CN114015662B - 具有强裂解作用的产气荚膜梭菌噬菌体及其应用 - Google Patents
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Abstract
本发明属于微生物技术领域，涉及一株具有强裂解作用的产气荚膜梭菌噬菌体及应用。具有强裂解作用的产气荚膜梭菌噬菌体，该噬菌体为噬菌体RKP‑CP‑21028，于2021年10月22日保藏在中国微生物菌种保藏委员会普通微生物中心，保藏编号为CGMCC No.23094。本发明发现并分离得到的噬菌体RKP‑CP‑21028，对养殖环境中的产气荚膜梭菌具有强裂解效用，为工业化生产噬菌体用于养殖环境中禽产气荚膜梭菌的防治提供了良好的噬菌体来源。
Description
技术领域
本发明属于微生物技术领域，涉及一株具有强裂解作用的产气荚膜梭菌噬菌体及应用。
背景技术
产气荚膜梭菌(Clostridium perfringens,Cp)也称魏氏梭菌(Clostridiumwelchii)，革兰氏阳性厌氧菌，广泛分布于土壤、饲料、污水、动物消化道和粪便中。产气荚膜梭菌是一种能够引起人类食物中毒和动物坏死性肠炎的食源性病原，在动物产品生产过程中引发疾病并通过加工环节传播，威胁人类健康，同时给养殖行业带来巨大经济损失。产气荚膜梭菌是引起动物坏死性肠炎(necrotic enteritis,NE)的主要病原，造成较大经济损失且不利于动物福利，NE广泛分布于世界各地，呈全球流行态势。
产气荚膜梭菌为条件性致病菌，导致的疾病多突然发生而且危害严重。因此，做好预防工作是防止该病发生的首要选择。近年来，治疗产气荚膜梭菌相关疾病所采用的方法主要有疫苗预防、抗生素治疗、噬菌体的预防和治疗。其中采用疫苗和抗生素防治产气荚膜梭菌病存在较多的弊端，例如疫苗可能导致一系列不良反应，抗生素预防和治疗导致多重耐药菌的产生和致动物道菌群紊乱以及环境污染等一系列问题。随着耐药问题的日益严峻，利用生物防控技术进行产气荚膜梭菌防治将在养殖业中发挥巨大作用。
噬菌体来源于环境，作为一类细菌依赖性病毒专门侵噬细菌，面对抗生素残留及细菌耐药现象的频发，噬菌体又成为了新型抗菌制剂的候选。由于其具有自我高效复制、特异针对病原、不影响正常菌群且不产生抗性等特点，目前在国外已有较多成功应用的报道，尤其在治疗多重耐药菌方面有着极大的优势。因此针对鸡坏死性肠炎主要病原产气荚膜梭菌因芽孢传播而难以清除等特点，利用噬菌体的特异性裂解优势，抑制并清除产气荚膜梭菌，不仅可以预防亦可用于NE的治疗。噬菌体在裂解细菌中发挥的作用方式更为直接。如Miller等的研究表明，噬菌体产品INF-401能够有效控制由产气荚膜梭菌引起的鸡NE，并且混合噬菌体往往优于单一噬菌体。用不同方式(灌胃、饮水和饲料)以2.5×109PFU连续饲喂5天，可显著降低NE鸡死亡率，同时还能提高肉鸡体重和饲料转化效率。有研究发现将产气荚膜梭菌噬菌体P4和P3组成“鸡尾酒”，并与细菌素结合应用，不仅能够抑制产气荚膜梭菌生长，当噬菌体加入后能够协同将产气荚膜梭菌完全清除。
发明内容
本发明的目的是针对现有技术中采用抗生素、疫苗等防治产气荚膜梭菌相关疾病存在的耐药性、药物残留、不良反应等缺陷，提供一种产气荚膜梭菌噬菌体，该噬菌体具有很强的裂解细菌的作用，并且采用噬菌体进行产气荚膜梭菌相关疾病的防治，具有安全无毒、不产生抗性等特点。
为实现上述目的，本发明采用的技术方案是：具有强裂解作用的产气荚膜梭菌噬菌体，该噬菌体为噬菌体RKP-CP-21028，于2021年10月22日保藏在中国微生物菌种保藏委员会普通微生物中心，保藏编号为CGMCC No.23094。
进一步地，所述噬菌体的基因组序列如SEQ ID No.1所示。
进一步地，本发明还提供所述噬菌体的应用，所述噬菌体可应用于制备预防和/或治疗产气荚膜梭相关疾病的抑菌制剂。
进一步地，本发明还提供一种预防和/或治疗产气荚膜梭相关疾病的抑菌制剂，包括所述的产气荚膜梭菌噬菌体或其培养物。
本发明的有益效果体现在：
(1)本发明发现并分离得到的噬菌体RKP-CP-21028，对养殖环境中的产气荚膜梭菌具有强裂解效用，为工业化生产噬菌体用于养殖环境中禽产气荚膜梭菌的防治提供了良好的噬菌体来源；
(2)本发明得到的噬菌体对产气荚膜梭菌的裂解谱较宽；对不同毒素型的产气荚膜梭菌均具有很强的裂解能力，此特性显示这株噬菌体具有很重要的生产应用价值和科学研究价值；
(3)噬菌体进行传代30次的条件下，仍然可以保持较高水平的效价，表明这株噬菌体具有较强的遗传稳定性，而高稳定性对于生产运输具有非常重要的意义；
(4)本发明所得噬菌体可以用于产气荚膜梭菌引起的疾病的防治和治疗，并且具有安全性极高的特性。
附图说明
图1为本发明实施例中产气荚膜梭菌在血琼脂基础平板上的培养特性；
图2产气荚膜梭菌在TSC板上的形态特征；
图3噬菌体RKP-CP-21028对宿主菌的噬菌斑；
图4噬菌体RKP-CP-21028的电镜观察照片；
图5噬菌体RKP-CP-21028对宿主的裂解试验效果；
图6为噬菌体RKP-CP-21028的遗传稳定性实验结果。
具体实施方式
为了便于理解本发明，下面结合附图和具体实施例，对本发明进行更详细的说明。附图中给出了本发明的较佳的实施例。但是，本发明可以以许多不同的形式来实现，并不限于本说明书所描述的实施例。相反地，提供这些实施例的目的是使对本发明公开内容的理解更加透彻全面。
除有特殊说明，本发明实施例中涉及到的实验方法、仪器、试剂等，均为实验室常规技术手段。
实施例1致病性产气荚膜梭菌的分离及其鉴定
从发病养殖场采样，取粪水于流体硫乙醇酸盐培养基(FTG)中37℃厌氧培养过夜，以三线分离法接种到血琼脂基础平板(5％绵羊血+1％葡萄糖)上，37℃厌氧培养24h，挑取草绿色、周围有双层溶血环的菌落(如图1所示)接种于胰月示-亚硫酸盐-环丝氨酸琼脂培养基(TSC)上，放置厌氧环境37℃厌氧培养18-24h，得到黑色菌落。如图2所示。
挑取典型的黑色菌落继续划线纯化3-5次，然后挑取单菌落接种于5mL流体硫乙醇酸盐培养基(FTG)中，37℃、厌氧箱培养4-6h至指数期，然后经16S rRNA基因测序技术和血清型鉴定，血清型为C型毒素型，命名为产气荚膜梭菌CP-21028；鉴定完成后菌液中加60％的甘油保存于-80℃。
实施例2噬菌体的分离及纯化、鉴定
1、噬菌体的分离及纯化
(1)粪便处理：从养殖场采取粪便，称取5g鸡粪便加入10mL SM缓冲液中浸泡过夜，然后将过夜后的浸出液10000rpm离心5min，取上清液用0.22μm滤器过滤，将滤出液4℃保存备用；
(2)噬菌体富集：取0.2mL产气荚膜梭菌菌悬液和1mL滤出液加入到5mL流体硫乙醇酸盐培养基(FTG)肉汤中，37℃、厌氧箱培养过夜，然后10000rpm离心10min，将上清液用0.22μm滤器过滤，滤出液备用；
(3)噬菌体分离：采用双平板法进行噬菌体的分离，取宿主产气荚膜梭菌菌悬液0.1mL与0.7％的TY软琼脂混合铺在TY固体平板上，待软琼脂凝固后将滤出液0.1mL点到平板上，静放至混合液吸收，放于37℃厌氧箱培养6-8h后，用接种环挑取透明的噬菌斑置于2mL SM缓冲液中，4℃保存过夜，让噬菌体完全释放，即得噬菌体。
(4)噬菌体纯化：采用双平板法进行噬菌体的分离，取混合菌悬液滤出液0.1mL和宿主产气荚膜梭菌菌悬液0.2mL混合均匀，加入冷却至50℃左右的软琼脂混合均匀，然后铺双平板，放于37℃厌氧培养箱培养6-8h后，挑取边缘光滑的单个透明斑于1mLSM缓冲液中，4℃过夜保存。取0.1mL过夜保存的浸出液和0.1mL菌悬液混合，加入冷却至50℃左右的软琼脂混合均匀，铺双平板，37℃厌氧培养箱培养4-6h，照此步骤纯化至噬菌斑为大小均一、边缘光滑的噬菌斑，即得纯化好的噬菌体，如图3所示。
(5)噬菌体的制备：取0.2mL产气荚膜梭菌菌悬液和1mL纯化好的噬菌体悬液加入到50mL流体硫乙醇酸盐培养基(FTG)肉汤中，37℃、厌氧箱培养过夜，然后10000rpm离心10min，将上清液用0.22μm滤器过滤，噬菌体滤出液备用；
(6)噬菌体的保存方式：将噬菌体菌悬液与60％的甘油+FTG按照1：1的比例混合后，于-80℃中保存，命名为RKP-CP-21018。
2、噬菌体的形态观察
取20μL含噬菌体粗制颗粒的液体滴于铜网上，自然沉淀15min，并用滤纸从侧面吸去多余液体，加一滴2％的磷钨酸(PTA)于铜网上对噬菌体染色10min，然后用滤纸从侧面吸去染色液，待样品干燥后用电子显微镜观察噬菌体形态，如图4所示。
噬菌体RKP-CP-21028有一个多面体立体对称的头部，包裹着核酸，直径约为28nm，有一个长约65nm的尾，颈部连着头部和尾部。根据国际病毒分类学组织病毒分类第九次报告，该噬菌体归类为有尾病毒目肌尾病毒科。
3、噬菌体的分子生物学鉴定
