物中的用途。
可选地,上述噬菌体通过拌料口服给药,0.5h即可在对虾肝胰腺中检测到噬菌体,能够在肝胰腺中存在3d以上。给南美白对虾注射致病性副溶血弧菌后,饲喂浸润了噬菌体的饲料,可有效控制对虾的死亡率,与对照相比,显著提高对虾存活率。
优选地,所述疾病为对虾急性肝胰腺坏死病。本发明的副溶血弧菌噬菌体PG07为控制由副溶血弧菌感染引起的急性肝胰腺坏死综合征提供环保新药。
本发明还提供所述新副溶血弧菌噬菌体在制备防治在水产品养殖、运输及保存过程中的细菌污染的制剂中的用途。
本发明还提供一种用于防治副溶血弧菌感染的杀菌组合物,其包括有效量为1×1011PFU/ml上述的副溶血弧菌噬菌体。
本发明还提供一种水体消毒剂,其有效成分为上述副溶血弧菌噬菌体。优选地,副溶血弧菌噬菌体的浓度为1×103PFU/ml以上。此浓度下,水体消毒剂的消毒效果最佳。上述水体消毒剂使用便利,可有效用于减少养殖环境中的副溶血弧菌及致病性弧菌的数量。
优选地,所述水体消毒剂可制备成不同剂型,对水体进行消毒或对病原菌的监测,剂型为粉剂、混悬剂、分散体或溶液剂。
优选地,所述水体消毒剂还包含其他相配合的杀菌活性成分或其他助剂。如能延长副溶血弧菌噬菌体的持效期的助剂。
本发明还提供一种对虾饲料添加剂,其包括上述副溶血弧菌噬菌体。上述噬菌体作为饲料添加剂添加入南美白对虾的饲料中,能有效预防对虾感染副溶血弧菌而导致的早期死亡综合征(偷死病),降低死亡率,提高虾苗成活率,降低养殖风险。优选地,饲料中副溶血弧菌噬菌体的含量为1×108PFU/g 以上。该条件下,对对虾因感染副溶血弧菌而导致的早期死亡综合征的防治效果最佳。
此外,上述噬菌体以浸浴的方式,作用于南美白对虾,再给南美白对虾注射致病性副溶血弧菌,结果显示浸浴噬菌体可有效控制对虾的死亡率,显著提高对虾保护率。由此可推断,噬菌体制剂可用于南美白对虾虾苗分塘前的处理,能够预防副溶血弧菌感染,减少发病率,提高对虾成活率。
本发明还提供一种检测试剂盒,其包括如上所述的副溶血弧菌噬菌体。本领域技术人员可根据本发明公开内容和本领域常识利用上述副溶血弧菌噬菌体制备检测试剂盒,用于检测其特异侵染的副溶血弧菌、或用于鉴定和防控由其宿主菌副溶血弧菌侵染导致的疾病。
本发明实施例提供的副溶血弧菌噬菌体及其应用具有以下有益效果:
1、上述噬菌体对副溶血弧菌具有强烈的裂解作用,可由宿主菌特异性扩增,且增殖时间短,发酵产物效价高,为工业化生产噬菌体及其应用提供了噬菌体来源。
2、该噬菌体可制备抗副溶血弧菌的药物,为控制由副溶血弧菌感染引起的急性肝胰腺坏死综合征提供新型药物。所述噬菌体单次泼洒养殖水体后,能够显著降低水体中副溶血弧菌的数量;将噬菌体拌料后饲喂南美白对虾,或者将对虾浸浴噬菌体,能够有效预防和治疗由副溶血弧菌引起的感染,提高对虾成活率。作为饲料添加剂使用时,能有效预防对虾感染副溶血弧菌而导致的早期死亡综合征(偷死病),降低死亡率,提高虾苗成活率,降低养殖风险。
附图说明
图1为本发明的噬菌体PG07的电子显微镜观察结果;
图2为噬菌体PG07的特异性引物扩增片段琼脂糖凝胶电泳图;其中,1:Marker2000;2:PG07;3~4:其他副溶血弧菌噬菌体;5~6:绿脓杆菌噬菌体; 7:葡萄球菌噬菌体;8~10:溶藻弧菌噬菌体;11:阴性对照;
图3为噬菌体PG07的热稳定性检测结果;
图4为噬菌体PG07的pH稳定性检测结果;
图5为噬菌体PG07对ATCC17802杀灭效果的时效图(体外裂解);
图6为噬菌体PG07降低水体中副溶血弧菌的效果图;
图7为拌料饲喂噬菌体PG07后对虾肠道中噬菌体的代谢结果;
图8为拌料饲喂噬菌体PG07后对虾肝胰腺中噬菌体代谢结果。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例一噬菌体的分离培养
(一)菌种的复苏和增殖
挑取副溶血弧菌的冻存菌液在TCBS平板上三区划线,于37℃恒温箱中培养16~24h。挑取单菌落,接种于5ml 2216E肉汤中,于37℃空气振荡器中增殖,170rpm振荡培养16h,得到单一的菌悬液。
(二)噬菌体的分离与纯化
取青岛市城阳区海鲜市场污水、养殖池水等样品先以10000rpm离心 5min,用上清液配制2216E肉汤,分装于三角瓶中,每个三角瓶中按照1%的量加入各株副溶血弧菌,搅拌均匀后,于37℃的空气振荡器中,170rpm振荡培养过夜,10000rpm离心5min,0.22μm的细菌滤器过滤除菌。将滤液和宿主菌混合,37℃孵育5min后倒双层平板,待凝固后放入37℃的恒温箱中倒置培养,6~8h后观察结果。若有噬菌体存在,则会在培养基上形成透明、规则的圆形空斑,即为噬菌斑。纯化时抠取单个噬菌斑,于1ml 2216E肉汤中, 37℃的空气振荡器中孵育30min,10000rpm离心5min,取上清再用双层平板法得到单个的噬菌斑,如此重复3~5次直至得到大小、形态一致的噬菌斑,并将该噬菌体命名为PG07。噬菌体PG07保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址:中国北京,保藏日期为2018年9月26日,保藏编号为CGMCC NO.16392。
实施例二噬菌体的菌种鉴定
1、电镜下噬菌体的形态观察
取20μl的1×109PFU/ml的该噬菌体样品滴于微孔铜网上,沉淀15min,用滤纸吸去多余的液体。在铜网上滴加15μl的2%的磷钨酸(PTA),染色5min,用滤纸吸去多余的染液,干燥后用透射电镜观察并拍照。噬菌体的形态结果参见图1。
观察结果为:PG07的头部为直径80nm左右的多面体,其非伸缩尾部长约150nm,根据国际病毒分类委员会(The International Committee on Taxonomy of Viruses,ICTV)第九次报告,可确定噬菌体PG07为长尾噬菌体科。
