CN114164182A - 一种基因vii型遗传拯救的新城疫病毒疫苗株 - Google Patents
一种基因vii型遗传拯救的新城疫病毒疫苗株 Download PDFInfo
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Abstract
本发明提供一种基因Ⅶ型新城疫病毒疫苗株，为基因Ⅶ型新城疫病毒弱毒株NDV‑Ⅶb株，是将保藏编号为CCTCC NO:V201968的新城疫病毒株的HN蛋白的340位、342位、347位和353位氨基酸分别突变为组氨酸、天冬酰胺、赖氨酸和精氨酸；再通过遗传拯救构建的。本发明提供的基因Ⅶ型新城疫NDV‑Ⅶb株用于制备疫苗。本发明所提供的新城疫病毒基因Ⅶ型弱毒株的免疫效果显著优于亲本株，可以对当前流行的基因Ⅶ型新城疫病毒提供良好的保护作用。
Description
技术领域
本发明属于兽用疫苗株技术领域，具体涉及一种基因VII型遗传拯救的新城疫病毒疫苗株。
技术背景
新城疫是由新城疫病毒引起的禽类的急性高度接触性传染病，可引起鸡、鸽子、鹅、野鸟等多种动物的发病。新城疫自1926年首次发现于印度尼西亚以来，在世界许多国家和地区迅速传播并流行，对养禽业危害巨大，是世界动物卫生组织要求的必须报告的A类疫病，我国农业部将其列为一类动物疫病。
刘华雷等通过对新城疫流行毒株的持续监测，发现我国目前流行的新城疫病毒以基因Ⅶ型和Ⅵ型为主，其中基因Ⅶ型主要存在于鸡群和鹅群中，基因Ⅵ型主要存在于鸽群中。刘秀梵等对江浙地区分离的新城疫病毒进行分析显示，多数分离株属于基因Ⅶ型。国外的研究表明，东南亚等地区的鸡群中新城疫的流行基因型也以Ⅶ型为主。
我国目前防控基因Ⅶ型新城疫病毒主要用灭活疫苗，市场上没有针对基因Ⅶ型新城疫病毒的活疫苗；而用于鸽子新城疫病毒的防控主要使用Lasota等传统的基因Ⅱ型的毒株，与鸽子流行的基因Ⅵ型的新城疫病毒毒株不匹配，免疫保护效果较差。
对NDV活疫苗的研制，主要是通过反向遗传的方法，将基因组中与毒力相关的基因进行改造，拯救出毒力减弱的毒株。国内外研究发现，与NDV毒力相关的基因包括F基因、HN基因、P基因和L基因等。直接对基因Ⅶ型或基因Ⅵ型强毒株的若干基因进行改造发现，病毒的毒力并不能完全致弱，免疫后仍会引起一定的副反应，有很大的安全风险。因此，能从健康鸡上分离到一株自然弱毒株就显得很有意义。
但是分离的自然弱毒株在使用的过程中往往会暴露出各种问题，包括免疫后的应激反应大，毒力有残存，免疫持续期短，免疫原性受母源抗体的影响或免疫原性弱等。
发明内容
本发明的目的是提供一种基因Ⅶ型新城疫病毒疫苗株，以及该疫苗株的遗传拯救方法，所提供的弱毒株用于制备疫苗。
本发明首先提供一株基因Ⅶ型新城疫病毒弱毒株NDV-Ⅶb株，其是将保藏编号为CCTCC NO:V201968的新城疫病毒株的HN蛋白的340位、342位、347位和353位氨基酸分别突变为组氨酸(H)、天冬酰胺(N)、赖氨酸(K)和精氨酸(R)；再通过遗传拯救构建的；
所述的基因VII型弱毒株NDV-VII株，于2019年10月15日保藏在位于武汉大学的中国典型培养物保藏中心，其保藏号为CCTCC NO:V201968。
所述的基因Ⅶ型新城疫病毒弱毒株NDV-Ⅶb株，其遗传拯救构建方法如下：
1)分别将基因Ⅶ型新城疫病毒的NP蛋白、P蛋白和L蛋白基因分别连接到载体构建辅助质粒；
所述的载体，一种实施例的具体记载为PCI-neo载体；
2)构建携带有基因Ⅶ型新城疫病毒的全基因组的重组载体，且HN蛋白的340位、342位、347位和353位氨基酸分别突变为H、N、K和R；
3)分别将全基因组载体与3个辅助质粒共转染宿主细胞，拯救获得新城疫病毒基因Ⅶ型弱毒株。
所述的宿主细胞为BSR细胞；
本发明提供的基因Ⅶ型新城疫NDV-Ⅶb株用于制备疫苗；
本发明再提供一种基因Ⅶ型新城疫病毒疫苗，所用的抗原为上述的新城疫病毒基因Ⅶ型弱毒株。
本发明所提供的新城疫病毒基因Ⅶ型弱毒株的免疫效果显著优于亲本株，可以对当前流行的基因Ⅶ型新城疫病毒提供良好的保护作用。
附图说明
图1：辅助质粒的PCR鉴定图；
图2：SPF鸡抗体效价测定图。
具体实施方式
发明人所在的实验室前期分离到一株基因Ⅶ型新城疫病毒弱毒株NDV-Ⅶ株(于2019年10月15日保藏在位于武汉大学的中国典型培养物保藏中心，其保藏号为CCTCC NO:V201968)，免疫SPF鸡后可提供100％的保护，但是用于商品肉鸡免疫还不能完全克服母源抗体的干扰。
为了解决这一问题，对NDV-Ⅶ株的基因序列分析后，选择将NDV-Ⅶ株的HN基因(其序列为SEQ ID NO:5)进行定点突变并构建全基因组载体，与辅助质粒共转染，获得改造后的基因Ⅶ型新城疫病毒重组毒株，筛选能够在鸡胚上繁殖效价高，毒力较弱的毒株。最终获得了本发明的基因Ⅶ型新城疫病毒疫苗株，使用该疫苗株免疫鸡后，与亲本株相比免疫原性有显著的提高。
下面结合实施例对本发明进行详细的描述。
实施例1基因Ⅶ型新城疫病毒弱毒株的构建
通过反向遗传学技术，分别构建了以NDV-Ⅶ株为亲本毒株的4个重组毒，其中HN蛋白基因进行了不同组合的点突变，包括E347K+Q353R+Y304H+D342N、E347K、E347K+Q353R以及Y304H+D342N。根据上述基因重组毒株的免疫效果，构建了1株基因Ⅵ型的新城疫病毒重组毒株。
下面就具体的步骤进行描述。
(1)新城疫病毒cDNA感染性克隆的构建
根据保藏编号为CCTCC NO:V201968NDV-Ⅶ株的全基因组序列(基因组的核苷酸序列为SEQ ID NO:1)设计引物，将全基因组分成5段，应用Red/ET同源重组技术一步克隆至载体PBRT。其中PBRT载体是以质粒PBR322为基础，连接上T7启动子、具有DNA剪切作用的丁肝病毒核酶序列以及T7终止子序列，NDV全基因组插入T7启动子之后、丁肝病毒核酶序列之前；片段NDV-3事先通过over-lap PCR技术进行点突变后，克隆至PMD 19-T载体进行测序，分别将用引物NDV F3/T-R1/T-F1/NDV R3、NDV F3/T-R2/T-F2/NDV R3、NDV F3/T-R3/T-F3/NDV R3和NDV F3/T-R4/T-F4/NDV R3扩增获得的克隆命名为NDV-3-T1、NDV-3-T2、NDV-3-T3和NDV-3-T4。所用引物见下表1。
表1：感染性克隆的构建引物表
注：斜体序列为设计的同源臂序列；下划线序列为设计的点突变序列；小写字母为酶切位点。
提取NDVⅦ株RNA并反转录成cDNA，以cDNA为模板，分别用引物NDV F1/NDV R1、NDVF2/NDV R2、NDV F4/NDV R4和NDV F5/NDV R5扩增并获得片段NDV-1、NDV-2、NDV-4和NDV-5；分别以NDV-3-T1、NDV-3-T2、NDV-3-T3和NDV-3-T4为模板，用引物NDV F3/NDV R3扩增获得片段NDV3-T1、NDV3-T2、NDV3-T3和NDV3-T4；以PBRT载体为模板，用引物NDV-V-F/NDV-V-R扩增获得载体片段NDV vector。
扩增获得的条带进行纯化后，测定DNA浓度。取NDV vector 200ng、NDV-1 400ng、NDV-2 400ng、NDV-4 400ng和NDV-5 400ng混合后，分别添加NDV3-T1 400ng、NDV3-T2400ng、NDV3-T3 400ng或NDV3-T4 400ng，混匀后电转到用L-阿拉伯糖诱导表达了Red/ET重组酶的GBdir大肠杆菌中，电压1250V，时间4-6ms，电转后迅速加入1ml的无抗LB培养基，复苏1h，涂在氨苄平板上，37℃过夜培养。
分别挑取单菌落，用氨苄LB培养基摇菌培养后进行PCR鉴定(图2)，全基因组载体的鉴定引物为NDV F1/NDV R1、NDV F2/NDV R2、NDV F3/NDV R3、NDV F4/NDV R4和NDV F5/NDV R5。鉴定正确的克隆送上海生工生物工程有限公司进行测序。构建的4个质粒分别命名为PBRT-Ⅶb、PBRT-Ⅶc、PBRT-Ⅶd和PBRT-Ⅶe。
以质粒pKD3为模板，用引物Cm-F/Cm-R扩增氯霉素抗性片段Cat基因，并引入Pac I酶切位点；DNA片段纯化后测定浓度。取Cat基因400ng与200ng的质粒PBRT-Ⅶb混合，电转到诱导表达Redα/β重组酶的大肠杆菌GB-red。电转后迅速加入1ml的无抗LB培养基，复苏1h，涂在氨苄/氯霉素双抗平板上，37℃过夜培养。挑取单菌落，摇菌后进行PCR鉴定，测序鉴定，构建的质粒命名为PBRT-NDV-Cat。
(2)辅助质粒的构建
设计引物，分别在NDV-Ⅶ株NP(编码基因的序列为SEQ ID NO:2)、P(编码基因的序列为SEQ ID NO:3)和L基因(编码基因的序列为SEQ ID NO:4)的上下游引入酶切位点，分别克隆至经相同核酸内切酶处理的PCI-neo载体。其中L基因片段较长，分成3段进行扩增，扩增的每一段DNA片段在首尾均有30bp左右的重叠，用NEB公司生产的
高保真DNA组装预混液进行一步法组装。引物由上海生工生物工程有限公司合成，引物序列见下表2。
表2：NDV辅助质粒构建用引物表
构建的3个辅助质粒分别用引物NP-F/NP-R、P-F/P-R、L-1/L-6进行PCR鉴定后测序并保存(图1)，分别命名为PCI-NP、PCI-P、PCI-L。
(3)病毒拯救
BSR细胞接种于6孔细胞培养板中，待细胞生长至60％-80％时进行转染。分别取5ug感染性克隆质粒PBRT-Ⅶb、PBRT-Ⅶc、PBRT-Ⅶd、PBRT-Ⅶe、，与3个辅助质粒(PCI-NP2.5ug、PCI-P 1.25ug、PCI-L 1.25ug)混合后进行共转染，按照磷酸钙转染试剂盒说明书进行操作。转染后3天，取培养液上清接种10日龄SPF鸡胚，培养96h后收取鸡胚尿囊液，测定HA效价。HA效价阳性样品进行冻存。将拯救获得的基因Ⅶ型的NDV毒株分别命名为NDV-Ⅶb(E347K+Q353R+Y304H+D342N)、NDV-Ⅶc(E347K)、NDV-Ⅶd(E347K+Q353R)和NDV-Ⅶe(Y304H+D342N)；
接胚后96h收取的鸡胚尿囊液，HA效价为24-26。尿囊液稀释后继续接种鸡胚，HA效价稳定在29-210。继续传代至20代，每隔5代取一次病毒尿囊液样品，提取RNA并反转录成cDNA。分别对NDV-Ⅶb株、NDV-Ⅶc株、NDV-Ⅶd株、NDV-Ⅶe株的HN突变位点进行测序，改造的位点没有发生任何插入、缺失等突变。表明拯救的毒株可以稳定遗传。
实施例2鸡胚平均致死时间(MDT)、脑内致病指数(ICPI)和静脉致病指数(IVPI)的测定
新城疫病毒毒力致病性的判定，可以根据MDT、ICPI和IVPI的测定结果进行划分。按照OIE的标准，分别对未改造的亲本毒株NDV-Ⅶ株、和改造毒株NDV-Ⅶb株、NDV-Ⅶc株、NDV-Ⅶd株、NDV-Ⅶe株进行上述3个指数的测定。结果见下表3所示。
表3：致病指数的测定表
结果表明，除YB17-Ⅵ株表现为中等毒力的特征外，其余毒株均为弱毒株，说明对HN基因进行突变改造或替换后，没有影响毒株致病力。
实施例3基因Ⅶ型NDV活疫苗的制备及安全性试验
分别将NDV-Ⅶ株、NDV-Ⅶb株、NDV-Ⅶc株、NDV-Ⅶd株和NDV-Ⅶe株制备成活疫苗并进行安全性试验。用5％蔗糖脱脂乳1:1加入至稀释的尿囊液中，定量稀释后的病毒含量为108.0EID50/0.1mL，充分混匀后冻干制备活疫苗。制备的活疫苗按2010年版《中国兽药典》附录进行无菌检验，结果符合标准，无细菌污染。