噬菌体全基因组测序及分析：采用Illumina TruSeqTMNano DNA Sample Prep Kit方法构建文库；具体步骤为：
(1)以1μg噬菌体基因组DNA起始量建库；
(2)Covaris M220超声打断DNA到300-500bp；
(3)补平3’端加A、连接index接头(TruSeqTMNano DNA Sample Prep Kit)；
(4)文库富集，PCR扩增8个cycles；
(5)2％琼脂糖胶回收目的条带(Certified Low Range Ultra Agarose)；
(6)TBS380(Picogreen)定量，按数据比例混合上机；
(7)cBot固相载体上进行桥式PCR扩增，生成clusters；
(8)Illumina Hiseq测序平台，进行2×150bp测序。
测序结果如SEQ ID No.1所示。
将噬菌体RKP-CP-21028于2021年10月22日保藏在中国微生物菌种保藏委员会普通微生物中心，保藏编号为CGMCC No.23094，保藏单位地址：北京市朝阳区北辰西路1号院3号。
实施例3噬菌体RKP-CP-21028对宿主产气荚膜梭菌CP-21028的裂解效果试验
将厌氧箱培养6h的宿主菌悬液以1:100的比例接种到100mL FTG流体培养基中，37℃厌氧培养2h(OD约0.15，约5.0×108cfu/mL，或者使用FTG液体培养基将菌液调整到OD为0.15)将菌悬液分装在6个无菌锥形瓶中，以最佳感染复数((MOI分别为0.1和1)向其中3个加入噬菌体悬液0.2mL(约2.5×109pfu/mL，经0.22μm膜过滤)，同时以不加噬菌体、只加同等量FTG的作为对照。分别在0h、2h、4h、6h、8h、10h后分别取0.2ml检测OD600值，比较加入噬菌体后的OD600变化。在细菌液中加入噬菌体，由于噬菌体对宿主的裂解作用，导致OD600的值产生变化，由此得到噬菌体对宿主的裂解曲线。结果如图5所示。
由图5可知，相比对照组，加入噬菌体的菌液OD600有所下降，尤其是MOI为1的体系噬菌体的对菌的裂解效果很明显。由此可见，噬菌体对宿主菌产气荚膜梭菌具有显著的裂解作用，而且不同作用浓度均能够有效抑制产气荚膜梭菌的生长。
实施例4噬菌体RKP-CP-21028的稳定性遗传实验
将噬菌体连续传代30次，每代培养基中加入等量的噬菌体和宿主菌，每代培养时间6h，然后铺双平板，将噬菌体连续传29代后，测定其效价。结果如图6所示。
由图6可知，噬菌体在传代过程中，效价整体稳定，说明噬菌体的生物遗传稳定性较好。
实施例5产气荚膜梭菌噬菌体RKP-CP-21028的裂解谱系分析
采用双层平板法点板法，测定本发明的产气荚膜梭菌噬菌体对菌株库保存的不同毒素型的产气荚膜梭菌的裂解效果，其中所用的产气荚膜梭菌A型20株、B型10株、C型40株、D型5株、E型5株。
制备不同毒素型的产气荚膜梭菌菌液，取100μL厌氧培养6h的待测菌菌液加入到0.7％的TY半固体培养基中混匀，倒入平皿中，静置凝固。取10μL噬菌体悬液滴加到软琼脂平皿上，37℃培养4-6h左右，观察有无噬菌斑出现。
由表1所知，本发明噬菌体对与宿主菌同毒素型的产气荚膜梭菌的裂解高达100％，对不同毒素型的产气荚膜梭菌的裂解率为92.1％。由此可见，噬菌体对同毒素型和不同毒素型的产气荚膜梭菌具有很好的裂解效果。
表1产气荚膜梭菌噬菌体RKP-CP-21028的裂解谱
实施例6噬菌体RKP-CP-21028的安全性检测
1、本实验以小鼠为实验对象，所用小鼠为7～8周龄雌性SPF级小鼠，重约25g左右，共30只。分为2组，各15只；实验组正常饲喂加噬菌体109pfu/0.1ml/只；对照组正常饲喂加相同体积的SM缓冲液。连续喂养7天，观察小鼠日常行为、内脏以及及消化黏膜的变化情况。
观察结果显示，以此量的噬菌体饲喂实验用小鼠，小鼠的日常行为没有变化、均正常，解剖检查小鼠的内脏和消化道无异常，且在饲喂结束两到三天后，在小鼠的粪便中检测不到噬菌体的存在。
2、噬菌体RKP-CP-21028在禽类养殖过程中的预防性抗/抑菌效果
本实验以雏鸡为实验对象，通过选20日龄雏鸡120只，分为6组，每组20只。其中第1组为对照组，进行正常的饲喂7天；第2组正常饲喂的情况下用噬菌体RKP-CP-21028灌服，1×108pfu/0.1ml/只，连续灌服7天；第3组正常饲喂的情况下用噬菌体RKP-CP-21028灌服，1×109pfu/0.1ml/只，连续灌服7天；第4组正常饲喂的情况下用噬菌体RKP-CP-21028灌服，1×1010pfu/0.1ml/只，连续灌服7天；第5组正常饲喂加恩拉霉素饮水(0.05％W/V)连续饮水7天；；第6组空白对照，进行正常的饲喂7天。
分别对前5组雏鸡在第8天以108pfu/0.1ml/只的量进行产气荚膜梭菌CP-21028感染，第10天对雏鸡进行解剖观察肠道健康情况、分析坏死性肠炎发生状况并统计结果(-为正常小肠，+为肠道轻度黏液覆盖，++为严重坏死性肠炎)，统计每组雏鸡的死亡率，结果参见表2。
表2噬菌体RKP-CP-21028在禽类养殖过程中的预防性抗/抑菌情况统计表
由表2可以看出，饲喂噬菌体组与饲喂抗生素组的肠道评分和死亡率均低于对照组，且肠道无显著的病理性损伤；仅饲喂噬菌体组的小鼠的健康情况与空白对照组比较，没有显著的差异，表明使用噬菌体RKP-CP-21028饲喂雏鸡无显著的副作用，具有安全无毒副作用的特性；而且噬菌体以1×108、1×109、1×1010pfu/0.1ml/只的剂量饲喂小鼠，均具有抗/抑制产气荚膜梭菌的效果，表明本发明提供的噬菌体RKP-CP-21028在养殖过程中具有预防性抗/抑菌的效果。
<110> 青岛润达生物科技有限公司
<120> 具有强裂解作用的产气荚膜梭菌噬菌体及其应用
<141> 2021-12-10
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19540
<212> DNA
<213> 产气荚膜梭菌噬菌体
<400> 1
accctccaca cgattcgaca tattccccag ttattaccag tagataaaac agcactatct 60
gttaacaaat tgtttacaaa tatatcttat aaaaccgttg aataacttaa cataatttgc 120
tataattaaa gtatcaatta ggagggtaat attatgaata aagaacaatt aaaattaatg 180
caaggttatg atttaaatat gaaagttgag cttacaaaaa agagattaag tgaatttgtt 240
tataaaatgg gtggagttga taatgtttac attgctttta gtggtgggaa agacagtaca 300
gttttattac acatagctag acaactatat ccaaatatta aggctgtttt ttcaaataca 360
gggcttgagt ttccagagat tgttgatttt gttaaatcaa tggataatgt tgatatagta 420
actccgaaaa aatcatttta tagagttatt aaagaggacg ggtatccaat ggtatctaaa 480
aaagtttcta atgctgttag attagcaaag agaaatataa agaatggaga ttatgatact 540
tttagagtta aacagttaac agaaggtttg gaaggctcta agtttagtat gaaaaaatgg 600
ggttatttat tagatgcacc ttttaatgta acagaaaaat gttgtgatta tttaaaaaaa 660
gaaccaatga aaacatacga aaaacacaca aataaaaaac ctattgttgc aacaatggca 720
tatgaaagtt tacagcgtga ggcatcatat ttaaaaactg gttgtaatac ttttaatgta 780
ggtaaagaaa tgtcaagacc tatgggtttt tggactgagc aagatgtatt aaaatatata 840
gctataaatg agatagaata ttgctctata tatggaaaga tagagtgtag aatggttaat 900
tctaatgttg aaattggttg ggattgggag tattatacta ctggcgaaaa aagaactggc 960
tgtatattct gtggttttgg tgttcattta gagaagggtg aaaatagata ccaaagatta 1020
caaaaaacgc atcctaaatt atatgattat tgcatgaata agttaggttt taaagatgtt 1080