PEG-NaCl法浓缩噬菌体,使用病毒基因组RNA提取试剂盒对噬菌体核酸进行提取,取5μl噬菌体核酸分别与5μl DNaseⅠ、5μlRNaseA以及5μl BAL31核酸酶和25μlBAL31buffer混合,将混合物置于37℃温箱中作用1h,取作用之后的产物进行1%的琼脂糖凝胶电泳。
实验结果显示该噬菌体的核酸是双链环状DNA。
2、噬菌体的分子生物学鉴定
(1)噬菌体PG07的基因组提取
取500μl已纯化噬菌体,加入DNaseⅠ和RNase A至终浓度1mg/L,37℃孵育1h,80℃加热15min,使DNA酶失活。然后加入EDTA(终浓度0.02mol/L),蛋白酶K(终浓度50mg/L),10%SDS(终浓度0.5%),56℃水浴1h裂解噬菌体。加入等体积平衡酚上下颠倒混匀,10000rpm离心10min,收集上层水相至新的1.5ml离心管中。加入等体积的酚:氯仿:异戊醇(25:24:1,体积比),温和混匀后10000rpm离心10min,以去除蛋白、糖类等杂质,收集上层水相至新的离心管中,加入等体积氯仿再抽提一次,最后收集上层水相并加入等体积无水乙醇沉淀核酸,-20℃放置2h后12000rpm离心20min,弃上清收集沉淀。用1ml 75%的冰乙醇洗涤上述DNA沉淀,12000rpm离心10min,室温干燥后,用30μl无核酸水溶解DNA。
(2)噬菌体PG07的全基因组测序
使用IlluminaMiseq(San Diego,CA,USA)测序仪对噬菌体进行全基因组测序。使用Miseq Reagent Kit v2试剂盒构建600bp测序文库,主要流程如下:对基因组DNA进行超声打断,末端补平,并加上特异接头后,扩增纯化筛选 DNA,即得到构建的测序文库。使用软件Newbler2.9对噬菌体原始测序数据进行组装和拼接。最后得到噬菌体基因组大小为112106bp,为双链DNA, G+C的含量为43.65%,含有158个预测的开放阅读框。使用BLAST在线工具(http://blast.ncbi.nlm.nih.gov/)对噬菌体基因组进行序列相似性比对分析。与其同源性最高的噬菌体为Vibrio phage pVp-1,噬菌体PG07与该噬菌体的同源性仅达到79.1%(覆盖率/相似度:2%/79.1%)。上述结果说明噬菌体PG07 是一株新的副溶血弧菌噬菌体。
使用在线工具BLASTp和保守域数据库(CDD)分别对噬菌体PG07的158 个ORFs蛋白序列进行比对,其中,噬菌体PG07含已知编码功能蛋白30个,其余ORFs为假想蛋白。此外,根据基因组测序结果进一步可得:该噬菌体基因组含有与噬菌体宿主识别相关的三个尾部纤维(tail fibers protein)蛋白基因序列分别见序列表SEQ ID No.1~3,位置分别为80818-81264bp、84527-87400bp 和93782-94921bp;保守的DNA连接酶亚基A(NAD-dependent DNA ligase subunit A)基因的序列见序列表中SEQ ID No.4,位置为60368-61318bp;DNA 聚合酶(DNA polymerase)基因的序列见序列表中SEQ ID No.5,位置为65932-68532bp;与裂解能力相关的裂解酶(lysozyme)基因序列见序列表中 SEQ ID No.6,位置为702-1028bp。
上述基因的具体信息见下表1。
表1噬菌体PG07的基因序列信息表
(3)噬菌体PG07的特异性扩增
本发明还提供一对特异性基因扩增引物为PG07-F/R(序列表中SEQ ID No.7和SEQID No.8),利用这一对引物扩增得到扩增产物,将分离纯化后的扩增片段进行测序,得到该扩增产物长度为527bp,序列具体见序列表中SEQ ID No9,为SEQ ID No2尾部蛋白基因中的一段序列,上述引物序列如下:
PG07-F:TGAGTCTCACCGAGGTCTTATC
PG07-R:CTGTAGGGTCTTGAGTAGGTGTAG
实验步骤:分别提取噬菌体PG07和2株其他副溶血弧菌噬菌体、2株绿脓杆菌噬菌体、1株葡萄球菌噬菌体、3株溶藻弧菌噬菌体的基因组,并分别用上述特异性基因扩增引物PG07-F和PG07-R进行目标基因的扩增,扩增条件为:94℃预变性5min,循环内94℃变性1min,58℃退火1min,72℃延伸 1min,35个循环,最后72℃延伸10min,反应产物经1.0%琼脂糖凝胶电泳检测。对PCR扩增产物进行回收并进行电泳检测,从电泳结果发现:仅噬菌体PG07的基因组中扩增出目标基因,其他噬菌体都未扩增出来(见图2)。这说明上述引物的特异性高,可通过这对引物对噬菌体PG07进行快速准确的分子鉴定。
实施例三噬菌体生物学特性的检测
(一)噬菌体的增殖和效价测定
各取100μl宿主菌和噬菌体抠斑浸出液加入到5ml 2216E肉汤中,于37℃的空气振荡器中,170rpm培养3~4h,待混合液变澄清,得到噬菌体增殖液。 10倍倍比稀释噬菌体增殖液,双层平板法测效价,每个稀释度分别做3个平行样。计数时,观察平板中的噬菌斑取30~300之间的平板来计数,计算效价。
取稀释度为10-7的3个平行样计数,结果分别为:107、112、109个噬菌斑,计算效价为1.09×1010PFU/ml。
(二)噬菌体PG07的裂解谱检测
选取实验室保存的副溶血弧菌123株,采用双层平板法检测PG07的裂解谱。这些菌株是2017~2019年内在江门、滨州、烟台、海口等地的患病南美白对虾肝胰腺和养殖池水中分离鉴定而来。其中有62株副溶血弧菌携带与致病性相关的基因,包括PriA、PriB、AP1、AP2、AP3、Tdh、Trh、Tlh、Ⅲ型分泌系统2 T3SS1:VP1670(vscP)、VP1686、VP1689(vscK)、VP1694 (vscF)、vscC2、vscD2以及orf8、toxRS、VopC2、vcrD2。
取100μl菌液与100μl PG07制备双层平板,置于37℃的恒温箱中倒置培养16h左右,观察PG07对各株菌的裂解情况。实验结果见表2。
