CN112111467B - 一种基因vii型新城疫标记疫苗株及其制备方法与应用 - Google Patents

一种基因vii型新城疫标记疫苗株及其制备方法与应用 Download PDF

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CN112111467B
CN112111467B CN202011036432.XA CN202011036432A CN112111467B CN 112111467 B CN112111467 B CN 112111467B CN 202011036432 A CN202011036432 A CN 202011036432A CN 112111467 B CN112111467 B CN 112111467B
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卢琴
温国元
王国康
邵华斌
罗青平
商雨
王红琳
罗玲
张蓉蓉
汪宏才
张腾飞
张文婷
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Institute of Animal Science and Veterinary of Hubei Academy of Agricultural Sciences
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Abstract

本发明公开了一种基因VII型新城疫标记疫苗株及其制备方法与应用。本发明利用反向遗传操作技术，以血清2型禽副粘病毒Y2株的全基因组转录质粒为骨架，将其F和HN基因的胞外域分别替换为基因VII型新城疫病毒株的F和HN基因的胞外域，通过病毒拯救，获得重组嵌合病毒。该疫苗株已保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:202052。该嵌合病毒可以用于我国主要流行的基因VII新城疫的防控。同时，该嵌合病毒不含有新城疫病毒NP蛋白，其免疫鸡后不会产生针对新城疫病毒NP蛋白的抗体。以常规的新城疫病毒NP抗体ELISA检测方法对rY2‑HB疫苗株免疫鸡群进行抗体检测，可实现特异检测临床野毒株产生的NP抗体，排除疫苗毒株产生的抗体干扰，有利于新城疫的监测与净化。

Description

一种基因VII型新城疫标记疫苗株及其制备方法与应用
技术领域
本发明属疫苗基因工程领域，具体涉及一种基因VII型新城疫标记疫苗株副粘病毒II型rY2-HB株及其制备方法。更具体地，本发明涉及到利用反向遗传操作技术，将血清2型禽副粘病毒Y2株的F和HN基因胞外域分别替换为基因VII型新城疫病毒株的F和HN基因胞外域，获得嵌合病毒rY2-HB株，以及该毒株在基因VII新城疫标记疫苗方面的应用。
背景技术
新城疫(Newcastle disease，ND)是由新城疫病毒(NDV)引起禽的一种高度接触性传染病，严重危害全球养禽业。其典型症状为高热、呼吸困难、下痢、神经紊乱、黏膜和浆膜出血。我国ND的防控以疫苗免疫预防为主。LaSota、V4、Clone-30等传统ND疫苗株在新城疫防控中起到了重要作用。但由于近年来在我国流行的ND以基因VII型为主，与当前广泛使用的LaSota株等疫苗在抗原匹配性方面存在明显的差异，以致临床带毒现象比较普遍。基因VII型ND疫苗的成功研发为我国基因VII ND的防控提供了有利支撑。但此类疫苗在接种后会对ND的血清学监测带来干扰，疫苗接种后动物体内会产生ND抗体，临床野毒感染后动物体内同样会产生特异性抗体。常规的血清学方法，如血凝抑制、ELISA、胶体金等方法，都不能很有效的区分疫苗免疫和临床野毒感染。因此，研发ND标记疫苗及配套鉴别诊断技术是当前ND防控技术研究的重要方向。
NDV属副黏病毒科、禽副粘病毒属。禽副粘病毒(Avian Paramyxovirus,APMV)的血清型有9个，APMV-I～APMV-IX，其中新城疫病毒是APMV-I的唯一成员。APMV基因组按照3'-NP-P-M-F-HN-L-5'，依次编码6种结构蛋白，即核衣壳蛋白(nucleoprotein，NP)、磷蛋白(phosphoprotein，P)、基质蛋白(matrix protein，M)、融合蛋白(fusion protein，F)、血凝素-神经氨酸酶蛋白(haemaggulatinin-neuraminidase，HN)和大分子量聚合酶蛋白(Largeprotein，L)。NP蛋白的基因编码区长度均为1467bp，编码489个氨基酸，相对分子质量为56kDa。NP蛋白与P和L蛋白结合包裹基因组RNA形成一个螺旋的核糖核蛋白(RNP)复合体，参与病毒基因转录和基因组复制。NP蛋白在APMV的各个血清型中高度保守，含量丰富，具有较好的免疫原性，且NP蛋白抗体不具有病毒中和效果。因此，NP蛋白是设计ND标记疫苗的首选靶标。