tgtaattata tgaatatacc atgtgaaagt gagagtgatt taaatattga ttaaaataat 1140
atgttactgg ttactattta atttattcgt tctatgttat ttattttatg taaagaggtg 1200
atattatggg tggtattcaa gtatactgtt ataatagaag gtcaataaat atgccttgtc 1260
gttgtaattg tctatatggt aatttatgtg acttatgtac taaaaaatat tacactttac 1320
taggtaattt gagtaataaa caaattgaaa atttaaaaag tgggttgtat aaaaatgatt 1380
aaaataatat gttactggtt actatttaat ttatttgttt tatgttattt attttatgta 1440
aagaggtgat attatgggtg gtattcaagt atactgttat aatagaaggt caataaatat 1500
gccttgtcgt tgtaattgtt tatatggtga tttatgtgac atatgtatta caaaatattg 1560
tactttatta agtaatttaa ataatgaaca aattgaaaat ttaaaaatcg agttgaataa 1620
aaatgattaa aataatatgt tactggttac tatttaattt attcgttata ggttatctat 1680
attatgtaaa gaggtgataa aaaaatgatt aaaaaactaa ttcaattaat actagtcatt 1740
ataatgggaa tattagttgg tatatcattt atgtttttat ataacaatac atttttaaca 1800
agttcaattt tcattatatc aagcttatta tttgtaatgt ttttaagctg tatattatat 1860
taagaggtga ataaaatgaa attaaaagaa ttggaaaaaa taattgattt tgagattaag 1920
cataatacat taattgaaaa ttatagtgaa agatatttat ttgattgtct aaagtcgatt 1980
attaaaaaat caagaactag aaatatttta attaataata ttaaagaacg taagtatatg 2040
gttgacaaat tcaacatcgt tttaaaagta aaaatagaag agttaggaaa aagaataaca 2100
catgaagaaa tgttttttac aaacttttcc gacacttctt atatgataga tatgtatata 2160
agaaagttag atttttatat tgacttgttg gatgacatta atgataaaaa agagggtaaa 2220
taaaccctct ttttatgtta caaaattact atttcattgt catggtcaat atagattttt 2280
acaggtgtat tacctccatt tattgttgtg aaaccaccat tctccataaa ctttgcatta 2340
ccagttttta ttaatgatgt tctatctttt cttatcttaa ataatccatg agtccatgaa 2400
aagtcaacaa ccaaaccaat acttgtaccg tctgatgtta aaaacgttgt tcctattaat 2460
ttattacctt gtccgtcttc ttgtaattca tacggaacgc taggtttatt taaatataca 2520
ttggtgtatg tgctaccatc ataagatgct gattgtatca tattttcaat agaatatttt 2580
tcaactctac cagtacttct aaagtcaacc attgtaccat tatagtcata atgtttttca 2640
atagatatat ttttattttt atatactggg tttgttatat ctgtgtcagt ttctatacca 2700
taagtatttg cgttaataac tctcacttta tgcttaccgt acatattatt attaccaatt 2760
ttaatgcctt caatccaacc ttttagtagt aaaccatttg aatataattc ttgattataa 2820
ggtcgccaca tattggattc aaattttaca ccttgacata ttttattatc gggactttct 2880
aagtataata aacctagggt ttcctctgaa tagttattac taaaatctat tgacccacta 2940
gtgaaaccac cttcaccctc aaccttcata taacaaccat aaactatatc attataatca 3000
gtacctttat tatttctcaa tgggtcactc atttctccat tttgctctaa gtcattattt 3060
ttaatagtat atgctctacc tttaacatcc aagtaaatac cgtggctttc atttctatta 3120
ataaagcatt tttctattat taaagcgttt gtttcacctg tgtaagttcc tttgttttta 3180
ccaattctta taccattgtt accattttga tacatccaac attgatttat tataggggtt 3240
attaatccat attctgcatt aattccacaa tttccacacc catggatttt aactttttct 3300
atttcaatcc agttacaaaa acctccgtcc atttgaccga taccattttg agttaatgct 3360
ctcctattcc caaaaatacc tatattttca gcgtatagac cattagtaaa tatattaata 3420
tttttatctg atgttattat actaccacga cacttattta aatatttttt atattcgtct 3480
aattttgagt ttggtcttgt atattcccat tctaaaaatg gaatataggg gtcacccatt 3540
cctatgattt ttacaccctt tgtgttaata ccctctgtaa tataaaattt tccagtagga 3600
attaacaaac cacgaacatt gttttcaaca catgaattta tagcattttc aaaggcttta 3660
ttgttgttag tttcaccatc accaacgccc ccaaaatcaa caacattaaa acatttaagg 3720
ttaccccaaa ctgttactag tttatctatt tcttttttat tgtttttaat attttctata 3780
ttagagttaa ttttattatt taaatctctg aatactgttt catttattat tgttgataaa 3840
gtaccatcaa ttaaccattg gttaagttgt ttggctactt cttcttttaa tccttgtcca 3900
actaaccatt ttgttaaatc tataacttga ttttgtgagt caattaattc atttatttta 3960
ctaaagaatt gacataaaat ttcttgcatg gataaacctg tgtaatcata aacggtaaag 4020
atgctacaat taaggtcaat aggatttaat ttatttatat ttaacattgt tataccccct 4080
tgtatggtaa tgtttgtatc atataagttt taaagttttc aaattcattt gttaaattat 4140
taaacttttc ttttaagtcc tctttttcaa atttcaaatc atttattaac ttattagtat 4200
ttactggttc tttaaatgaa cttgtaccat ttatggaatt ttcaaagttt acgtaagttt 4260