结果显示:针对临床分离的123株副溶血弧菌VP001~VP123,PG07能够裂解其中的95株,裂解率高达77.2%,其中62株带有毒力基因的副溶血弧菌能被裂解50株,裂解率为80.6%,说明该噬菌体宿主谱较宽,能够在南美白对虾养殖中起到杀菌的作用,并能够净化水体中的副溶血弧菌。
表2噬菌体PG07裂解谱
(三)噬菌体对温度和pH稳定性检测
将2.18×1010PFU/ml的噬菌体PG07增殖液分别于40℃、50℃、60℃、70℃、 80℃,水浴中作用20min、40min、60min,每个温度设两个平行组。双层平板法测定噬菌体的效价。
具体结果参见图3,噬菌体PG07在40℃和50℃保存3h后基本保持原活性;60℃作用20min后仍保持在1.26×109PFU/ml以上;70℃与80℃的高温作用下,20min噬菌体基本失活。因此,噬菌体PG07的热稳定性较好。
取无菌试管中加入不同pH值(2、3、4、5、6、7、8、9、10、11、12、 13)的2216E肉汤4.5ml,各三支,置于37℃的水浴锅中,待温度稳定之后,各加入500μl 2.18×1010PFU/ml的噬菌体增殖液,混匀37℃水浴作用1h、2h、 3h。作用结束之后,向混合液中加入适量的HCl或者NaOH使混合液的pH 值约为7,双层平板法测定噬菌体的效价。
具体结果参见图4,结果可见:在pH4~9范围内噬菌体PG07效价几乎没变或略有降低,仍然在109PFU/ml以上;当pH为3,处理3h时,噬菌体PG07 的效价下降不到1个数量级;当pH为10、11时,处理3h后,噬菌体PG07 的效价下降1个数量级,可见该噬菌体对pH的适应范围较广。
(四)噬菌体的最佳感染复数(MOI)测定
按照常规方法增殖副溶血弧菌噬菌体PG07和宿主菌副溶血弧菌 ATCC17802,测定噬菌体及宿主菌效价,并将噬菌体PG07和宿主菌进行适当的稀释。按照感染复数为100、10、1、0.1、0.01、0.001、0.0001、0.00001的比例各取100μl PG07和宿主菌加入到NB肉汤中。于37℃,170rpm振荡培养至液体变澄清,记录液体变澄清时间。10000rpm离心5min,双层平板法测定噬菌体的效价,结果如表3。
表3噬菌体的最佳感染复数(MOI)测定结果
结果可见噬菌体的最佳感染复数是0.1,该条件下噬菌体感染宿主菌产生子代噬菌体的滴度为3.92×1011PFU/ml,在9个感染复数中噬菌体滴度最高。
实施例四噬菌体体外裂解试验和水体消毒试验
(一)PG07对副溶血弧菌菌株体外裂解试验(OD值法)
按照一定的比例加入副溶血弧菌菌株ATCC17802和噬菌体PG07,副溶血弧菌菌株的终浓度为5.00×105CFU/ml,噬菌体的终浓度分别为 5.00×107PFU/ml,5.00×106PFU/ml,5.00×105PFU/ml,5.00×104PFU/ml。对照组加入与噬菌体溶液相同量的无菌2216E培养基,菌液和噬菌体混合后在 37℃,170rpm的摇床内振荡培养。每隔一定的时间测定OD值直至混合液变清亮,涂布平板法测定作用一定时间后各组菌的残留量。
结果可见PG07对副溶血弧菌菌株的裂解效果很好,4个不同浓度的噬菌体对副溶血弧菌菌株的裂解效率都可以达到99.90%以上,只是时间长短不同,但是4h以内都能够达到较好的杀灭效果,具体数据见表4和表5。噬菌体PG07 对ATCC17802杀灭效果的时效结果见图5。
表4各时间段OD值变化
表5 OD值测定结束后测定菌残留量
(二)PG07水体消毒试验
3L无菌海水,每桶放入5尾南美白对虾,28℃水浴,小气泵通氧。向体系中加入一定体积的副溶血弧菌ATCC17802,调整终浓度至1×106CFU/ml, 1h后分别向海水中加入噬菌体PG07,使其终感染复数为1:1和0.1:1,每组设置平行,攻毒组加入等体积2216E培养基,详细分组情况见表6。于1、7、 10、25h后用涂布平板法检测水体中副溶血弧菌的数量,具体残留副溶血弧菌数据见表7。水体消毒试验效果图见图6。
表6水体消毒实验分组
表7水体消毒试验副溶血弧菌残留量CFU/ml
噬菌体在养殖水体中能够显著降低副溶血弧菌的活菌数量,高达3个数量级。所以噬菌体可以作为水体消毒剂(改良剂)杀灭副溶血弧菌。
实施例五噬菌体在南美白对虾肠道和肝胰腺中的代谢动力学及安全性实验
体重5g左右健康的南美白对虾,空腹过夜。按照体积与质量比为5%的添加量,将噬菌体与对虾饲料均匀混合,噬菌体拌料后效价为1.36×108PFU/g,将饲料阴干,再以对虾体重3%的剂量饲喂,喂食后0.5、6、24、48、72、96、 120、144h检测虾肠、肝胰腺以及空白对照组正常对虾体内噬菌体的含量。结果见图7-8。
实验结果说明:饲喂浸泡过噬菌体的虾料0.5h后,虾体肠道、肝胰腺内即检测到噬菌体的存在,效价可达5.15×106PFU/g,说明噬菌体可以通过喂食进入虾体内,且噬菌体可持续存在3天以上,这对噬菌体作为饲料添加剂具有指导作用,且可用于预防和治疗因感染副溶血弧菌引起的急性肝胰腺坏死综合征。实验结果还表明,噬菌体对南美白对虾的健康没有影响,不影响对虾正常活动,说明噬菌体安全性很好。
实施例六噬菌体预防副溶血弧菌感染南美白对虾效果测定
(一)噬菌体拌料饲喂南美白对虾预防副溶血弧菌效果测定
体重5g左右健康的南美白对虾,空腹过夜。每组50只,试验组设置双平行,且按照体积与质量比为5%的添加量,将噬菌体与对虾饲料均匀混合,阴干后以对虾体重3%的剂量饲喂。对照组按照同样剂量用2216E培养基浸泡饲料后饲喂。1h后给所有对虾注射副溶血弧菌,剂量为1.31×106CFU/只。记录各组48h内死亡对虾数目,并计算噬菌体的保护率。具体数据见表8。
表8噬菌体拌料对急性肝胰腺坏死综合征的预防效果
噬菌体拌料能够很好的预防南美白对虾因感染副溶血弧菌而导致的急性肝胰腺坏死综合征,噬菌体可作为饲料添加剂添加入南美白对虾的饲料中,能有效预防对虾感染副溶血弧菌而导致的早期死亡综合征(偷死病),降低死亡率,提高虾苗成活率,降低养殖风险。