用1日龄SPF鸡进行安全性试验。每个毒株用20只雏鸡，每10只一组，分别免疫107.0EID50和108.0EID50剂量的病毒液，PBS对照组5只，免疫方式为滴鼻点眼，在相同的条件下饲养，连续观察14日，记录试验鸡采食、饮水及临床表现。结果显示，免疫后鸡只没有出现精神沉郁、食欲不振、嗜睡麻痹及全身不良反应，鸡只健康，证明制备的5株NDV活疫苗对雏鸡安全。
表4：基因Ⅶ型NDV活疫苗对SPF雏鸡的安全性试验
实施例4：基因Ⅶ型NDV活疫苗免疫效果评价
分别将制备的基因Ⅶ型NDV活疫苗免疫SPF鸡和肉鸡，评价5株NDV毒株的免疫效果。其中SPF鸡免疫组观察至42天，每7天采血并分离血清，测定HI抗体水平；肉鸡免疫后采血至21天后攻毒:评价免疫效果。
下面就具体的步骤进行描述。
(1)SPF鸡抗体持续性试验
19日龄SPF鸡随机分成5组，每组10只，分别免疫107.0EID50剂量的基因Ⅶ型活疫苗，另设1组PBS阴性对照组5只，滴鼻点眼，0.1mL/只。分别在免疫后7天、14天、21天、28天、35天和42天测定HI抗体，评价疫苗免疫效果。结果见下表5所示。
表5：SPF鸡抗体持续时间表
结果显示，免疫后NDV-Ⅶb的免疫效果最好，具体表现在，在相同的采血时间内诱导的抗体水平更高。因此，确定HN蛋白基因的E347K+Q353R+Y304H+D342N突变后所遗传拯救的基因Ⅶ型新城疫病毒弱毒株与亲本毒株和HN基因其它位点突变的毒株相比，免疫动物后抗体效价更高，持续时间更长，具有最好的免疫效果。
(2)肉鸡攻毒保护试验
1日龄商品肉鸡随机分成5组，每组10只，分别免疫107.0EID50剂量的基因Ⅶ型活疫苗；另设一组Lasota对照组10只鸡，免疫相同剂量的Lasota活疫苗；一组PBS对照组5只；每只鸡进行编号。免疫途径为滴鼻点眼。免疫后14天和21天采血测定HI抗体。21天采血后，所有鸡进行攻毒，攻毒毒株为实验室保存的基因Ⅶ型NDV强毒株JS 02/06株，攻毒剂量104.0ELD50/只，攻毒方式为肌肉注射，连续观察14日。攻毒后5日，分别采集每只鸡的喉头和泄殖腔拭子，经处理后，每个样品尿囊腔内接种10-11日龄非免疫鸡胚5枚，每胚0.2ml，孵育观察5日，无论死胚、活胚均应测定鸡胚液红细胞凝集价，每个拭子样品接种的5枚鸡胚中，只要有1枚鸡胚液的凝集价不低于1:16(微量法)，即可判为病毒分离阳性，对病毒分离阴性的样品，盲传一次后再进行判定。保护率＝(动物总数-死亡动物数-未死亡但排毒动物数)/动物总数。具体结果见下表所示：
表7肉鸡攻毒保护性试验
综上所述，本发明所拯救的NDV-Ⅶb株免疫效果最好，诱导动物机体产生的抗体水平较高，持续时间更长，最重要的是可以克服母源抗体的干扰，对商品肉鸡可以达到100％的保护水平。
序列表
<110> 青岛易邦生物工程有限公司
<120> 一种基因VII型遗传拯救的新城疫病毒疫苗株
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15186
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
accaaacaga gaatccgtaa ggtacgatag aaggcgaagg agcaatcgaa gtcgtacggg 60
tagaaggtgt gaatctcgag tgcgagcccg aagctcaaac tcgagagagc cttctgccaa 120
aatgtcttct gtattcgatg agtacgagca gctcctcgcg gctcagactc gccccaatgg 180
agctcatggc ggaggagaga aggggagcac cttaaaggta gaagtcccgg tattcactct 240
caacagtgat gacccagaag atagatggaa ctttgcagtg ttttgtcttc ggattgctgt 300
tagcgaggat gccaacaaac cacttaggca aggtgctctc atatctctct tatgttccca 360
ctctcaagtg atgaggaacc atgttgccct tgcggggaaa cagaatgagg ccacactggc 420
tgttcttgag atcgatggtt ttaccaacgg cgtgccccag ttcaacaaca ggagtggagt 480
gtctgaagag agagcacaga gatttatgat gatagcaggg tctctccctc gggcatgcag 540
caacggtacc ccgttcgtca cagctggggt tgaagatgat gcaccagaag acattactga 600
taccctggag aggatcctct ctatccaggc tcaagtatgg gtcacggtgg caaaggccat 660
gactgcatat gagacagcag atgagtcaga aacaagaaga atcaataagt acatgcagca 720
aggcagggtc cagaagaagt acatcctcca ccccgtatgc aggagcgcaa tccaactcac 780
aatcagacag tctctggcgg tccgcatctt tttggttagc gagcttaaga gaggccgcaa 840
cacggcaggt gggacctcca cctattacaa cttggtgggg gatgtagact catacatcag 900
gaacactggg ctaactgcat tcttcctgac acttaaatat ggaattaaca ccaagacatc 960
agcccttgca cttagcagcc tctcaggcga tatccagaaa atgaagcagc tcatgcgctt 1020
gtatcggatg aaaggagata atgcgccgta catgacattg ctcggtgaca gtgaccagat 1080
gagctttgca cctgccgagt atgcacaact ttactccttt gccatgggta tggcatcagt 1140
cctagataaa ggaactagca aataccaatt tgccagggac tttatgagca catcattctg 1200
gagacttgga gtagagtacg ctcaggctca aggaagtagc atcaatgagg atacggccgc 1260
cgagctaaag ctaaccccag cagcaaggag aggcctggca gctgctgccc aaagagtgtc 1320
tgaggagacc agcagcatgg acatgcccac ccaacaagcc ggggtcctca ctggactcag 1380
cgacggaggc tcccaagccc cccaaggtgc actgaacaga tcacaagggc aaccggacac 1440
cggggatggg gagacccaat ttctggatct gatgagagcg gtggcaaata gcatgagaga 1500
agcgccaaac tctgcgcagg gcacccctca accggggcct cccccaaccc ctgggccctc 1560
tcaagacaat gacaccgact gggggtactg accgacagca cccagtttgc ttctatgagg 1620
tcatcccaat tcctctgccc acaccccacc cctcaatccg caatcccgca tggccaaacc 1680
cacaaacgaa cccccctgtc tccctcctct cccccagccc cacaacccca cctgcccagg 1740
gcaacatagg tacaatgcga cccactaata atcaatacag ggccaaagaa attagaaaaa 1800
agtacgggta gaagggagac attcagagat cagggcgagt cacccgggtc tctgctctcc 1860
cttctaccta gtggattagg atggagatgg ccacctttac agatgcggag atcgacgagc 1920
tatttgagac cagtggaact gtcattgaca gcataattac ggcccaggga aaaccagtag 1980
agactgttgg aaggagtgca atcccacaag gcaaaactaa ggctttgagc gcagcatggg 2040
agaagcatgg gagcatccag tcaccagcca gccaagacac ccctgatcga caggacagat 2100
cagataaaca actgtccaca cccgagcaag cgagtccaaa cgacagcccc ccagccacat 2160
ccactgacca gcctcccact caggctgcag atgaggccgg cgatacacag ctcaagaccg 2220
gagcaagcaa ctctctgctg tcgatgcttg ataaactcag caataagtca tctaatgcta 2280
aaaagggccc agggtcgagc cctcaagaaa ggcatcatca acgtctgact caacaacagg 2340
ggagtcaaca aagccgcgga aacagccaag agagaccgca gaaccaggcc aaggccatcc 2400
ctggaaacca ggtcacagac gcgaacacag catatcatgg acaatgggag gagtcacaac 2460
tatcagctgg tgcaacccat catgctctcc gatcagagca gagccaagac aatactcctg 2520
cacctgtgga tcatgtccag ctacctgtcg actttgtgca ggcgatgatg tctatgatgg 2580
aggcgatatc acagagggta agtaaagttg actatcagct ggaccttgtc ttgaaacaga 2640
catcttctat ccccatgatg cggtctgaaa tccagcagct gaaaacgtct gttgcggtca 2700
tggaagccaa tttgggcatg atgaagatcc tggaccctgg ttgtgccaac gtttcatctc 2760
taagtgatct acgggcagtt gcccgatccc acccggtttt aatttctggc cccggagacc 2820
catctcctta tgtgacccaa gggggcgaaa tggcactcaa taaactttcg caaccggtgc 2880
aacacccctc tgaattgatt aaacccgcca cggcaagcgg gcctgatata ggagtggaga 2940
aagacactgt ccgtgcattg atcatgtcac gccctatgca tccgagctct tcagctaggc 3000
tcttgagcaa actggacgca gccggatcga ttgaggaaat cagaaaaatc aagcgccttg 3060