tatcgttaaa tgtttttaca tttatatttt catttaaagg cgtttctatc agttcatcta 4320
aaacataata agtatttact ttatttgtaa ttacattatc taatagtaaa tccccatctc 4380
tatattctct tttaccgatt cttttaatag caacttttct tatatagtta atctcgtctg 4440
atatactatt tattcctcta agtccttcat caaaacctaa attttgcaat aaaatatctt 4500
ttttatcttg tttgtattct tcataacttg attttgatgt tccagtttca atttgtatat 4560
ctttaaacct tacagtatta ttattttcaa caacactact ggttgatata taacaagtag 4620
tgtttctagg taatactatt tttccccaaa ccttatcatc tttcttgtcc ccattcaaac 4680
ctatgtaaat aaagtattct tcttgattta atgttgtaaa ttgtttttca ccagttcctc 4740
cagcaactaa aattttttga gataaggagt tattttctgt gaaaatgaca aaataactat 4800
taattcttga attaccatct gctttaaact ctaattcacc tttttcattt attttttctg 4860
aaaattcatt tatgttaaaa aaattcttac catgacttaa aatactaatt ttattccctt 4920
ctacttctcc aaaggatttt attccctcaa aaaaactgtt taacacaaca ttattacctt 4980
ctacaactat agctttatct gcattaacta aatcattgtt aacatcccat ccatttgtat 5040
tatgcaatgt tagtctaaaa actataggat aatcttcttc tacagtaaat gtattagtag 5100
tgctacttaa aataatattt tttgttactc cttctttatt tttgacaaca ataattattt 5160
gtttattatt tctattagga atatttactg tatatgttgt cccaacttta aacatttcag 5220
atttaagtaa tttttcaatt tggacattag cagcattttt agctggagta ttggaagcta 5280
aattctgtaa agttttaccc ctaattacca tatcagttgt cataccctca acagtattaa 5340
gtatcgtatg actttctgct tcttcttgtt gtaacataga aacttctatc tttttattaa 5400
gtctttcaac ttcacaatca attctttcgt ctaaagtgtt gaacttctct ccagttgtag 5460
ctgtacgggc ttgtactact tcatcttttg tatcattatt ttttatggat tcacttagtt 5520
ttttaaagtc gctatctaat ctttctttta acttagaaaa tgtcttacca gttgtatcaa 5580
ctctactttc tttcacttca tttaatatta aatcagaaat attgaaaggt tgagggtttt 5640
ctaagttgcc attattactc catgataata ttccatcttc tgaaatgtga ggcgtataaa 5700
aatatccctt atcaccttta atatttactg ttggcggatt ttcaagttct ccattatttg 5760
accacgataa attaccttca ctatcaacgt gaggaataaa cataaagccc tgtttaccat 5820
gtaaatttac tggtaatgga ttttctaaat taccgttgtt tgtccaatac aaaattccgt 5880
caactgtaac gtgtggttca aacgtataac cagtatcacc ttttaacact tctttataag 5940
taacttttcc tgttatttta ctcaactata tcacctcaca tatactgtta ataatttccg 6000
ttctaacaac acctttttgc aatattgaca cagaataatt gaaagtacct tgctcatcag 6060
ttgggaatac taaatcagta tcattataat ttttttcttc ttttatagaa ttaaatacaa 6120
attctatttt atcaccttct tgtaatttat atccacaaac atttaaatct aaaatacaag 6180
tatcaaattg attaaccttt attttatttc tatcaattat aatcattatt tcaccaccta 6240
ataaacatac ataaataaac ttctagcacc tagtataatc tgttcatcta tgttatttaa 6300
gcaactttgc catttctctt ttaattctgc tgatgaagtt acgccaatat taccttgcga 6360
aattaattca gttttttctt tttgtttgtt gtttttatcc ccagtattat ttatatttaa 6420
attactctta ttatctgttt tacttattgt actactatta ctatcataag tgtctgtaga 6480
ttgacttgaa tttgtcaaat aaccgtcagc aatttttgaa ctagatacac catcagcaag 6540
tgaactttct tttgtattat taattgagtt attactttca ttattattta tatttgtaga 6600
agtattacca acttcatttt gtgatgttaa taaattattt acctcatttt cttctagttc 6660
cctaataaaa gtttctttta agtctttatt taacataaag tcaatgtctt tacatcttaa 6720
ttctatttca taaagttgct gataatatgg cataactctt aaaagatatg cccttagttg 6780
ttgtttaaac cttgctatag tttcaaatcc tatttcatga aagtaataat gatttataaa 6840
tttttcttca aagtaacatt ttgcatcttc ttcatagtaa ggataatcaa agtcaaataa 6900
attattctcc aatgtcctca attctagtgt gtacttcgcc attttcgcca cctccaaagc 6960
tatcaacaac cttagttact tttatattag tcttaaactt ttcatttatc attttacaag 7020
ctaataacct agttttatat tccatatcca aattagtttc aatatattca ttattggcgt 7080
taacttcatc aactaaaagt cgttctcttt tttctgtatt tgcattatct aaacccaaaa 7140
atgaaagtag ttcactctca acagcctttc tttcatcttc taatttatca actaagtaaa 7200
taacaccagt ctgtagcatc ttaaaaccgt caaaaccttg ttcccctaag tctttatcta 7260
atataacaac ctctttacca ttatcaacat cattaacaat atttttaact gataattcat 7320
tattttttgt agctgtaaca aagaatggtt tcatttgttg ctttaaattc cttttaataa 7380
cagtttctat atcatccatt ttttgagcat aatgagatat aaaatgtgat gtcggtaaac 7440