NDV反向遗传学的快速发展使得对NDV进行点突变修饰、插入外源基因、基因片段互换等基因操作成为可能。关于新城疫标记疫苗和反向遗传操作技术的文献有一定的报道，但均未涉及到通过基因替换的方法研发新城疫标记疫苗的报道。
发明内容
本发明为解决上述技术问题提供一种基因VII型新城疫标记疫苗株及其制备方法与应用。
为实现上述发明目的，本发明的技术方案如下：
一种基因VII型新城疫标记疫苗株副粘病毒II型rY2-HB株，该毒株保藏于中国典型培养物保藏中心，保藏地址是，中国，武汉，武汉大学，保藏编号为CCTCC NO：V202052，保藏时间为8月28日。
优选地，所述血清2型禽副粘病毒Y2株的转录质粒的构建方法包括以下步骤：a.对12株禽副黏病毒2型的全基因组序列进行比对分析，选取较为保守的F8株为模板，将该毒株基因组中10处特有的核苷酸位点突变为其他毒株共有的一致序列，获得一株新的禽副粘病毒Y2株的全基因组序列；b.通过基因合成和克隆的方法，获得Y2株的全基因组转录质粒。
优选地，所述12株禽副黏病毒2型为在GenBank中下载，登录号分别为KT071756、KT071757、KT071755、HQ896023、HQ896024、EU338414、HM159993、HM159994、HM159995、LC187305、LC187306和NC_039230。
优选地，F8株特有的10处氨基酸突变具体为T1440C，C2142T，T2709C，C5552T，A6397G，A6868G，G7768T，A11753C，G14409A，T14480C。
优选地，所述的禽副粘病毒2型Y2株的全基因组基因序列为SEQ ID NO：1。
所述的基因VII型新城疫标记疫苗株副粘病毒II型rY2-HB株在基因VII型新城疫标记疫苗方面的应用。
优选地，利用新城疫病毒NP抗体ELISA检测试剂盒对所述VII型新城疫标记疫苗株副粘病毒II型rY2-HB株免疫鸡群后进行抗体检测。
本发明的有益效果是：
1)本发明以血清2型禽副粘病毒Y2株的全基因组转录质粒为基础，将其F和HN基因的胞外域分别替换为基因VII型新城疫病毒HB0901株的F和HN基因胞外域，通过病毒拯救，获得基因VII型新城疫标记疫苗株副粘病毒II型rY2-HB株。由于该毒株含有基因VII型新城疫病毒HB0901株的F和HN基因胞外域，这两个区域为新城疫病毒的关键免疫保护区域。因此，该疫苗株可以用于预防基因VII型新城疫。
2)与其他传统疫苗株相比，rY2-HB株不含有新城疫病毒NP蛋白，其免疫鸡后不会产生针对新城疫病毒NP蛋白的抗体。利用新城疫病毒NP抗体ELISA检测试剂盒对rY2-HB株免疫鸡群进行抗体检测，可特异检测出临床野毒株产生的NP抗体，排除rY2-HB疫苗株免疫产生的抗体干扰。因此，rY2-HB株可作为基因VII新城疫标记疫苗，实现疫苗免疫与野毒感染的鉴别诊断，可用于临床新城疫的监测与净化。
附图说明
图1是Y2株的全基因组转录质粒pY2的BamHI酶切鉴定结果。
图2是辅助质粒pVAX-NP和pVAX-P的HindIII酶切鉴定结果。
图3是辅助质粒pcDNA-L的SalI酶切鉴定结果。
图4是基因VII型新城疫标记疫苗株副粘病毒II型rY2-HB株的基因组结构示意图。
图5是全基因组转录质粒pY2-HB的HindIII酶切鉴定结果。
具体实施方式
以下结合附图和实施例进一步对本发明进行说明，但本发明的内容不仅仅局限于下面的实施例。
本发明提供一种基因VII型新城疫标记疫苗株副粘病毒II型rY2-HB株，该毒株保藏于中国典型培养物保藏中心，保藏地址是，中国，武汉，武汉大学，保藏编号为CCTCC NO：202052，保藏日期为2020年8月28日。
所述基因VII型新城疫标记疫苗株副粘病毒II型rY2-HB株是以血清2型禽副粘病毒Y2株为母本株，将其F和HN基因的胞外域分别替换为基因VII型新城疫病毒株的F和HN基因胞外域，得到所述的rY2-HB株。本发明选用的是HB0901株，可以理解的是，还可以为ZJ1株、NA-1株或其他基因VII型新城疫病毒株。
本发明还提供所述的基因VII型新城疫标记疫苗株副粘病毒II型rY2-HB株的制备方法，包括以下步骤：a.构建血清2型禽副粘病毒Y2株的转录质粒；b.将所述Y2株转录质粒中F和HN基因的胞外域分别替换为基因VII型新城疫病毒HB0901株的F和HN基因胞外域；c.将胞外域替换后的转录质粒与三个辅助质粒共同转染宿主细胞，获得基因VII型新城疫标记疫苗株副粘病毒II型rY2-HB株。所述的禽副粘病毒2型Y2株的全基因组基因序列为SEQID NO：1。
本发明还提供所述的基因VII型新城疫标记疫苗株副粘病毒II型rY2-HB株在基因VII型新城疫标记疫苗方面的应用。