cactgaatgg ctaatcacca ccgcaacccg cagcagatcc ctgtccaccc agcaccacac 3120
ggtatctgca ccaagctcct ctctgcaaac ccaaggtcca acaccccgag cgacaaccct 3180
gtcctgcttc ctctgcccca ctaaatgatc gcgcagctgc aatcaattca gctatattaa 3240
ggattaagaa aaaatacggg tagaatcgga gtgccccgat tgtgccaaga tggactcatc 3300
taggacaatc gggctgtact ttgattctac ccttccttct agcaacctgc tagcattccc 3360
gatagtccta caagacacag gggacgggaa gaagcaaatc gccccgcaat acaggatcca 3420
gcgtcttgac tcgtggacag acagcaaaga agactcggta ttcatcacca cctatggatt 3480
catctttcag gttgggaatg aagaagccac tgtcggcatg atcaatgata atcccaagcg 3540
cgagttactt tccactgcca tgctatgcct agggagtgta ccaaatgtcg gagatcttgt 3600
tgagctggca agggcctgcc tcactatggt ggtaacatgc aagaagagtg caactaacac 3660
cgagagaatg gtcttctcag tagtgcaggc accccaggtg ctgcaaagct gtagggttgt 3720
ggcaaacaaa tactcgtcgg tgaatgcagt caagcacgtg aaagcaccag agaagattcc 3780
tgggagcgga accctagagt acaaagtgaa ctttgtctct ctgaccgtgg tgccaagaaa 3840
ggacgtctac aagataccaa ctgcagcact taaggtctct ggctcaagtc tgtacaatct 3900
tgcgctcaat gtcactattg atgtggaggt agacccgaag agcccgttgg tcaaatccct 3960
ttccaagtcc gacagtgggt actatgctaa tctcttctta catattgggc ttatgtccac 4020
tgtagataag aaggggaaga aagtgacatt tgacaagctg gaaaggaaga taaggagact 4080
tgatctatct gtagggctta gtgacgtgct cggaccttcc gtgcttgtaa aggcgagagg 4140
tgcacggact aagctgctgg cacctttctt ctctagcagt gggacagcct gctatcccat 4200
agcaaatgcc tctcctcagg tggccaagat actctggagc caaaccgcgt acctgcggag 4260
tgtaaaagtc attatccaag cgggcaccca gcgtgctgtc gcagtgaccg ccgaccacga 4320
ggttacctct actaagctgg agaaggggca taccattgcc aaatacaatc ccttcaagaa 4380
ataggctgca tctctgagat tgcactccgc ccatcttccc ggatcaccat gacactaaat 4440
aatgatctgt cttgattact tatagttagt tcgcctgtct atcaaattag aaaaaacacg 4500
ggtagaagag tctggatccc gaccggcaca ttcaggacac agcatgggct ccaaaccttc 4560
taccaggatc ccagcacctc taatgctgat cactcggatt atgctgatat tgagctgtat 4620
ccgtctgaca agctctcttg acggcaggcc ccttgcagct gcaggaattg tagtaacagg 4680
agataaggca gtcaatgtat acacctcgtc tcagacaggg tcaatcatag tcaagttgct 4740
cccgaatatg cccagagata aggaggcatg tgcaaaagcc ccattggagg catataacag 4800
aacactgact actctgctca ctcctcttgg cgactccatc cgcaagatcc aagggtctgt 4860
gtccacgtcc ggaggaggga gacaaggacg ccttataggt gctgttattg gcagtgtagc 4920
tcttggggtt gcaacagcgg cacagataac agcagctgcg gccctaatac aagccaaaca 4980
gaatgccgcc aacatcctcc ggcttaagga gagcattgct gcaaccaatg aagctgtgca 5040
tgaagtcacc gacggattat cacaactatc agtggcagtt gggaagatgc agcagtttgt 5100
caatgaccag ttaaataata cggcgcgaga attggactgt ataaaaatca cacaacaggt 5160
cggtgtagaa ctcaacctat acctaactga attgactaca gtattcgggc cacagatcac 5220
ctcccctgca ttaactcagc tgaccatcca ggcactttat aatttagctg gtggcaatat 5280
ggattactta ttaactaagt taggtatagg aaacaatcaa ctcagctcat taattggtag 5340
cggcctgatc actggttacc ctatactgta tgactcacat actcaactct tgggcataca 5400
agtaaatctg ccctcagtcg ggaacttaaa taatatgcgt gccacctatt tggagacctt 5460
atctgtaagt acaaccaaag gatatgcctc agcacttgtc ccgaaagtag tgacacaagt 5520
cggttctgtg atagaagagc ttgacacctc atactgtata gagtccgatc tggatttata 5580
ttgtactaga atagtgacat tccccatgtc cccaggtatt tattcctgtt tgagcggcaa 5640
cacatcagct tgcatgtatt caaagactga aggcgcactc actacgccgt atatggccct 5700
tagaggctca gttattgcca attgtaagat aacaacatgc agatgtacag accctcctgg 5760
tatcatatcg caaaattacg gagaagctgt atccctgata gatagacatt catgcaatgt 5820
cttatcatta gacggaataa ctctgaggct cagtggggaa tttgatgcaa cttatcaaaa 5880
gaacatctca atattagatt ctcaagtcat cgtgacaggc aatcttgata tatcaactga 5940
acttggaaac gtcaacaatt caatcagcaa tgccttggat aggttggcag aaagcaacag 6000
caagctagaa aaagtcaatg tcagactaac tagcacatct gctctcatta cctatattgt 6060
tctaactgtc atttccctaa ttttcggtgc acttagtctg gttttagcgt gttacctgat 6120
gtacaaacag aaggcacaac agaagacctt gctatggctt gggaataata ccctcgatca 6180
gatgagagcc accacaagag catgaatgca gataagaggt ggacagatac ccaacagcag 6240
cctgtgtgtc aattccgata acctgtcaag tagaagactt aagaaaaaat tactgggaac 6300
aagcaaccaa agagcaatgc acgggtagaa cggtcgggga ggccgtccct caatcgggag 6360
ccgggcctca caacatccgt tctaccgcat caccaatagc agttttcagt catggaccgc 6420
gcagttagcc aagttgcgct agagaatgat gaaagagagg caaagaatac atggcgcttg 6480
gtattccgga tcgcaatcct actctcaacg gtggtgacct tagccatctc tgcagccgcc 6540
cttgcatata gcatggaggc cagcacacct agcgatcttg taggcatacc gactgcgatc 6600
tctagagcag aggaaaagat tacatctgca ctcggttcca atcaagatgt agtagatagg 6660
atatataagc aggtggccct cgaatctcca ctggcattgc taaacaccga atctacaatt 6720
atgaacgcaa taacgtctct ctcttatcga atcaatgggg ccgcaaatag cagcggatgt 6780
ggagcaccca ttcatgatcc agattatatt ggaggaatag gtaaagaact tattgtagat 6840
gatgctagcg acgtcacatc atactatccc tctgcgttcc aagaacacct gaactttatc 6900
ccggcgccta ctacaggatc aggttgcact cggataccct catttgacat gagcgctacc 6960
cactactgtt atactcacaa tgtgatatta tctggctgca gagatcactc gcactcacat 7020
caatatttag cacttggtgt gcttcggaca tctgcaacag ggagggtatt cttttccact 7080
ctgcgttcca tcaatctgga tgacacccaa aatcggaagt cttgcagtgt gagtgcaacc 7140
cccttgggtt gtgatatgct gtgctctaaa gtcacagaga ctgaagaaga ggattataac 7200