(二)浸浴噬菌体预防副溶血弧菌感染南美白对虾效果测定
体重5g左右健康的南美白对虾,正常饲喂。每组50只,试验组设置双平行,浸浴噬菌体,效价为1×108PFU/ml。对照组加入等体积2216E培养基,2h后给所有对虾注射副溶血弧菌,剂量为1.31×106CFU/只。记录各组48h内死亡对虾数目,并计算噬菌体的保护率。具体数据见表9。
表9浸浴噬菌体对急性肝胰腺坏死综合征的预防效果
结果显示,浸浴噬菌体能有效预防南美白对虾感染副溶血弧菌引起的偷死病,可与拌料同时应用,以降低对虾的死亡率,降低养殖成本,提高养殖成活率。
实施例七噬菌体治疗南美白对虾感染副溶血弧菌效果测定
体重5g左右健康的南美白对虾,空腹过夜。每组50只,试验组设置双平行,给所有对虾注射副溶血弧菌,剂量为1.31×106CFU/只。攻毒1h后,按照体积与质量比为5%的添加量,将噬菌体与对虾饲料均匀混合,阴干后以对虾体重3%的剂量饲喂。对照组按照同样剂量用2216E培养基浸泡饲料后饲喂。记录各组48h内死亡对虾数目,并计算噬菌体的保护率。具体数据见表10。
表10噬菌体拌料对急性肝胰腺坏死综合征的治疗效果
给南美白对虾注射致病性副溶血弧菌1h后,饲喂浸润了噬菌体的饲料,可有效控制对虾的死亡率,与对照相比,显著提高对虾存活率。为噬菌体制剂制备抗副溶血弧菌的药物,从而治疗由副溶血弧菌感染引起的急性肝胰腺坏死综合征提供新的理论依据和实践经验。
可以理解的是,对本领域普通技术人员来说,可以根据本发明的技术方案及本发明构思加以等同替换或改变,而所有这些改变或替换都应属于本发明所附的权利要求的保护范围。
序列表
<110> 青岛诺安百特生物技术有限公司
<120> 具有宽裂解谱的新副溶血弧菌噬菌体、其特异性引物及其应用
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 447
<212> DNA
<213> 副溶血弧菌噬菌体PG07的尾部蛋白(tail protein of Vibrio parahaemolyticusPG07)
<400> 1
atgagcgttt tagaccaaaa aacatacgat attatactag aggaaggagc agacttcggg 60
ctgaggcttc gtctaaaaga aacagagttg cagtgtactc aacatcctgt tacaggcaat 120
gatgagaact gcgtggaggt agaaactcct attaatttaa catatcaaga gttcactgga 180
agtatagcag actctcttga ggattccgct actgatatcg gtagcttcac tttccagaaa 240
accgacgcag ttaatggggt tgtggatatg agccttggta gggcggaagt aactgcgatg 300
cttccttatg cgcaaccaac tcgagataaa tacagcccaa gaatcaggtt cttaggctat 360
tacgacgtct tggtaagaga tacccaatca gacgtggtga ctagaataat gcaaggtaaa 420
gtatacttta gtgatggggt agtataa 447
<210> 2
<211> 2874
<212> DNA
<213> 副溶血弧菌噬菌体PG07的另一个尾部蛋白(tail protein of Vibrioparahaemolyticus PG07)
<400> 2
atgccgagta agattactca ggcactaaaa gactacttaa acaataatga taaggtacag 60
ctagcgcacc tagttagaat agaactaccg ggagaactag ctagctttgc ctactacaca 120
gattattcta gagaaatcca gtacgacgga caggagtttg tccctggtaa ggtcaagaat 180
atcagtgacg ttaagcagtc taataggctt agtgcccata acgtgacaat taagataact 240
ggtgcatcct ccgaggaggt agaccgtgca gtacgctcac agcagtacct gaacaagaag 300
atttctatat ggagagtagt actagataac acaactggtg aggtagttcc ctactacgca 360
gatggtagta cactactatt cttcgagggt accataactg aggtgtctat cgacgaaaac 420
cgctcagcta cttctagggg cgaatccacc atctcttgga agtgtgctaa tgagttctat 480
gacttagagc gagtaaatgg taggttaacg gacgatgagt ctcaccgagg tcttatcgta 540
gaccaagacg gagaagaggt tccctccaac gcggctaaga gatttgagta tcagacggac 600
ctaggcttct tccacgccaa caagtccgta aacatactag ccaaatacca aggtctagag 660
caagcttaca agctgaagaa aaagtcttca ggcctttttg gtattaagaa atcttacgac 720
cttgtagagt actgggaaac tgtggaaaaa gaggttgata tgcggttcaa ccttgcagca 780
aagaatatcc ctactattta tggggttcaa aagacccaag gtatcccagt attcgcagac 840