下面以具体实施例进行说明。
实施例1禽副粘病毒2型(APMV-2)Y2株的全基因组转录质粒构建
在GenBank中下载12株禽副粘病毒2型的全基因组序列，登录号分别为KT071756、KT071757、KT071755、HQ896023、HQ896024、EU338414、HM159993、HM159994、HM159995、LC187305、LC187306和NC039230。对这些序列进行核苷酸序列比对分析后，选取较为保守的F8株(HQ896023)为模板，将该毒株基因组中特有的10处氨基酸突变为其他毒株共有的一致序列(T1440C，C2142T，T2709C，C5552T，A6397G，A6868G，G7768T，A11753C，G14409A，T14480C)，获得一株新的副粘病毒Y2株的全基因组序列。通过基因合成和克隆的方法，构建获得Y2株的全基因组质粒。该质粒包含了Y2株的全基因组序列、T7 RNA聚合酶启动子序列、T7 RNA聚合酶终止子序列、丁型肝炎病毒核酶序列和低拷贝质粒序列等，其主要功能是转录表达并准确切割出Y2株的全基因组RNA序列。其具体构建策略为：首先将全基因组序列共分为编号从A到I的9个小片段，进行人工合成。其中在A片段的上游加入了T7启动子，I片段的下游加入了丁型肝炎病毒核酶序列和T7终止子。通过Overlap PCR将9个小片段按顺序组合成3个中片段，通过In-fusion PCR克隆的方式，将3个中片段依次连入克隆质粒中，获得转录质粒pY2。
1.1转录质粒的连接与鉴定
将人工合成并鉴定正确的9个PCR片段分先后以overlap PCR克隆的方式按顺序组合成3个中片段。反向PCR的方式线性化载体，同时通过PCR在插入片段上加入同源臂，参照In-fusion克隆试剂盒的操作说明将3个中片段依次插入载体，最终获得质粒pY2。酶切结果见图1。
1.2辅助质粒的构建
以转录质粒为PCR模板进行PCR扩增，分别得到核蛋白NP、磷蛋白P和大聚合酶蛋白L的3个基因全序列，以酶切连接的方式，将3个片段分别克隆至真核表达质粒。为增加蛋白的表达量，在起始密码子前加入Kozak序列。对PCR检测为阳性的菌液进行质粒提取及酶切鉴定。结果表明，所构建的三个辅助质粒pcDNA-NP、pcDNA-P和pcDNA-L均正确，结果见图2、图3。
实施例2rY2-HB毒株的全基因组转录质粒构建与病毒拯救
以重组质粒pY2为模板，通过In-fusion的方法将Y2株的F和HN基因胞外域替换为NDV基因VII型HB0901株的F和HN基因胞外域。具体替换的位置如下：NDV HB0901株F基因的胞外域(1-1506nt)替换Y2株F基因的胞外域(1-1455nt)，NDV HB0901株HN基因的胞外域(139-1713nt)替换Y2株HN基因的胞外域(130-1740nt)。构建示意图见图4。具体构建方案如下。
2.1新城疫病毒HB0901株F、HN基因胞外域的PCR扩增
以新城疫病毒HB0901株鸡胚尿囊液为对象，参照试剂盒说明书进行病毒基因组RNA的提取，反转录为cDNA。以cDNA为模板，采用设计好的引物，PCR扩增出F、HN基因的胞外域。琼脂糖凝胶电泳检测，将特异的阳性条带以DNA纯化试剂盒进行纯化回收，以相应的内切酶进行酶切鉴定，鉴定正确的PCR片段保存于-20度。
2.2新城疫病毒HB0901株F基因改造
由于新城疫病毒HB0901株为强毒株，需要将HB0901病毒F蛋白裂解位点GRRQKR↓F突变为LaSota病毒的F蛋白裂解位点GGRQGR↓L。通过重叠PCR的方法将F裂解位点进行突变改造。具体使用的引物为，上游引物：gggagacaaggacgccttATAGGTGCAGTTATTGGCAGTGTAG，下游引物：aaggcgtccttgtctccctTCCTCCGGACGTGGATACAG。
2.3重组质粒pY2-HB的构建以及病毒rY2-HB的拯救
以重组质粒pY2为模板，通过In-fusion的方法，用获得的HB0901株F和HN基因胞外域替换pY2质粒的相应区域。对阳性质粒进行酶切鉴定，结果见图5。将重组质粒pY2-HB质粒与三个辅助质粒共转染宿主细胞，收获细胞培养物并进行病毒鉴定，最终获得重组病毒rY2-HB。
实施例3新城疫病毒rY2-HB株的安全性及免疫原性试验
1日龄SPF鸡共20只，随机分成2组，分别为rY2-HB组和PBS组(磷酸盐缓冲溶液)，进行隔离饲养。将rY2-HB株病毒以滴鼻/点眼的方式对鸡进行免疫，免疫剂量0.1ml，浓度为107.0EID50/ml。对照组接种同样剂量的PBS。观察免疫鸡的健康状态。在免疫后2周采集血清进行HI效价测定。同时以新城疫VII型HB0901株以滴鼻/点眼的方式对免疫鸡进行攻毒，攻毒剂量为104.0EID50/只。攻毒后，每天观察记录鸡的发病、死亡情况，持续观察2周，计算攻毒保护率。试验结果如表1所示，免疫后两周内所有试验鸡均健康，未出现发病症状。免疫两周后rY2-HB组的NDV HI抗体水平为6.5log2，对照组没有HI抗体效价。免疫两周进行基因VII型NDV强毒攻击，rY2-HB组的存活率为100％，而对照PBS组则为0％。结果表明，rY2-HB株病毒免疫试验鸡安全、无毒副作用，且可100％抵抗基因VII型NDV强毒的攻击。