ataagtcatt cgctcttatt tttattccat ctttgccatc ttcaagtata ttctggtatt 7500
taccatttct tgaaactata cttaatttaa tattgtcacc atagatgttt aaatcaccta 7560
aaccatgaca aggtaaacac attaaaccca acttatcatc tttaaagaaa aaacattgtc 7620
cattttcgaa taaaaagtcc tcaatatgtc tactttctaa tcctgttggt aagttctccc 7680
atttaaacct attacaagcc ataagtttat aatgtaaaaa caaccttgtt tggttgtcac 7740
atattgtatt ttcattaagc aaaatattag gattttgaga tattatagat tttccttttc 7800
ccatctaatc accacctttt aaaaagggga aatttattcc ccttgttttt gattatgctt 7860
taagctgtca atatctgatt gtatctttgt taatgttatg ttgagttctg aaattgcatt 7920
tgatagtttg ctattttgat taaacataat catacacgca actatgggaa acccaacact 7980
atttattata tttattattt cttgcattaa ttacactcct ctacaaaaca ataccatctt 8040
ttagcaattt cctgttgaca attttcagca tcttccttgc tgttaaaata ttggacaaca 8100
actctataaa gttcattttc cttaatagtt gggtcaatag catttgcaat tagtaaagcc 8160
attttctcgt atggtgtagg tgaccaaact tcgtttatgt catgttcatt atcacaaaac 8220
atagtttcaa atattatgtt aggcgctttt acgtttttca tttcatataa attaacttct 8280
tttacacctc tatttactaa acctaaacta gcaaagtttt tacataatct tgttgcaatt 8340
tcttttatag aatttcttgc tccaactgca acaagtgact ctgttccttg agcttgtcca 8400
ttaaaactat tcatgtgtag tgaaataaat atatcaacat ttgagttatt agcttttctc 8460
acaccttctg aaagttcacc attttgtgta cttgcatcac taccacaata aactacctca 8520
tgaccatgtt tttctaatat ttctataact tctttacata aaactctcat acatgattgt 8580
tcatcacgta aaccaatagc acctttacaa tttggactat gtccatctct aatacctatt 8640
ttcatttttt aaacctccct gttatccata gaataattac caatttcaac accttcatta 8700
tcaatatgcc atattgtagt accattgtca aaaatacctt taaggatatt taagtaatta 8760
ttaggaattt tattagtttt tatattaatt cctattgttt ttatatagtt ataataatac 8820
cttgaattaa tattaatttt catcatttta ttttgtttat acccaaactg tgcaaaataa 8880
tctcctattt tttcataata tctttctgtt aaaccatagc gataaagtct taattcttga 8940
ccaccattac gcaaaccata gattatattt gacccttgac taagtagagt gttaggtgtt 9000
gacctcatgt cgtttgctgt agctaaagct gaactgatag ccgattccct agttaattga 9060
ctctgcatat ttgtatgttt tttatccata tatgagccaa caccaccaaa gacactatct 9120
attacacccc caacattacc agtaaacgca ctagcaacac ccccaacaac actatttgcg 9180
atacctaaat tcatgttatt tttcgctatt ttatcattta aaatcgtttg gtttacggta 9240
ttttgtattg attgtgctgt ctgatttttt gaagttgcaa cccaatttgc atatgctgaa 9300
ctagtgcaag gcaattcaag tgcatcattt gacaccaaag cttctgtcat tccgtctgta 9360
tcgcccttat aagtttgtac aaatatacca taggaacatc tgtctgaaac agataatttt 9420
ataccaacag aacagctagg aattgaacaa aattggggct ttaaaatcat agggtcattt 9480
ataccatcac ttaacattaa ataagtgtac ggataattat ataatttact ttcatttctc 9540
cagtttcttt taccacccac actctttttt actttgtaag tttcaaactc gccaacaaat 9600
ttagaacctt ttaataattt tttgattctg aaaacgtgag ggtttgcttt aatctcgggt 9660
ctaatttcag aaatattccc aaatctttta ttatcatatg gaatttcttc taactctaaa 9720
tcagaaatat caagaaatgg ggtgtataca acgctaaaaa cattatcacc acttgcacca 9780
ataacagatg tattttcacc cgctactgta gagtcaatat aataactata tacggcatat 9840
ggtaacccgt acattttgga ttcccacgta tcataatctt tattagttat cgtcatactt 9900
tgtcacccac cttataattt ggcatagggt taacagtttt aacaccattt gcaacaactg 9960
ttcccttttc gtttctaatc tcccaatgta aatggtcacc actactatta ccagtattcc 10020
cacttaatgc aattaattgt ccttgtttta ctgtgtcacc ttcatttact tttaactctg 10080
aattatgagc gtaaacaact tgcgaatcac catgctgtat aattaaaaat ttaccatagc 10140
tagtagttaa ttctcttctt ttaataacag ttccgtcttt acttgcatat actggtgtac 10200
cagttggggt tgcaatatca acaccattat gagggttgcc acttggataa gttgggtaat 10260
atgcacttat ttgtcctgtt gttggtatta gccaatttgt acccgcatca ttatcaacat 10320
atttaccgtc ttttttacca ctaggcaacc aaccgttacc atctacatat ccaactattt 10380
ggccattttg tccatacttt aaaatttgtc tttgaggata aacaccactt tcaaataatg 10440
ccccctctct ttgtctacgc tgtggattag cttttaaatt ttttattgct gttgctatat 10500
ttttagtgtc ttttgggtta gctaaaaaca tttgccacgc atttaatcca ctcaatccac 10560
ccataccata attaaatgct atagataccc aaacatcaaa ttgataaatg ggaatatcac 10620