tcagctatcc ccacgtcgat ggtacatgga aggttagggt tcgacggcca ataccacgag 7260
aaggacctag atgtcacaac actattcgag gactgggtgg caaactaccc aggagtaggg 7320
ggcgggtctt ttattgacaa ccgcgtatgg ttcccagttt acggagggct aaaacccaat 7380
tcgcccagtg acaccgcaca agaagggaaa tatgtaatat acaagcgata caatgacaca 7440
tgtccagatg agcaagatta tcagattcaa atggctaagt cttcatataa gcctgggcgg 7500
tttggaggga aacgcgtaca gcaggccatc ttatctatca aagtgtcaac atccttgggc 7560
gaggacccgg tactgactgt accgcccaac acagtaacac tcatgggggc cgaaggcaga 7620
gttctcacag tagggacatc tcatttcctt tatcagcgag ggtcatcata cttctcccct 7680
gccctactat atcctatgat agtcagcaac aaaacagcca ctcttcatag tccttataca 7740
ttcaatgcct tcactcgacc aggtagtgtc ccttgccagg cttcagcaag atgccctaac 7800
tcatgtgtta ccggagtcta tactgatcca tatcccttgg tcttctatag gaaccacacc 7860
ttgcgagggg tattcgggac gatgcttgat gataaacaag caagactcaa ccctgtatct 7920
gcagtatttg acagcatatc ccgcagtcgc ataacccggg tgagttcaag cagcaccaag 7980
gcagcataca caacatcaac atgttttaaa gttgtaaaga ccaataaaac ctattgtctc 8040
agcattgccg aaatatccaa taccctcttc ggggaattca gaatcgtccc tttactagtt 8100
gagattctca aggatgatgg ggttagagaa gccaggtcta gccggttgag tcaactgcga 8160
gagggttgga aagatgacat tgtatcacct atcttttgcg acgccaagaa tcaaactgaa 8220
taccggcgcg agctcgagtc ctacgctgcc agttggccat aatcagctag tgctaatgtg 8280
attagattaa gtcttgtcgg tagtcacttg attaagaaaa aatgtgggtg gtagcgggat 8340
ataaggcaaa acaactcaag gaggatagca cgggtaggac atggcgagct ccggtcccga 8400
gagggcggag catcagatta tcctaccaga gtcacacctg tcttcaccat tagtcaagca 8460
caaactactc tattactgga aattaactgg gctaccactc cctgacgagt gtgacttcga 8520
ccacctcatt ctcagccgac aatggaagaa aatacttgaa tcggcctccc ctgacactga 8580
gagaatgata aaacttggaa gggcagtgca ccagactctc aaccacaatt ccaagataac 8640
cggagtactc catcccaggt gtttagaaga attggctagt attgaggttc ctgactcaac 8700
caacaagttt cggaagatcg agaagaaaat ccaaattcac aacacaaggt atggagaact 8760
gttcacaaga ctgtgcacgc atgtagagaa gaaattgttg ggatcatctt ggtctaataa 8820
tgtcccccgg tcagaagagt tcaacagcat ccgtacagat ccggcattct ggtttcactc 8880
aaaatggtcc acaactaagt ttgcatggct ccatataaaa cagattcaaa ggcatctgat 8940
tgtggcagca agaacaaggt ccgcagccaa caaattggtg acgctgaccc ataaggtagg 9000
ccaagtcttt gttactcctg agcttgtcat tgtgacacat acagatgaga acaagttcac 9060
gtgtcttacc caggaacttg tgttgatgta tgcagatatg atggagggca gagatatggt 9120
caacataata tcatccacgg cggcacatct caggagccta tcagagaaaa ttgatgacat 9180
tctgcggtta gtagatgccc tggcaaaaga tctgggtaat caagtctacg atgttgtagc 9240
actcatggag ggatttgcat acggcgccgt ccagctgctt gagccgtcag gtacattcgc 9300
aggggatttc ttcgcattca acctgcagga gctcaaagac actttgatcg gcctccttcc 9360
taaggatata gcagaatctg tgactcacgc aatagccact gtattctctg gcttagaaca 9420
aaatcaagcg gctgagatgc tgtgcctgtt gcgtctatgg ggccacccat tacttgagtc 9480
ccgtattgcg gcaaaagcag taaggagcca aatgtgcgca ccaaaaatgg tagactttga 9540
tatgatcctc caggtattgt ctttctttaa aggaacaatc atcaacggat acagaaagaa 9600
gaatgcaggt gtttggccac gtgtcaaagt agatacgata tacgggaagg tcattgggca 9660
gctacacgct gattcagcgg agatttcaca cgatatcatg ttgagagagt acaagagttt 9720
atctgcgctt gaattcgagc catgtataga atacgaccct atcaccaatc tgagcatgtt 9780
tctaaaagac aaggcgatcg cacacccgaa agacaactgg ctcgccgcgt ttaggcgaaa 9840
ccttctctct gaggaccaga agaaacatgt aaaggaggca acctctacta accgtctctt 9900
gatagagttc ttagagtcaa atgattttga tccatataag gagatggaat atctgacgac 9960
ccttgagtac ctaagagatg acaatgtggc agtatcatac tcgctcaagg agaaggaagt 10020
gaaggttaat gggcggattt ttgctaagct aacaaagaaa ttaaggaact gtcaagtgat 10080
ggcggaaggg atcttagctg accagattgc acctttcttt caagggaatg gggtcattca 10140
ggatagcata tctttaacca agagtatgct agcgatgagt caattgtctt tcaacagcaa 10200
taagaaacgt atcactgact gcaaagaaag agtagcctca aaccgcaatc acgatcaaaa 10260
gagcaagaat cgtcggagag ttgccacttt tataacgact gacctgcaaa agtactgtct 10320
taattggaga tatcagacaa tcaaactgtt cgctcatgcc atcaatcagc tgatgggctt 10380
acctcacttc ttcgaatgga ttcatctaag actaatggat actacgatgt ttgtaggaga 10440
ccctttcaat cccccaagtg acccaactga ctgtgatctc tcaagagtcc caaatgatga 10500
catatatatt gtcagtgcta gagggggtat tgagggatta tgtcagaagc tatggacaat 10560
gatctcaatt gctgcaatcc aacttgctgc agcaagatca cattgtcgcg tcgcctgtat 10620
ggtacagggt gacaatcaag taatagctgt aacgagagag gtaaggtcag atgactcccc 10680
ggaaatggtg ttaacacaat tgcatcaagc cagtgataat ttcttcaagg aattgattca 10740
tgttaatcat ttgattggcc ataatttgaa ggatcgtgaa acaatcagat cagacacatt 10800
cttcatatac agcaaacgaa tattcaaaga tggagcaata ctcagtcaag tcctcaaaaa 10860
ttcatctaaa ttagtgctaa tatcaggcga ccttagtgaa aacaccgtaa tgtcctgtgc 10920
caacattgca tctactatag cacggctgtg cgagaacggg cttccaaagg atttctgtta 10980
ttacttaaac tacctgatga gttgcgtgca gacatacttt gattctgagt tttccatcac 11040
taacagctcg caccccgatt ctaaccagtc gtggattgaa gacatctctt ttgtgcactc 11100
atatgtcctg acccctgccc agctaggggg actgagcaac ctccaatact caaggctcta 11160
cacgaggaac atcggtgacc cgggaactac tgcttttgca gagatcaagc gattagaagc 11220
agtggggtta ctaagtccta gtattatgac taacatctta actaggccgc ctggaaatgg 11280
agattgggcc agtctgtgta acgaccctta ctctttcaat tttgagactg tcgcgagtcc 11340