表1.rY2-HB株病毒的免疫及攻毒保护试验
Figure GDA0002958528320000061
实施例4新城疫病毒NP抗体ELISA检测方法的建立
采用原核表达纯化的方法，获得了NDV的NP蛋白。将其作为包被抗原，通过反应条件的优化，建立了NDV间接ELISA抗体检测方法，对其敏感性、重复性、特异性进行了测定分析。该方法的检测灵敏度是血凝抑制试验(HI)的50倍，与H9亚型禽流感病毒等其他禽源病毒或细菌的阳性血清无交叉反应，组间重复性试验的变异系数小于6％。200份养殖场采集的鸡血清检测结果表明，ELISA方法与HI试验的检测符合率为97％。表明该方法可用于临床鸡血清的新城疫抗体检测。
实施例5rY2-HB株免疫鸡血清的NP抗体ELISA检测
采集rY2-HB株免疫试验鸡两周后的血清，分别以建立的新城疫病毒NP抗体ELISA检测方法和NDV HI方法对其进行抗体水平测定。NDV HI抗体平均值为6.5log2。ELISA检测结果如表2所示，5份rY2-HB株免疫鸡血清的ELISA检测结果均为阴性。
表2.rY2-HB株免疫鸡血清的NP抗体ELISA检测结果
Figure GDA0002958528320000071
结果表明，新城疫病毒NP抗体ELISA检测方法对rY2-HB疫苗株免疫鸡后的血清抗体检测结果为阴性。rY2-HB株可以作为基因VII新城疫标记疫苗候选株。
序列表
<110> 湖北省农业科学院畜牧兽医研究所
<120> 一种基因VII型新城疫标记疫苗株及其制备方法与应用
<140> 202011036432X
<141> 2020-09-27
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 14904
<212> DNA
<213> 禽副粘病毒2型(Avian Paramyxovirus serotype 2)
<400> 1
accaaacaag gaataggtaa gcaacgtaaa tcttagataa aaccatagaa tccgtggggg 60
cgacatcgcc tgaagccgat ctcgagatcg ataactccgg ttaattggtc tcagcgtgag 120
gagcttatct gtctgtggca atgtcttctg tgttttcaga ataccaggct cttcaggacc 180
aactggtcaa gcctgccact cgaagggctg atgtggcatc gactggattg ttgagagcgg 240
agataccagt ttgtgtaacc ttgtctcagg acccaactga tagatggaac ctcgcatgtc 300
tcaatctgcg atggctgata agtgagtcct ctactactcc catgagacaa ggggcgatcc 360
tgtcactgct gagcttgcac tctgacaaca tgcgagctca cgcaaccctt gcagcgagat 420
ccgctgatgc tgccatcact gtgcttgagg ttgacgccat agacatggcg gatggcacaa 480
tcacttttaa tgccagaagt ggagtatccg agaggcgcag cacacagctc atggcaatcg 540
caaaagatct gccccgctct tgttccaatg actcaccatt caaagatgac actatcgagg 600
atcgcgaccc ccttgacctg tccgagacta tcgatagact gcaggggatt gctgcccaaa 660
tctggatagc ggccatcaag agcatgactg ccccggatac tgctgcggag tcagaaggca 720
agaggcttgc aaagtaccaa caacaaggcc gcttggtgcg acaggtgtta gtgcatgatg 780
cggtgcgtgc ggaattccta cgtgtcatca gaggcagcct ggtcttacgg caattcatgg 840
tatcagaatg taagagggca gcatccatgg gtagcgagac atctaggtac tatgccatgg 900