acactgctag acgacccttc aactgtatac gtagtatacg ctttctgtga aggagagata 900
gatggattct tagacatatg gatggaagat aaacctttag tatgtaatga tactaacgat 960
gctgatgagc gagcttgttt tggtgtaaaa agaggtaacg gggacactat ctctgttgct 1020
acacctactc aagaccctac agcgccttct gtacatgggc aagaatacat cgttcaggat 1080
ggtttagggc aagtaagctt ctggacattt catggtaaaa gagaccagac agcatgctct 1140
aagctagtag acatagcagc taacggaaac ttctatctac agaacaacgg gccaacccct 1200
atgggaccag agtactggga ctctaggtat aagctactag acactgcgta tgtggtagcc 1260
gagtttaaaa taaccgacca aagaacagaa atacctacta tctatgcaga gatacaggga 1320
agaaaagtag ctgtctatga tgagaatggg ctagtaaggg atgatagaac aagcaccaac 1380
ccagcatggc agatgctaga ctacttaaat agccctatct tcggggccgc tgtaggtatg 1440
gaccgaatag acttgaatac ttttgtagag gtagcaaacc tactaggtac agtagacaac 1500
acctatgagt tatcgtgggt accattctgg agatacatcg gttgggagga caataccact 1560
gaggcgaata aggctatttt gcaaaccaac ccactgttga atggagagac ttctctcttt 1620
aaaaacatga agagtatgtt agagcaagcc gagtcctctt tgaatatcat tgaaggtaag 1680
tacgcactta ctgtagagtc cctaaaagac cctgtggcgg acctaaccga ggcagattta 1740
gttggtggac gacttcaagt ctcagatatt acgtccaagg aaaagtacaa cacagtgcag 1800
gcagatatta gagaccctgg catagggtgg gaatccaacg acattatttt ctttaatgca 1860
gacttcaaag cggaagacag tcaattagag aaaaaagcca atatctcctt tccacatata 1920
actaactact actgtgctag ggctttagcg gaacgagttc tcaagaagtc tagatacaac 1980
cgagaggttt ctattacagt gccttacaag tatgtagatt tacctattaa ctccccaata 2040
actttaacgt atgagcgttt tggttgggac aagaagcagt tcctaatcag ggagaccgta 2100
tggcaatcta ctggtaaggt taagctgaaa cttcgagaat acgaggatgg ggtatttatt 2160
aactcccctc aggcggatat aggagactct caaattccag agataggtac taaggtactt 2220
cctcctaggg atttgaacta taggccttct atacctgggg accctgaagg agtgaatgga 2280
tatttaagat ggcttcctag tttctctcct gacgtaacgt actacgcaat caactatact 2340
ggagtagctt cgactatcac agtcaacgcc tcctccacgg acgagcctag tacgtacata 2400
gagcttcctg tagacactat tactgaggat aggatttata ctttcgaagt ccgagcggta 2460
gcaggcaata aaggattatc ttcctctccg gctatactct ccttgcaact agggcccgac 2520
ggtacaaaaa accttactat ggtgacggga tttgacctag tgaacaatat tacaggggat 2580
gagacaatat ggaaaggtgc ggaggttagc ctacaatgga atcctatatt cgaagaagac 2640
ttcatggata atatatatta taatttggaa ttttgggacg gcgatttcga cacgggtgac 2700
ctaattcgtt cgctagagat acgtaatgct tatgcttaca tatacgacct ccctgctaat 2760
aagtcagact acttagctgc taaaggcact aaaggtatgt ttaggcagtt aggggttagg 2820
atacgagcag aatcagatga cggagctaaa tccgtgggat ggacaaaaat atga 2874
<210> 3
<211> 1140
<212> DNA
<213> 副溶血弧菌噬菌体PG07的主体尾部蛋白(major tail protein of Vibrioparahaemolyticus PG07)
<400> 3
atggcagtta aattactccg taatacccgt ctgtgggtaa gtacggttct atcaggtcac 60