aaatattgtc cttaagaaac atacacaaag agtcctattt gaaacttgtt caaatccctt 11400
attatctggc gtgcatacag aggataatga ggcagaagag aaggcgttgg ctgaattttt 11460
actcaatcaa gaagtaattc atccacgtgt cgcacatgct atcatggaag caagctctat 11520
aggtaggagg aagcagattc aagggcttgt tgacacaaca aacaccgtaa tcaagattgc 11580
attgactagg aggccacttg gcatcaagag gctgatgcgg atagttaact actcgagcat 11640
gcatgcaatg ctgtttagag acgatgtttt ctcatctaac aggtctaacc accccttagt 11700
ttcctctaat atgtgttctc tgacgctagc agactatgca cggaatagaa gctggtcacc 11760
attgacgggg ggtagaaaga tactgggtgt atctaatcct gatactatag aacttgtaga 11820
gggtgagatc cttagcgtca gcggaggatg cacaagatgt gacagcggag atgaacaatt 11880
cacttggttc catcttccga gcaatataga actgaccgat gacaccagca agaatcctcc 11940
gatgagagtg ccgtacctcg ggtcaaagac tcaagagagg agggccgcct cgcttgcgaa 12000
aatagctcat atgtcaccac atgtgaaagc tgctctaagg gcatcatccg tgttgatctg 12060
ggcttatgga gacaacgaag taaattggac tgctgctctt aaaattgcaa gatctcggtg 12120
caatataaac tcagagtatc ttcgactatt gtccccctta cccacagctg ggaatctcca 12180
acatagactg gatgacggca taactcagat gacattcacc cctgcatctc tctacagggt 12240
gtcaccttat attcacatat ccaatgattc tcaaaggtta ttcacggaag aaggagtcaa 12300
agagggaaat gtagtttatc agcaaatcat gctcttgggt ttatctctaa tcgaatcact 12360
cttcccgatg acgacaacca ggacatacga tgagatcaca ttgcacctcc acagtaaatt 12420
tagctgctgt atcagggaag caccggttgc agttcctttc gagttactcg ggatggcacc 12480
agaactaagg acagtgacct caaataagtt tatgtatgat cctagtcctg tatcggaggg 12540
tgactttgcg agacttgact tagctatctt taagagttat gagcttaatc tagaatcata 12600
tcccacaata gagctaatga acattctttc aatatccagc gggaagttaa tcggccagtc 12660
tgtggtttct tatgatgaag atacctccat aaagaatgac gccataatag tgtatgacaa 12720
cacccggaat tggatcagcg aagctcagaa ttcagatgtg gtccgcctat tcgagtatgc 12780
agcacttgaa gtgcttctcg actgttctta tcagctctac tatctgagag taagaggcct 12840
agacaatatc gtgttgtata tgagtgactt atataagaat atgccaggaa ttctactttc 12900
caacattgca gctacaatat ctcatcccat cattcattca agattgcatg cagtaggcct 12960
ggtcaatcac gacgggtcac accaacttgc agacacagat ttcatcgaaa tgtctgcaaa 13020
actattagtc tcttgcactc gacgcgtggt ctcaggttta tatgcaggga ataagtatga 13080
tctgctgttc ccgtctgtct tagatgataa cctgagtgag aagatgcttc agctgatatc 13140
tcggttatgc tgcctgtata cggtgctctt tgctacaaca agagagatcc cgaaaataag 13200
aggcttatct gcagaagaga agtgttcagt acttactgag tacctactgt cagatgctgt 13260
gaaaccatta cttagttctg agcaagtgag ctctatcatg tctcctaaca tagttacgtt 13320
cccagctaat ctatattaca tgtctcggaa gagccttaat ttgattaggg aaagagagga 13380
cagggacact atcttggcat tgttgttccc ccaagagcca ctacttgagt tccccttagt 13440
acaagatatt ggcgctcgag tgaaagatcc attcacccga caacctgcgg cgtttttaca 13500
agaattagat ttgagcgctc cagcaaggta tgacgcattt acacttagtc aggttcattc 13560
tgaacacaca tcaccaaatc cggaggacga ctacttagta cgatacctgt tcagaggaat 13620
agggaccgcg tcctcctctt ggtataaggc atctcacctt ctttctgtac ctgaggtcag 13680
atgtgcaagg cacgggaatt ccttatactt ggcagaagga agcggagcca ttatgagtct 13740
tctcgaactg catgtgccgc atgagactat ctattacaat acgctcttct caaacgagat 13800
gaacccccca cagcggcatt tcggaccgac cccaacacag tttctgaatt cagttgttta 13860
taggaatcta caggcggagg taccatgtaa ggatggattt gtccaggagt tccgtccatt 13920
atggagagag aatacagaag aaagcgatct gacctcagat aaagcagtgg gttacatcac 13980
atctgcagtg ccctaccggt ctgtatcatt gctgcactgt gacattgaga ttcctccagg 14040
atccaatcaa agcttactgg atcaactggc taccaatctg tctctgattg ccatgcattc 14100
tgtaagggag ggcggggtcg tgatcatcaa agtgttgtat gcaatgggat attacttcca 14160
tctactcatg aacttgttca ctccgtgttc tacgaaagga tatattctct ctaatggcta 14220
tgcatgtaga ggggatatgg agtgttacct ggtatttgtc atgggctatc gaggtgggcc 14280
tacatttgta catgaggtag tgaggatggc aaaaactcta gtgcagcggc acggtacact 14340
tttgtccaaa tcagatgaga tcacactgac taggttattt acctcacagc ggcagcgtgt 14400
aacagacatc ctatccagtc ctttaccgag actaataaag ttcttgagaa agaatatcga 14460
tactgcgcta attgaagccg ggggacaacc cgtccgtcca ttctgtgcag agagcttggt 14520
gaggacacta gcggacacaa ctcagatgac ccagatcatc gctagtcaca ttgacacagt 14580
cattcgatct gtgatctaca tggaggctga gggtgatctc gccgacacag tgttcttatt 14640
taccccctac aatctctcta cagacggtaa aaagagaaca tcacttaaac agtgcacaag 14700
gcagatctta gaggtcacaa tattgggtct tagagttgaa aatctcaata aagtaggtga 14760
tgtagtcagt ctagtactta aaggtatgat ttctctggag gacctgatcc ctctaagaac 14820
atacttgaag cgtagtacct gccctaagta tttgaagtct gttctaggta ttactaaact 14880
caaagaaatg tttacagaca cctctttatt atacttgact cgtgctcaac aaaaattcta 14940
catgaaaact ataggcaacg cagtcaaggg atactacagt aactgtgact cttaaagata 15000
atcacatatt aataggctcc ttttctagtt aactgagccc ttgttgattt aatgatacta 15060
tattagaaaa aagttgcact ccgatccttt aggactcgtg ttcgaattca aataattgtc 15120
ttagaaaaaa gttgcgcgta attgttcttg aatgtagtcc tgtcattcac caaatctttg 15180
tttggt 15186
<210> 2
<211> 1470
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgtcttctg tattcgatga gtacgagcag ctcctcgcgg ctcagactcg ccccaatgga 60
gctcatggcg gaggagagaa ggggagcacc ttaaaggtag aagtcccggt attcactctc 120
aacagtgatg acccagaaga tagatggaac tttgcagtgt tttgtcttcg gattgctgtt 180
agcgaggatg ccaacaaacc acttaggcaa ggtgctctca tatctctctt atgttcccac 240