ttaaatttat attagctttt aacatagcat ttttaacggg tattccaaac tcattattta 10680
ataaatcata agtaacttga ctagcttttt cctctgatac tgggaatggt tctagttgtc 10740
tataatattt agtttggtaa ttttctgtta caccatagcc accagttttg aaagattcac 10800
cattaaaata tgctccatat tctgcaaaac cctcttcctg ttttacaaat aaaagtccat 10860
ttgctgatat aataccattt gctatatcac catttgctga accagttccg tcagaaccac 10920
ccccaacatt tgtttcaact tttcccaaag gtactgtaga agtaacaaca acaccacgac 10980
ccatatcagc tattttttca tattctagca tgatattttc accgtatgaa atatcttctg 11040
tttcatattc atttgttgga ttatctccat cccatctatt aacatgacat ctgtcaacaa 11100
aactttctaa aatttcaaag tcaaattggt aagtttgaaa tacatcaacg gatattacta 11160
ataatgttga gttttctgtt ttataaacaa agtcgtctat gaaataataa atatttttca 11220
tgttagtgtc ataagctgaa atataattga tattattctc cataaaccac ccataaggct 11280
tgtctaccac taagcttaca cgggaaggct ctacctttat gttatattga cccacccaca 11340
ccactttaga agaaaaataa ttattttgat tacttatact tttaaagtta aaaaggtttt 11400
gatatgaggg gtcaacataa ggtaaagaat aaaaatatac gtttttattc acttgttaga 11460
attgcaaaat ttgaaaaagt atttactggt agactataat tttcatagta ataataattt 11520
gttgttaaac tatttgggtc gtcaattcta tctaaaccat tataatttaa cattataggc 11580
attaatcttt tatccataat taacgctact ggtttaattg tatcattcgt tgataaatta 11640
gtaatatcag tcggtaaatc agtaattgac ctaatattat attcacctct taaaagtatg 11700
ctatctgtac ttactaactt tgcataaatt tcagttttca caaatattat tatgtcactt 11760
tttaagcatt gagtattaaa tttgctaggg ttgtattttg tacttggtag tgtaaactct 11820
tctacaattt taacaatgct ctcaattaag tttgaactta caacttcttt atatgggact 11880
tgtttaaaat taagattttc tccgttggag tctaaaccct tagcaaattc ctttaaagct 11940
ttttctattt ctttgtattt atctttactc atagaagttt ttaaattaga taacatttta 12000
gaaattaaat cgtataatcc aaactctttt aaaaatgctg actttaatct ttcaacacta 12060
agagtcattt taactttttt atcaaatcta ctatttgcaa ctaatccatt tagtcttggt 12120
aaagaagaat tgaaaacttc attttcatca ccatctaata atgattgaac agttgagtcc 12180
tctaaaaata attcttctat tgaataaccg aatggcattt caccataaaa aagttcattg 12240
aaagggtttg aaaaatgttg ctctatatat aacattttac ctattttatt gcatatactt 12300
tcaataaaac tattcatatc aatatttatt aaattcaacc ttaaagcctc ttcataagaa 12360
ggaaaagagt tgcccaaatc cttgggcatg gttgaaatta atttatcata aatttcttta 12420
ttagttaact tttccatttt aaatcacctc attatgatgc tgatttagtg tatattatag 12480
catttaaaaa tggtactata ccacataacc cttgtttgtg taagaagtag ttagtagata 12540
agttgtttgg gttattaaaa taccttgttt caaatacaaa atccttaaat tttaaaaggt 12600
ctttatcaac taacattcca atacaagttt ctcctgtatg aacattagtt ccattatcac 12660
cactcttaat agttgttggt agttcatcca ttaatataat tctttgctga acctctgatt 12720
ttgaaaggtt aaaaacatct gctaaataat caacatctat ttctgctttc atttcaggtg 12780
ttgttataaa aacacaatca tttaaagatg aaaattgcat tactttagca ctattatatt 12840
tatcactttc aaattgtagt ctgtcattca tagcttttaa tgatttacaa agatttttta 12900
caagactagc atcatcattt aatttaataa cttgggtgtc ttgaatatat tgagtttttg 12960
taggtgaact actcattcca acatcttgat tagttaaacc cttacaatat ttaaaaaaca 13020
tatctttcat atatttatat tcatcacgat aagcccctct taagttagcc tctaataatt 13080
tattaactaa gtttgccaaa ccgtattcat ttctaaacgc tgttcttaac tgaatatctg 13140
atattgttat tttatattta tcagcaaaat ttcttgaaag ataattagtg tataccttag 13200
gtactaaagt agctactatg tcctttactt catcaccact attaaaatgt tctttaaacc 13260
caactttttg tgatgcctca acatacatat cttctatagt ataaccgtat tcttccattc 13320
caccatgaaa tatttttaaa ggatttttcc aaacttttga atgtatttga gtcataaata 13380
ttttatttat aagggaactt ataaactcat tttttacaac ttgataattg aataaagcat 13440
ctgaaacagc tctactgtca gataaatcta cttgacttaa aactttttta gtatcttcgg 13500
ttgctccttc taaaaaagcc tccatgattt ctttgtttgc cttaacagcc attttatcac 13560
tcccattctt ttaataactt gtccattttt gactcattat tttctttaat ttcagtatta 13620
ttttcttcct ttggtttact agtaacttta aggaataatt ccatgttact tttttgtaat 13680
ttatcaattt cttttgaatt agtttccttt tcagaattta atgtgctgat tacattattc 13740
tgattttcta tagtttcctc taattcaaca attcttaaaa gggcatcatc aagagtcatt 13800