gacacaacca atacttggga aatccaagtg caggacgacc tatcctttaa ccagaaccct 120
acgagttctg atattgaatt agaagaagca ggtgcaacac ctacacgtgg ttccgcccgc 180
ttcaatgacg cactagagcc agcggattgg acattctcca cttacattcg ttcgtattta 240
gtagacccag atggcactcc agatagtggt gacgagtatc agtttactcc tgatgcactt 300
ctatggcact ccctagcttc tggctctcca ttcgacgtta ctagtgcagc aggtgttcat 360
tctaaccctg ttaacatgct tctagacttt aaagattctc aacaccacga actattgaag 420
ttccatgttt acatgaatgt agatggtgca tggtacgtta ttgagaactg tcaagtaggt 480
gaagcaacaa tctcaaacga tattgatggt attggttcta ctacatggac tggacaaggt 540
actttactat cagagctacc ttcgcaacca tttgacccag caaacgttct ttctgtagac 600
tgtaacttac aagcttctta catccgtaac aagctgacag tcctacgagt tactgataac 660
agaaattcta acacagcgta tgacatcgct atcactggtg gttctattac atttaccaac 720
aacatcacgt tcctaactcc tagtaccctt agctgtttag acgttcctat cggttcgtac 780
accgggtctc tatctattac aggtgagatg acagcgtacc tagacgacaa gactaacggg 840
tctaagaagt tactagcgga catgttgcaa caaaaatcag taacatctag ttttgagatt 900
gcggtgatta tgggtggtgt ggctagctcc ggtacggctc cagcagctgt tatcgttcta 960
ccgacagcgc acctagacgt acctagtatc gaatccgctg acgttatcag taccagccta 1020
tcctttaagg gtattcctac tgacttctca gcaggggacg aggccttctt aggcttctct 1080
gaccgattca ctagtgcaga aattgaccgt ctaatcaaca ctggagacgg tgaagcttaa 1140
<210> 4
<211> 951
<212> DNA
<213> 副溶血弧菌噬菌体PG07的NAD依赖的DNA连接酶亚基A(DNA ligase subunit A ofVibrio parahaemolyticus PG07)
<400> 4
atgcaagagt atatccgtta cctacaaaag tgctactacg aaggcaaccc agtaatctct 60
aatgaggagt acgatgcact agtctctcgc tttggagagg atggattggg tgtgggcggc 120
gaattcaagc atatgtatcg tatgtactca ctagacaagg tgtatcctgg tcgaggtgag 180
gaattaccac tacctgtaga aaaatgcata gaaactccga aattggacgg atgtgcaatt 240
tctttgtttt acattggtaa taagctagtt caaggtaaca ctcgtggcga cggaataaca 300
tccccaacca cttttgagcc gtggaagctg gcgttgcttg taccaggtga actatctagt 360
agctctccta tccaaattac tggtgaggta gttactacca atcctgtaaa aaacatgcgt 420
aactacgcgt ctggagctat gcagctaaaa gacgaaaaag agtttcgaga gcgtgtttta 480
gaaggcgggc ttatcttcgt agcttatggg attcaggaag cggaagacca cgcaggtatt 540
ttccctcact atgagcagga tatggcctgg ctatctggtg aaggtttctg tacggtagtt 600
gactcagact gggaagactg cccgcaagac gggcgagtgt tccgcctctc tagcaacaga 660
gatttcttcg atgctggatt tacccacaaa catcctaaag gtgcattcgc cgtaaaagaa 720
gacgaagaag gtatctggac taagttagta gacctaacat ggcagactgg taagtctggt 780
aaagtgactc cagtagcaat ctttgagcca atctttatcg acgatgcaga aatcacccga 840
gcaacattaa acaatgctgg ttatgtggaa aacctaggag tctacgaagg ctgcgaggtt 900
tgtgttatcc gctcaggcgg cattatccct aagattatag acgtaaggta a 951
<210> 5
<211> 2601