tctcaagtga tgaggaacca tgttgccctt gcggggaaac agaatgaggc cacactggct 300
gttcttgaga tcgatggttt taccaacggc gtgccccagt tcaacaacag gagtggagtg 360
tctgaagaga gagcacagag atttatgatg atagcagggt ctctccctcg ggcatgcagc 420
aacggtaccc cgttcgtcac agctggggtt gaagatgatg caccagaaga cattactgat 480
accctggaga ggatcctctc tatccaggct caagtatggg tcacggtggc aaaggccatg 540
actgcatatg agacagcaga tgagtcagaa acaagaagaa tcaataagta catgcagcaa 600
ggcagggtcc agaagaagta catcctccac cccgtatgca ggagcgcaat ccaactcaca 660
atcagacagt ctctggcggt ccgcatcttt ttggttagcg agcttaagag aggccgcaac 720
acggcaggtg ggacctccac ctattacaac ttggtggggg atgtagactc atacatcagg 780
aacactgggc taactgcatt cttcctgaca cttaaatatg gaattaacac caagacatca 840
gcccttgcac ttagcagcct ctcaggcgat atccagaaaa tgaagcagct catgcgcttg 900
tatcggatga aaggagataa tgcgccgtac atgacattgc tcggtgacag tgaccagatg 960
agctttgcac ctgccgagta tgcacaactt tactcctttg ccatgggtat ggcatcagtc 1020
ctagataaag gaactagcaa ataccaattt gccagggact ttatgagcac atcattctgg 1080
agacttggag tagagtacgc tcaggctcaa ggaagtagca tcaatgagga tatggccgcc 1140
gagctaaagc taaccccagc agcaaggaga ggcctggcag ctgctgccca aagagtgtct 1200
gaggagacca gcagcatgga catgcccacc caacaagccg gggtcctcac tggactcagc 1260
gacggaggct cccaagcccc ccaaggtgca ctgaacagat cacaagggca accggacacc 1320
ggggatgggg agacccaatt tctggatctg atgagagcgg tggcaaatag catgagagaa 1380
gcgccaaact ctgcgcaggg cacccctcaa ccggggcctc ccccaacccc tgggccctct 1440
caagacaatg acaccgactg ggggtactga 1470
<210> 3
<211> 1188
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggccacct ttacagatgc ggagatcgac gagctatttg agaccagtgg aactgtcatt 60
gacagcataa ttacggccca gggaaaacca gtagagactg ttggaaggag tgcaatccca 120
caaggcaaaa ctaaggcttt gagcgcagca tgggagaagc atgggagcat ccagtcacca 180
gccagccaag acacccctga tcgacaggac agatcagata aacaactgtc cacacccgag 240
caagcgagtc caaacgacag ccccccagcc acatccactg accagcctcc cactcaggct 300
gcagatgagg ccggcgatac acagctcaag accggagcaa gcaactctct gctgtcgatg 360
cttgataaac tcagcaataa gtcatctaat gctaaaaagg gcccagggtc gagccctcaa 420
gaaaggcatc atcaacgtct gactcaacaa caggggagtc aacaaagccg cggaaacagc 480
caagagagac cgcagaacca ggccaaggcc atccctggaa accaggtcac agacgcgaac 540
acagcatatc atggacaatg ggaggagtca caactatcag ctggtgcaac ccatcatgct 600
ctccgatcag agcagagcca agacaatact cctgcacctg tggatcatgt ccagctacct 660
gtcgactttg tgcaggcgat gatgtctatg atggaggcga tatcacagag ggtaagtaaa 720
gttgactatc agctggacct tgtcttgaaa cagacatctt ctatccccat gatgcggtct 780
gaaatccagc agctgaaaac gtctgttgcg gtcatggaag ccaatttggg catgatgaag 840
atcctggacc ctggttgtgc caacgtttca tctctaagtg atctacgggc agttgcccga 900
tcccacccgg ttttaatttc tggccccgga gacccatctc cttatgtgac ccaagggggc 960
gaaatggcac tcaataaact ttcgcaaccg gtgcaacacc cctctgaatt gattaaaccc 1020
gccacggcaa gcgggcctga tataggagtg gagaaagaca ctgtccgtgc attgatcatg 1080
tcacgcccta tgcatccgag ctcttcagct aggctcttga gcaaactgga cgcagccgga 1140
tcgattgagg aaatcagaaa aatcaagcgc cttgcactga atggctaa 1188
<210> 4
<211> 6615
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggcgagct ccggtcccga gagggcggag catcagatta tcctaccaga gtcacacctg 60
tcttcaccat tagtcaagca caaactactc tattactgga aattaactgg gctaccactc 120
cctgacgagt gtgacttcga ccacctcatt ctcagccgac aatggaagaa aatacttgaa 180
tcggcctccc ctgacactga gagaatgata aaacttggaa gggcagtgca ccagactctc 240
aaccacaatt ccaagataac cggagtactc catcccaggt gtttagaaga attggctagt 300
attgaggttc ctgactcaac caacaagttt cggaagatcg agaagaaaat ccaaattcac 360
aacacaaggt atggagaact gttcacaaga ctgtgcacgc atgtagagaa gaaattgttg 420
ggatcatctt ggtctaataa tgtcccccgg tcagaagagt tcaacagcat ccgtacagat 480
ccggcattct ggtttcactc aaaatggtcc acaactaagt ttgcatggct ccatataaaa 540
cagattcaaa ggcatctgat tgtggcagca agaacaaggt ccgcagccaa caaattggtg 600
acgctgaccc ataaggtagg ccaagtcttt gttactcctg agcttgtcat tgtgacacat 660
acagatgaga acaagttcac gtgtcttacc caggaacttg tgttgatgta tgcagatatg 720
atggagggca gagatatggt caacataata tcatccacgg cggcacatct caggagccta 780
tcagagaaaa ttgatgacat tctgcggtta gtagatgccc tggcaaaaga tctgggtaat 840
caagtctacg atgttgtagc actcatggag ggatttgcat acggcgccgt ccagctgctt 900
gagccgtcag gtacattcgc aggggatttc ttcgcattca acctgcagga gctcaaagac 960
actttgatcg gcctccttcc taaggatata gcagaatctg tgactcacgc aatagccact 1020
gtattctctg gcttagaaca aaatcaagcg gctgagatgc tgtgcctgtt gcgtctatgg 1080
ggccacccat tacttgagtc ccgtattgcg gcaaaagcag taaggagcca aatgtgcgca 1140
ccaaaaatgg tagactttga tatgatcctc caggtattgt ctttctttaa aggaacaatc 1200
atcaacggat acagaaagaa gaatgcaggt gtttggccac gtgtcaaagt agatacgata 1260
tacgggaagg tcattgggca gctacacgct gattcagcgg agatttcaca cgatatcatg 1320
ttgagagagt acaagagttt atctgcgctt gaattcgagc catgtataga atacgaccct 1380
atcaccaatc tgagcatgtt tctaaaagac aaggcgatcg cacacccgaa agacaactgg 1440
ctcgccgcgt ttaggcgaaa ccttctctct gaggaccaga agaaacatgt aaaggaggca 1500
acctctacta accgtctctt gatagagttc ttagagtcaa atgattttga tccatataag 1560
gagatggaat atctgacgac ccttgagtac ctaagagatg acaatgtggc agtatcatac 1620
tcgctcaagg agaaggaagt gaaggttaat gggcggattt ttgctaagct aacaaagaaa 1680