tgggtgacat cagcctctac atcaagaatg caggacttac cgccttcttc ttgacactca 960
gatttggtat tgggacacac taccccactc ttgccatgag tgtgttctct ggagaactga 1020
agaagatgtc gtccttgatc aggctgtata agtcaaaagg ggaaaatgct gcatacatgg 1080
cattcctgga ggatgcggac atgggaaact ttgcgcctgc taactttagt actctctact 1140
cctatgcaat gggggtaggt acagtgctgg aagcatcagt tgcgaaatac cagttcgctc 1200
gagagttcac cagtgagaca tacttcaggc ttggggttga gaccgcacag aaccaacagt 1260
gcgctctaga tgaaaagacc gccaaggaga tggggcttac tgatgaagcc agaaagcagg 1320
tgcaagcatt ggctagcaac atcgagcagg ggcaacattc aatgcccatg caacaacagc 1380
ccacattcat gagtcagccc taccaggatg acgatcgtga ccagccaagc accagcagac 1440
cagagccaag accatcgcaa ttgacaagcc aatcagcagc acaggacaat gatgcggcct 1500
cattagattg gtgaccgcaa tcagctcagc caagccattg ttggacgcag gacattcaaa 1560
tcatacattg ccctaagagt attaaagtga tttaagaaaa aaggaccctg ggggcgaagt 1620
tgtcccaatc caggcaggcg ctgaaaccga atccctccaa cctccgagcc ccaggcgacc 1680
atggagttca ccgatgatgc cgaaattgct gagctgttgg acctcgggac ctcagtgatc 1740
caagagctgc agcgagccga agtcaagggc ccgcaaacaa ccggaaagcc caaagttccc 1800
ccggggaaca ccaagagcct ggctactctc tgggagcatg agactagcac ccaagggagt 1860
gcattgggca cacccgagaa caacacccag gcacccgatg acaacaacgc aggtgcagat 1920
acgccagcga ccaccgacgt ccatcgcact ctggatacca tagacaccga cacaccaccg 1980
gaagggagca agcccagctc cactaactcc caacccggtg atgaccttga caaggctctt 2040
tcgaagctag aggcgcgcgc caagctcgga ccagataggg ccagacaggt taaaaagggg 2100
aaggagatcg ggtcgagcac agggacgagg gaggcagcca gtcaccacat ggaagggagc 2160
cgacagtcgg agccaggagc gggcagccga gcacagccac aaggccatgg cgaccgggac 2220
acaggaggga gtactcattc atctctcgag atgggagact ggaagtcaca agctggtgca 2280
acccagtctg ctctcccatt agaagcgagc ccaggagaga aaagtgcaca tgtggaactt 2340
gcccagaatc ctgcatttta tgcaggcaac ccaactgatg caattatggg gttgacaaag 2400
aaagtcaatg atctagagac aaaattggct gaggtattgc gtctgttagg aatactcccc 2460
ggaataaaga atgagattag tcagctgaaa gcaaccgtgg ctctgatgtc aaatcagatt 2520
gcctccattc agattcttga tcctgggaat gccggagtca aatcccttaa tgagatgaaa 2580
gccctgtcaa aagcagccag catagttgtg gcaggtccag gagtccttcc tcctgaggtc 2640