<212> DNA
<213> 副溶血弧菌噬菌体PG07的NAD依赖的DNA聚合酶I(DNA polymerase I of Vibrioparahaemolyticus PG07)
<400> 5
atgtcaaaaa tagcactaat tgtacctaaa cctactaaaa ccaacttttc taagttgttc 60
ggtagggaag tggatgtctt tccacttacc actcaatcag taaaacgagt tttggtgaag 120
cacgttgata tagatattga cgtgtatgag tatgactggg ttatcctagt tggttctgaa 180
ccattaaagt atttttctaa ggaaacctct gtagtagata tgactggtat gcgtgtagac 240
gctaaaccta agaaagactg gtcagactac gagaacttcc tttgttctat gaaccctgcg 300
gcgcttttct ttaagccgga gggcaagcct gtgttcgacc aaacggttga gcgtctaaat 360
tctcacatcg acggaaccta cgtagagcca gagaaagtaa tctacaatat cttctgcaac 420
cacactactc aagaggagtt ggacgagcag gaagataact tagttattgg ctccatgact 480
gagttcaaga tgtacttgcg ttccgtacta gggatgcgtc ctaacgtagt tgctctcgac 540
tcggaaacag gaggctttta cgccagaaat gctgagatgc taggaatatc aatgtctcac 600
tccgaaggcc aaggggtcta cgtaaacgca gactacttcg acgaagaagt atttaacctc 660
atccagaagg tagtggattg tgaagacatt catatagtat tccataatac caagttcgac 720
tggcactggt ttactcatca catgggtata agctttgaga agtgtttcaa agagaaacgc 780
ctacacgatt ccatgattca gcactatgtt ctggacgaac gtaaaggaca cggcttaaaa 840
cctctagcaa tcaagtacac cgatatgggt aactatgata aaaagctaga cgaattcaag 900
tctgagtact gtaaggtgca caagattaag aaagagcagt tcacctatga cctgattcct 960
ttcccaatcc tcgcagctta cgctgcgaaa gatacggacg ctaccatacg tctacacaac 1020
aagttctacc caatcgtaat gaaaaaccct cgtctgaagg aactttacga gaacttaatg 1080
attccaggta tgcgattcct taagcgtatg gaagaccgag gagttccggt ctctaagaag 1140
agattacgtg cagctcaaaa attactgcat gaagcaatct tcgaggctga agaagagctt 1200
tactcacacc cagcagttat cgagttcgaa aagcaggaag gtaagaaatt taatcctcaa 1260
tcccctatac agctacgtgt tttgctgttt gatatagcag ggtaccagcc tacagggatt 1320
atgacagaca caggacagga ctccactaac gcagaagttc taaccaaact gtcagagttc 1380
ggggacttac ctaagttact actaaaagtg cgtaaaaaca ccaaactact atcttctttc 1440
attaacaaaa tgattgataa cgtggattgg gatggtcgtg tacgtacagg ttttggtatc 1500
actacaacta cctcaggccg tctatcgtcc tctggtacaa ttaacttgca acaactgcct 1560
cgtgataacc caatagtcaa gggttgtatt gtagctcctc caggctacaa gattgtggca 1620
aaagacttga caactgccga ggtttattac gcggccgtac tgtctggaga caaagcccta 1680
caacaagtgt tcgttaacat gactaacgag cctgataagt acgcggactt ccactccact 1740
atcgcacaca tggtgttcaa cctaccttgt gagcctaacg aggttaagaa gctatatcca 1800
gcgatgcgtc aagcatctaa ggctatcacc ttcggtatct tgtacggttc tggccctgcg 1860
aaagtaatgg agtcggtaaa cgaagctcta ctagagcaac acatcgaaat gggtactccg 1920
tacaacccat gtactctagg agatgcaaaa gggtacatcg aaacttactt cggtcgattc 1980
cctcaactta agaagtggat tgaagaatct cacgaccaaa tacgtcagta cgggtttatc 2040
tacagcttct acggacgtaa gcgtagactg cacaacatca agtctgatga ccgtggagtt 2100