ttaaggaact gtcaagtgat ggcggaaggg atcttagctg accagattgc acctttcttt 1740
caagggaatg gggtcattca ggatagcata tctttaacca agagtatgct agcgatgagt 1800
caattgtctt tcaacagcaa taagaaacgt atcactgact gcaaagaaag agtagcctca 1860
aaccgcaatc acgatcaaaa gagcaagaat cgtcggagag ttgccacttt tataacgact 1920
gacctgcaaa agtactgtct taattggaga tatcagacaa tcaaactgtt cgctcatgcc 1980
atcaatcagc tgatgggctt acctcacttc ttcgaatgga ttcatctaag actaatggat 2040
actacgatgt ttgtaggaga ccctttcaat cccccaagtg acccaactga ctgtgatctc 2100
tcaagagtcc caaatgatga catatatatt gtcagtgcta gagggggtat tgagggatta 2160
tgtcagaagc tatggacaat gatctcaatt gctgcaatcc aacttgctgc agcaagatca 2220
cattgtcgcg tcgcctgtat ggtacagggt gacaatcaag taatagctgt aacgagagag 2280
gtaaggtcag atgactcccc ggaaatggtg ttaacacaat tgcatcaagc cagtgataat 2340
ttcttcaagg aattgattca tgttaatcat ttgattggcc ataatttgaa ggatcgtgaa 2400
acaatcagat cagacacatt cttcatatac agcaaacgaa tattcaaaga tggagcaata 2460
ctcagtcaag tcctcaaaaa ttcatctaaa ttagtgctaa tatcaggcga ccttagtgaa 2520
aacaccgtaa tgtcctgtgc caacattgca tctactatag cacggctgtg cgagaacggg 2580
cttccaaagg atttctgtta ttacttaaac tacctgatga gttgcgtgca gacatacttt 2640
gattctgagt tttccatcac taacagctcg caccccgatt ctaaccagtc gtggattgaa 2700
gacatctctt ttgtgcactc atatgtcctg acccctgccc agctaggggg actgagcaac 2760
ctccaatact caaggctcta cacgaggaac atcggtgacc cgggaactac tgcttttgca 2820
gagatcaagc gattagaagc agtggggtta ctaagtccta gtattatgac taacatctta 2880
actaggccgc ctggaaatgg agattgggcc agtctgtgta acgaccctta ctctttcaat 2940
tttgagactg tcgcgagtcc aaatattgtc cttaagaaac atacacaaag agtcctattt 3000
gaaacttgtt caaatccctt attatctggc gtgcatacag aggataatga ggcagaagag 3060
aaggcgttgg ctgaattttt actcaatcaa gaagtaattc atccacgtgt cgcacatgct 3120
atcatggaag caagctctat aggtaggagg aagcagattc aagggcttgt tgacacaaca 3180
aacaccgtaa tcaagattgc attgactagg aggccacttg gcatcaagag gctgatgcgg 3240
atagttaact actcgagcat gcatgcaatg ctgtttagag acgatgtttt ctcatctaac 3300
aggtctaacc accccttagt ttcctctaat atgtgttctc tgacgctagc agactatgca 3360
cggaatagaa gctggtcacc attgacgggg ggtagaaaga tactgggtgt atctaatcct 3420
gatactatag aacttgtaga gggtgagatc cttagcgtca gcggaggatg cacaagatgt 3480
gacagcggag atgaacaatt cacttggttc catcttccga gcaatataga actgaccgat 3540
gacaccagca agaatcctcc gatgagagtg ccgtacctcg ggtcaaagac tcaagagagg 3600
agggccgcct cgcttgcgaa aatagctcat atgtcaccac atgtgaaagc tgctctaagg 3660
gcatcatccg tgttgatctg ggcttatgga gacaacgaag taaattggac tgctgctctt 3720
aaaattgcaa gatctcggtg caatataaac tcagagtatc ttcgactatt gtccccctta 3780
cccacagctg ggaatctcca acatagactg gatgacggca taactcagat gacattcacc 3840
cctgcatctc tctacagggt gtcaccttat attcacatat ccaatgattc tcaaaggtta 3900
ttcacggaag aaggagtcaa agagggaaat gtagtttatc agcaaatcat gctcttgggt 3960
ttatctctaa tcgaatcact cttcccgatg acgacaacca ggacatacga tgagatcaca 4020
ttgcacctcc acagtaaatt tagctgctgt atcagggaag caccggttgc agttcctttc 4080
gagttactcg ggatggcacc agaactaagg acagtgacct caaataagtt tatgtatgat 4140
cctagtcctg tatcggaggg tgactttgcg agacttgact tagctatctt taagagttat 4200
gagcttaatc tagaatcata tcccacaata gagctaatga acattctttc aatatccagc 4260
gggaagttaa tcggccagtc tgtggtttct tatgatgaag atacctccat aaagaatgac 4320
gccataatag tgtatgacaa cacccggaat tggatcagcg aagctcagaa ttcagatgtg 4380
gtccgcctat tcgagtatgc agcacttgaa gtgcttctcg actgttctta tcagctctac 4440
tatctgagag taagaggcct agacaatatc gtgttgtata tgagtgactt atataagaat 4500
atgccaggaa ttctactttc caacattgca gctacaatat ctcatcccat cattcattca 4560
agattgcatg cagtaggcct ggtcaatcac gacgggtcac accaacttgc agacacagat 4620
ttcatcgaaa tgtctgcaaa actattagtc tcttgcactc gacgcgtggt ctcaggttta 4680
tatgcaggga ataagtatga tctgctgttc ccgtctgtct tagatgataa cctgagtgag 4740
aagatgcttc agctgatatc tcggttatgc tgcctgtata cggtgctctt tgctacaaca 4800
agagagatcc cgaaaataag aggcttatct gcagaagaga agtgttcagt acttactgag 4860
tacctactgt cagatgctgt gaaaccatta cttagttctg agcaagtgag ctctatcatg 4920
tctcctaaca tagttacgtt cccagctaat ctatattaca tgtctcggaa gagccttaat 4980
ttgattaggg aaagagagga cagggacact atcttggcat tgttgttccc ccaagagcca 5040
ctacttgagt tccccttagt acaagatatt ggcgctcgag tgaaagatcc attcacccga 5100
caacctgcgg cgtttttaca agaattagat ttgagcgctc cagcaaggta tgacgcattt 5160
acacttagtc aggttcattc tgaacacaca tcaccaaatc cggaggacga ctacttagta 5220
cgatacctgt tcagaggaat agggaccgcg tcctcctctt ggtataaggc atctcacctt 5280
ctttctgtac ctgaggtcag atgtgcaagg cacgggaatt ccttatactt ggcagaagga 5340
agcggagcca ttatgagtct tctcgaactg catgtgccgc atgagactat ctattacaat 5400
acgctcttct caaacgagat gaacccccca cagcggcatt tcggaccgac cccaacacag 5460
tttctgaatt cagttgttta taggaatcta caggcggagg taccatgtaa ggatggattt 5520
gtccaggagt tccgtccatt atggagagag aatacagaag aaagcgatct gacctcagat 5580
aaagcagtgg gttacatcac atctgcagtg ccctaccggt ctgtatcatt gctgcactgt 5640
gacattgaga ttcctccagg atccaatcaa agcttactgg atcaactggc taccaatctg 5700
tctctgattg ccatgcattc tgtaagggag ggcggggtcg tgatcatcaa agtgttgtat 5760
gcaatgggat attacttcca tctactcatg aacttgttca ctccgtgttc tacgaaagga 5820