tctttactca ttttaaaacc tccttacaaa atattatacc cccttacttc actatacaac 13860
gcccacggtc ataggtggaa aaatgttagg cacgttctac cgtgtgactt cctaatcatt 13920
tacgttatat agctaagtaa gggggtatac tttactttat gctaattgaa atattattct 13980
ctttttagtt tctgttactt taaattctat tcctaaagaa gttaaaagcg tatcaaacat 14040
tacagcctca tgttctttag attttggaaa ctctttttct tcttgattta taaaagaaac 14100
tactctatta agttgtaatc tcttttcatt aactttttct aaagctaatt cctttctgat 14160
ttctaattct gttaactctc tattttcttc cattttttat tcacctctat ataaacttta 14220
gtaggtggga attaacccac ctttaaatta tttatttctt tcaaatattt tttcatcacc 14280
agtaattaaa aagttctcta agtctttatt tgatacttca taatatactg tttcattttt 14340
cttgattaac tctaaacctt caaatacttt cttatctgat gcacctaaat actcaaattc 14400
atatgaagta ttagtatcac ccttagcacc taatcttgtt acctcataag tgtggttaat 14460
aaggtctggt cttttttcag ctaagaaaat taatttttct attaaagtct tgcctagttg 14520
ccaaatttgt acactcttag tagtatggtt ataaacatta acagctacct tttctgacat 14580
cttaaaacct tcaccacata aaggacaagt atctttaatt acttttaagt cgttgtctaa 14640
ttcagccaca cacttaacat ttacttctct accatctgct aattttactt ggtgaactaa 14700
atctagctca acctcatcaa tgttattgat taagaaagtt ataacagcct tttctctgtt 14760
atcctttaat ctaaaatata ccccacttga aacgttgttt cctaaattga aaattgataa 14820
ttttttcatg ttaaaatctc tcctttaatt ttaaatattt tgttgttttg gtttgttttt 14880
tccttatgag ttaattatac attacattta tatattatgt caacactttt ttgcgttatt 14940
ttttaaaaaa taatccaatt tgattgaagt aaaattttgt ttcagcatta ttaaagaaaa 15000
cagcaccaac tttattatta aaattgattc tatttataag ccctttatta cataaagata 15060
tatgcttatt ttcatctgtt tggtaatcta aatctaaaaa gtattcgtca cattttcctt 15120
tattatgatt atcaacccag taataaaaca ttgtatcact ttgacatttc catattttaa 15180
ttaccttgtc cacatctaca cgaaccttaa aaactggttc tagcttatca gtatcccata 15240
attctttttt actacaaacg ttaaatctat cattactgat attttcatta tctatattat 15300
agcgtgatga accactcatt acagttaatt taccaacctt agaatttaag gcatactctt 15360
taaactcttt aggtgtttca agtgttttaa tcgccataag ttcattttta acaaccctat 15420
tcggtttggt tgaaggatat aactttaaag tgtcaaaaat agggttataa taacttgtat 15480
tattaccaac acatatcact tctattctat cctcaaatct atcaactgtt acaattatag 15540
attctaattt cttatattca tcttttataa ttttctcccc aggctcaggc ataacttcct 15600
cataaataat ataataaacg ttgggaaatg atgaaccttt agctaaacta tttccattta 15660
accctgtaaa taagcaaaat ggttctccat ttataaaccc catatcaccc tttaactcat 15720
attcaatagt tggaaattcc ttttgtaata catctgtaaa gtatgtcgtt gccatgcttt 15780
cagcttgatt tttatatctc cttaatatca acgcttgttt tccatattcc aaaaactcct 15840
ctattattaa tcgcttagtt gaatatgatt tcccaacacc ccttgcacct ataacacagt 15900
gtattaaatg gtttgggtat tcttctataa actcttttat ttcaaaaaat ttactcatgt 15960
tacctcctta aacttaaatc ttttattata tattgaactt ctactaaatc aagaccaccg 16020
tcacatttta tttgtgctaa tttacccttt acttctttac ctaatttaaa ctcttcaaaa 16080
ccttcattac ataatatctt ttgtgcatct tttggaacac ctgcacattt acaagttatt 16140
ttaccgtcat tagttctaag catatacttt ttagccccta tggttttaaa ctctttaaag 16200
tttttctcat tatccatagc ccctaaattg ttagggtgaa cttcaacacc cattttcttt 16260
aaaccttcaa ttacttgttc tttaggtata gaacagtaaa agctgtcagt atcgctgtat 16320
aaaaaatttt caactccaca accttctatt atcattgatt ggagatatac tctagcatat 16380
gctgtaacaa aactggcgaa tggtttataa aaaggttcac tttcataagt ttctatatat 16440
tgtccacttg ctaaaatttg atttcttctt ttttcatttt ttaatttttt aaccctttct 16500
aaatttttct ctttaccccc tctatcgaat gtaaatattt tatctttttt tactaaatag 16560
tctatagtac gcgtagttct tgtcccaaac ttaccataaa aagaattgag tatagttttt 16620
attgctgaat attgagcgtc taacttttca cccttatcta ttctatgctt tatttctgat 16680
tttaaattat aaaagtgatt tacaaagttt gagaaaatac ctttttcagc tttatacact 16740
aagctagtac caaactttaa accattaaat ttaatcttat ctttttcttt aaataaaaac 16800
ccatcaacct caacattttc ataataccca aagtcataat attttgttaa atgttcaagt 16860
tctacacttg taatactcat attataatta cataaatttt cacaaccatt acgataaatt 16920