acagaaggag gactgattgt gaaagatgag ctagcaaggc ctatccccat ccaaccgcaa 2700
cgagattcca aacccaaaga cgacccgcac acatcaccaa atgatgtcct tgctgtacgc 2760
gctatgatcg acacccttgt ggatgatgag aagaagagaa agagattaaa ccaggccctt 2820
gacaaggcaa agaccaagga tgacgtctta agggtcaagc ggcagatata caatgcctag 2880
gagtccattt gtctaaagaa cctccaatca tatcaccagt ttcgtgccac atgcttccct 2940
gccgagaatc tagccgacac aaaaactaaa tcatagttta acaaaaaaga agtttggggg 3000
cgaagtctca catcatagag cacccttgca ttctaaaatg gctcaaacaa ccgtcaggct 3060
gtatatcgat gaagctagtc ccgacattga actgttgtct tacccactga taatgaaaga 3120
cacaggacat gggaccaaag agttgcagca gcaaatcaga gttgcagaga tcggtgcatt 3180
gcagggaggg aagaatgaat cagttttcat caatgcatat ggctttgttc agcaatgcaa 3240
agttaaaccg ggggcaaccc aattcttcca ggtagatgca gctacaaagc cagaagtggt 3300
cactgcaggg atgattataa tcggtgcagt caagggggtg gcaggcatca ctaagctggc 3360
agaagaggtg ttcgagctgg acatctccat caagaagtcc gcatcattcc atgagaaggt 3420
tgcggtgtcc tttaatactg tgccactatc actcatgaat tcgaccgcat gcagaaatct 3480
gggttatgtc acaaacgctg aggaggcgat caaatgcccg agcaaaatac aagcgggtgt 3540
gacgtacaaa tttaagataa tgtttgtctc cttgacacga ctgcataacg ggaaattgta 3600
ccgtgtcccc aaggcagtgt atgctgtaga ggcatcagct ctatataaag tgcaactgga 3660
agtcgggttc aagcttgacg tggccaagga tcacccacac gttaagatgt tgaagaaagt 3720
ggaacggaat ggtgagactc tgtatcttgg ttatgcatgg ttccacctgt gcaacttcaa 3780
gaagacaaat gccaagggtg agtcccggac aatctccaac ctagaaggga aagtcagagc 3840
tatggggatc aaggtttcct tgtacgactt atgggggcct actttggtgg tgcaaatcac 3900
aggtaagacc agcaagtatg cacaaggttt cttttcaacc acaggtacct gctgcctccc 3960
agtgtcgaag gctgcccctg agctggccaa acttatgtgg tcctgcaatg caacaatcgt 4020
tgaagctgca gtgattatcc aagggagtga taggagggca gtcgtgacct cagaggactt 4080
ggaagtatac ggggcagttg caaaagagaa gcaggctgca aaaggatttc acccgttccg 4140
caagtgacac gtggggccgc acacctcatt accccagaag cccgggcaac tgcaaattca 4200
cgcttatata atccaattac catgatctag aactgcaatc gatactaatc gctcattgat 4260
cgtattaaga aaaaacttaa ctacataact tcaacattgg gggcgacagc tccagactaa 4320
gtgggtggct aagctctgac tgataaggaa tcatgaatca agcactcgtg attttgttgg 4380