tatattctct ctaatggcta tgcatgtaga ggggatatgg agtgttacct ggtatttgtc 5880
atgggctatc gaggtgggcc tacatttgta catgaggtag tgaggatggc aaaaactcta 5940
gtgcagcggc acggtacact tttgtccaaa tcagatgaga tcacactgac taggttattt 6000
acctcacagc ggcagcgtgt aacagacatc ctatccagtc ctttaccgag actaataaag 6060
ttcttgagaa agaatatcga tactgcgcta attgaagccg ggggacaacc cgtccgtcca 6120
ttctgtgcag agagcttggt gaggacacta gcggacacaa ctcagatgac ccagatcatc 6180
gctagtcaca ttgacacagt cattcgatct gtgatctaca tggaggctga gggtgatctc 6240
gccgacacag tgttcttatt taccccctac aatctctcta cagacggtaa aaagagaaca 6300
tcacttaaac agtgcacaag gcagatctta gaggtcacaa tattgggtct tagagttgaa 6360
aatctcaata aagtaggtga tgtagtcagt ctagtactta aaggtatgat ttctctggag 6420
gacctgatcc ctctaagaac atacttgaag cgtagtacct gccctaagta tttgaagtct 6480
gttctaggta ttactaaact caaagaaatg tttacagaca cctctttatt atacttgact 6540
cgtgctcaac aaaaattcta catgaaaact ataggcaacg cagtcaaggg atactacagt 6600
aactgtgact cttaa 6615
<210> 5
<211> 2002
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
acgggtagaa cggtcgggga ggccgtccct caatcgggag ccgggcctca caacatccgt 60
tctaccgcat caccaatagc agttttcagt catggaccgc gcagttagcc aagttgcgct 120
agagaatgat gaaagagagg caaagaatac atggcgcttg gtattccgga tcgcaatcct 180
actctcaacg gtggtgacct tagccatctc tgcagccgcc cttgcatata gcatggaggc 240
cagcacacct agcgatcttg taggcatacc gactgcgatc tctagagcag aggaaaagat 300
tacatctgca ctcggttcca atcaagatgt agtagatagg atatataagc aggtggccct 360
cgaatctcca ctggcattgc taaacaccga atctacaatt atgaacgcaa taacgtctct 420
ctcttatcga atcaatgggg ccgcaaatag cagcggatgt ggagcaccca ttcatgatcc 480
agattatatt ggaggaatag gtaaagaact tattgtagat gatgctagcg acgtcacatc 540
atactatccc tctgcgttcc aagaacacct gaactttatc ccggcgccta ctacaggatc 600
aggttgcact cggataccct catttgacat gagcgctacc cactactgtt atactcacaa 660
tgtgatatta tctggctgca gagatcactc gcactcacat caatatttag cacttggtgt 720
gcttcggaca tctgcaacag ggagggtatt cttttccact ctgcgttcca tcaatctgga 780
tgacacccaa aatcggaagt cttgcagtgt gagtgcaacc cccttgggtt gtgatatgct 840
gtgctctaaa gtcacagaga ctgaagaaga ggattataac tcagctatcc ccacgtcgat 900
ggtacatgga aggttagggt tcgacggcca ataccacgag aaggacctag atgtcacaac 960
actattcgag gactgggtgg caaactaccc aggagtaggg ggcgggtctt ttattgacaa 1020
ccgcgtatgg ttcccagttt acggagggct aaaacccaat tcgcccagtg acaccgcaca 1080
agaagggaaa tatgtaatat acaagcgata caatgacaca tgtccagatg agcaagatta 1140
tcagattcaa atggctaagt cttcatataa gcctgggcgg tttggaggga aacgcgtaca 1200
gcaggccatc ttatctatca aagtgtcaac atccttgggc gaggacccgg tactgactgt 1260
accgcccaac acagtaacac tcatgggggc cgaaggcaga gttctcacag tagggacatc 1320
tcatttcctt tatcagcgag ggtcatcata cttctcccct gccctactat atcctatgat 1380
agtcagcaac aaaacagcca ctcttcatag tccttataca ttcaatgcct tcactcgacc 1440
aggtagtgtc ccttgccagg cttcagcaag atgccctaac tcatgtgtta ccggagtcta 1500
tactgatcca tatcccttgg tcttctatag gaaccacacc ttgcgagggg tattcgggac 1560
gatgcttgat gataaacaag caagactcaa ccctgtatct gcagtatttg acagcatatc 1620
ccgcagtcgc ataacccggg tgagttcaag cagcaccaag gcagcataca caacatcaac 1680
atgttttaaa gttgtaaaga ccaataaaac ctattgtctc agcattgccg aaatatccaa 1740
taccctcttc ggggaattca gaatcgtccc tttactagtt gagattctca aggatgatgg 1800
ggttagagaa gccaggtcta gccggttgag tcaactgcga gagggttgga aagatgacat 1860
tgtatcacct atcttttgcg acgccaagaa tcaaactgaa taccggcgcg agctcgagtc 1920
ctacgctgcc agttggccat aatcagctag tgctaatgtg attagattaa gtcttgtcgg 1980
tagtcacttg attaagaaaa aa 2002
Claims (7)
1.一株基因Ⅶ型新城疫病毒弱毒株，其特征在于，所述的基因Ⅶ型新城疫病毒弱毒株是将保藏编号为CCTCC NO:V201968的新城疫病毒株的HN蛋白的340位、342位、347位和353位氨基酸分别突变为组氨酸H、天冬酰胺N、赖氨酸K和精氨酸R；再通过遗传拯救构建的。
2.权利要求1所述的基因Ⅶ型新城疫病毒弱毒株的构建方法，其特征在于，所述的方法包括如下的步骤：
1)分别将基因Ⅶ型新城疫病毒的NP蛋白、P蛋白和L蛋白基因分别连接到载体构建辅助质粒；
2)构建携带有基因Ⅶ型新城疫病毒的全基因组的重组载体，且HN蛋白的340位、342位、347位和353位氨基酸分别突变为H、N、K和R；
3)分别将全基因组载体与3个辅助质粒共转染宿主细胞，拯救获得新城疫病毒基因Ⅶ型弱毒株。
3.如权利要求2所述的构建方法，其特征在于，所述的1)中的载体为PCI-neo载体。
4.如权利要求2所述的构建方法，其特征在于，所述的2)中的辅助质粒为携带有基因Ⅶ型新城疫病毒的NP基因、P基因和L基因的重组质粒。
5.如权利要求2所述的构建方法，其特征在于，所述的3)中的宿主细胞为BSR细胞。
6.权利要求1所述的基因Ⅶ型新城疫病毒弱毒株在制备疫苗中的应用。
7.一种基因Ⅶ型新城疫病毒疫苗，其特征在于，所述的基因Ⅶ型新城疫病毒疫苗所用的抗原包含有权利要求1所述的基因Ⅶ型新城疫病毒弱毒株。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5733556A (en) * | 1995-10-18 | 1998-03-31 | Akzo Nobel N.V. | Newcastle disease virus combination vaccine |
| CN103585643A (zh) * | 2009-04-03 | 2014-02-19 | 梅里亚有限公司 | 运载新城疫病毒的禽疫苗 |
| CN107158369A (zh) * | 2017-05-27 | 2017-09-15 | 山东信得科技股份有限公司 | 一种使用构建的基因vii型新城疫病毒弱毒株制备的疫苗 |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5733556A (en) * | 1995-10-18 | 1998-03-31 | Akzo Nobel N.V. | Newcastle disease virus combination vaccine |
| CN103585643A (zh) * | 2009-04-03 | 2014-02-19 | 梅里亚有限公司 | 运载新城疫病毒的禽疫苗 |
| CN107158369A (zh) * | 2017-05-27 | 2017-09-15 | 山东信得科技股份有限公司 | 一种使用构建的基因vii型新城疫病毒弱毒株制备的疫苗 |
Non-Patent Citations (1)
| Title |
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| DENNIS V UMALI等: "Characterization of complete genome sequence of genotype VI and VII velogenic Newcastle disease virus from Japan", 《VIRUS GENES》, vol. 49, no. 1, pages 89 - 99 * |
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