tcaataactc tatcatctat tatattagta tgtataaaag agttaccatt cataagaacc 16980
atattcaatt tatctctata aagtaaaggt aaatcagtat tagaaaagtt ctttgctcct 17040
aatctaatca tttgtaattc atactcttta ctcttaggtt taaaataatc aaagcctata 17100
ttataaattg ctacttcttc gccatcaact ggtttaacat atcctttgta ttcagttggc 17160
atcccataag gtagcaaggc actttcaacc tgaaatggat agcttgaatt tatatcaaag 17220
cttgacccat tacattttac aactttatta actattttag ggttgtacca tgtccaccca 17280
ccttcataac ttttattctc catttcccta gccattttgc ttaaaagcat ctgtctttct 17340
tcaatttcga actgatttat aaaggctttt ttcttgttac gctcattgaa atatgttatc 17400
tctaaagctg atttaaatga tattgatgaa cttgttttac aatttgaact tatgtaaatc 17460
tcttcatcat tatattcaaa ctttaaaagt gagttgtaaa aataatcatt tataacttct 17520
tttagtatcc taatatcatt atatagatac cttctttcta tatctgttag tatatggtta 17580
tcttctctta ttttatcata atcaaactct tctttcaatt tataaaacat ttcatctaag 17640
ttattacaaa acttataagc tttatctaaa gaacatggaa ctatttttaa agtatcccaa 17700
aacttcacct ttacgctaaa tgtattaaca tgaccgtctt tgtcagtaag ttctaaagta 17760
tttggtaaat ttacttctaa accataataa acattatcag tttgtactat agaaaaagtc 17820
attggtttaa aattctcaat ggttctgcta attcctaatt cgttagtctg taaactagga 17880
ttaacataag tgaacccatt gtcagttaac cagtattgca tataactaat ttcataagca 17940
acgttatgaa cagcaacact taaagttatt actctcttat ttttattctt ttttatattc 18000
tcttttttag cttgttcaaa tcctatcatc tgaaaggtat aaaaagcctc gtctaaatca 18060
caaccaaata ttacatcatt tgtatttgta gcacctaaac caaacgccca aacttttctc 18120
ttcttgtctt tttgtggata atctttattt aacatttttc cgtcaacgta gcaagtttca 18180
atgtcaaata ttaaatcttt actatctatt ttggtatatt cagttacttg taaactatgt 18240
tcgttgacat aagttaaatg tttattcatt aattccacat ccttatattt tattgtttca 18300
atcccgtttt aaatgttgct aataagtcct ctaatatata acccatttca acatcatgca 18360
caccatgggt ataataacct ttttcaacca tattttcttc atagataaac tcaaagtatt 18420
tatctatatt ttcataaaaa tcagatactt ttgatacagc atcatttgat gacataataa 18480
atttattaac ttcatttaca aagttgtcta ttttgtcttt tggtaaattc ctttgaaatg 18540
caccttttaa tatcccttta ccgctaccat caccaaaaaa taaatcagta accttattaa 18600
ctaattcctt tctgatgttt tcttctgcat cttctttccc aacaaacttt aataacccta 18660
aatgtgtgtc acttataatc ttttgtttac tctcatgtgt taaatctaat ttgtttaaat 18720
tttcgactat ctttttgcgt tcttttgttt tctctctctt ttttcttttt tcttcttttt 18780
cttttctttt ctgttcattc ttaacaaacc tgtcagcatt tcttttatag tctgtatctc 18840
ttgccatatc aactattata ttaagtgtag atttgtcata acctaataat ttaatagcct 18900
tcttttgaat atccttgtcc attaaataga catatgctcc attttttaca gctttgttaa 18960
attgtttttc aaagttcttt ttctctattt gttttttctt actttttaat gttattcttt 19020
ttcttgccat gttttctcac tctcctaaca tttttacgtg gtatgatgtc tataacatca 19080
ctctctaaaa gatacttatc acctagaata tcaattacaa atatatcatc accaatatta 19140
taatttttag aatttaattc ataataaaaa ggttcaacta ccacagcgtg aaaagtttct 19200
aatcccatgt tatgtttatt cctcactatc caccacaccc cttatcttat tagctaggtc 19260
ataaagtttc tcaagttcat ctatgctctt agcttgtacc attctagtat ctaagcataa 19320
atcaatagta cttaattcat ctagtgaaag ctttacaata tattctttca aatttgacac 19380
ctctctttta ttttggtata atttccccta acataattct agcattttat acctattatt 19440
tcaaggtttt tattaattta tttgtaaaca atttgttaac agatagtgct gttttatcta 19500
ctggtaataa ctggggaata tgtcgaatcg tgtggagggt 19540
Claims (3)
1.具有强裂解作用的产气荚膜梭菌噬菌体，其特征在于：该噬菌体为噬菌体RKP-CP-21028，于2021年10月22日保藏在中国微生物菌种保藏委员会普通微生物中心，保藏编号为CGMCC No.23094；所述噬菌体的基因组序列如SEQ ID NO.1所示。
2.一种如权利要求1所述噬菌体的应用，其特征在于：所述噬菌体应用于制备裂解产气荚膜梭菌的抑菌制剂。
3.一种裂解产气荚膜梭菌的抑菌制剂，其特征在于：包括如权利要求1所述的噬菌体。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111524540.6A CN114015662B (zh) | 2021-12-14 | 具有强裂解作用的产气荚膜梭菌噬菌体及其应用 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111524540.6A CN114015662B (zh) | 2021-12-14 | 具有强裂解作用的产气荚膜梭菌噬菌体及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114015662A CN114015662A (zh) | 2022-02-08 |
| CN114015662B true CN114015662B (zh) | 2024-06-25 |
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