gtgggtggcg aaatccgttc aggattcaac gctatcattc agtcagcgtc ttctgattcc 2160
ctgttgatgg gttgtataga tgcggataat gagattgctg agcgtggaat ggacgcagaa 2220
attgtggcgc tagtacacga ctcgattgtc gcagttgttc gtgaagactt agtggacgag 2280
tactctgagc ttctagatag aaatatccag aaagctagat tcaatagtct tggagagttc 2340
ttaggtattg agggtacccc tatcggtatc gaaagcgact ctgagaaagg aggctctgta 2400
gactatgggt gtggcaagct caagaagatg tggccagatt tggctgtgct agaagacgag 2460
aagttcgcta cggaagtaac gagctggtta tctaagtacg aaaccgaccc tatcccagag 2520
gatgacttgc agcctcacga aaaggtctgg gttgagagag ccgaaaggat aaagacagtg 2580
gtagaggagg agtatgctta a 2601
<210> 6
<211> 327
<212> DNA
<213> 副溶血弧菌噬菌体PG07的裂解酶(lysozyme of Vibrio parahaemolyticus PG07)
<400> 6
atgggcgaat tagtttggat tgcactaacc actttacaac cttacccagt gttcgttccc 60
ctagaggaac acatcccact gagagctatc aaactctctg aagacaagat gcaagtaagc 120
ctagagcagg tgactagaat ctatcctgct gttgcggaca tcttcactaa tgaaaacttt 180
gttatcttct ctggagcgga gttcactgaa ctaccagtag cacacgatgg agaaatagta 240
gagagcttag agcggttagc tgaagcttcc cgaaaagggg cagaagctat ccaatccctg 300
tcagcggcta ttgcggaact tcgttag 327
<210> 7
<211> 22
<212> DNA
<213> 人工合成序列 PG07-F(Synthetic sequence PG07-F)
<400> 7
tgagtctcac cgaggtctta tc 22
<210> 8
<211> 24
<212> DNA
<213> 人工合成序列 PG07-R(Synthetic sequence PG07-R)
<400> 8
ctgtagggtc ttgagtaggt gtag 24
<210> 9
<211> 527
<212> DNA
<213> 扩增产物(DNA PCR Product)
<400> 9
tgagtctcac cgaggtctta tcgtagacca agacggagaa gaggttccct ccaacgcggc 60
taagagattt gagtatcaga cggacctagg cttcttccac gccaacaagt ccgtaaacat 120
actagccaaa taccaaggtc tagagcaagc ttacaagctg aagaaaaagt cttcaggcct 180
ttttggtatt aagaaatctt acgaccttgt agagtactgg gaaactgtgg aaaaagaggt 240
tgatatgcgg ttcaaccttg cagcaaagaa tatccctact atttatgggg ttcaaaagac 300
ccaaggtatc ccagtattcg cagacacact gctagacgac ccttcaactg tatacgtagt 360
atacgctttc tgtgaaggag agatagatgg attcttagac atatggatgg aagataaacc 420
tttagtatgt aatgatacta acgatgctga tgagcgagct tgttttggtg taaaaagagg 480
taacggggac actatctctg ttgctacacc tactcaagac cctacag 527
Claims (8)
1.具有宽裂解谱的新副溶血弧菌噬菌体,其特征在于,拉丁名为Vibrio parahaemolyticus,被命名为PG07,保藏编号为CGMCC NO.16392,于2018年9月26日被保藏于中国微生物菌种保藏管理委员会普通微生物学中心。
2.如权利要求1所述的新副溶血弧菌噬菌体在制备疾病药物的用途,其特征在于,所述疾病为对虾急性肝胰腺坏死病。
3.如权利要求1所述的新副溶血弧菌噬菌体在制备防治在水产品养殖、运输及保存过程中的细菌污染的制剂中的用途。
4.一种用于防治副溶血弧菌感染的杀菌组合物,其特征在于,包括有效量为1×1011PFU/ml的如权利要求1所述的副溶血弧菌噬菌体。
5.一种水体消毒剂,其特征在于,有效成分包括如权利要求1所述的副溶血弧菌噬菌体。
6.如权利要求5所述的水体消毒剂,其中还包含其他用于环境中病毒、细菌抑制或消灭的活性成分。
7.一种对虾饲料添加剂,其特征在于,包括如权利要求1所述的副溶血弧菌噬菌体。
8.一种检测试剂盒,其特征在于,包括如权利要求1所述的新副溶血弧菌噬菌体。
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