tatctttcca gctcggcgtt gccttagata actcagtgtt ggctccaata ggagtagcta 4440
gcgcacagga gtggcaactg gcggcatata caacgaccct cacagggacc atcgcagtga 4500
gatttatccc ggtcctgcct gggaacctat caacatgtgc acaggagacg ctgcaggaat 4560
ataatagaac tgtgactaat atcttaggcc cgttgagaga gaacttggat gctctcctat 4620
ctgacttcga taaacctgca tcgaggttcg tgggcgccat cattgggtcg gtggccttgg 4680
gggtagcaac agctgcacaa atcacagccg ccgtggctct caatcaagca caagagaatg 4740
cccggaatat atggcgtctc aaggaatcga taaagaaaac caatgaggct gtgttggaat 4800
tgaaggatgg acttgcaacg actgctatag ctttggacaa agtgcaaaag tttatcaatg 4860
atgatattat accacagatt aaggacattg actgccaggt agttgcaaat aaattaggcg 4920
tctacctctc cttatactta acagagctta caactgtatt tggttctcag atcactaatc 4980
ctgcattatc aacgctctct taccaggcgc tgtacagctt atgtggaggg gatatgggaa 5040
agctaactga gctgatcggt gtcaatgcaa aggatgtggg atccctctac gaggctaacc 5100
tcataaccgg ccaaatcgtt ggatatgacc ctgaactaca gataatcctc atacaagtat 5160
cttacccaag tgtgtctgaa gtgacaggag tccgggctac tgagttagtc actgtcagtg 5220
tcactacacc aaaaggagaa gggcaggcaa ttgttccgag atatgtggca cagagtagag 5280
tgctgacaga ggagttggat gtctcgactt gtaggtttag caaaacaact ctttattgta 5340
ggtcgattct cacacggccc ctaccaactt tgatcgccag ctgcctgtca gggaagtacg 5400
acgattgtca gtacacaaca gagataggag cgctatcttc gagattcatc acagtcaatg 5460
gtggagtcct tgcaaactgc agagcaattg tgtgtaagtg tgtctcaccc ccgcatataa 5520
taccacaaaa cgacattggc tccgtaacag ttattgactc aagtatatgc aaggaagttg 5580
tcttagagag tgtgcagctt aggttagaag gaaagctgtc atcccaatac ttctccaacg 5640
tgacaattga cctttcccaa atcacaacgt cagggtcgct ggatataagc agtgaaattg 5700
gtagcattaa caacacagtt aatcgggtcg acgagttaat caaggaatcc aacgagtggc 5760
tgaacgctgt gaacccccgc cttgtgaaca atacgagcat catagtcctc tgtgtccttg 5820
ccgccctgat tattgtctgg ctaatagcgc tgacagtatg cttctgttac tccgcaagat 5880
actcagctaa gtcaaaacag atgaggggcg ctatgacagg gatcgataat ccatatgtaa 5940
tacagagtgc aactaagatg tagagaggtt gaataagcct aaacatgata tgatttaaga 6000
aaaaattgga aggtgggggc gacagcccat tcaatgaagg gtgtacactc caacttgatc 6060
ttgtgacttg atcatcatac tcgaggcacc atggatttcc catctaggga gaacctggca 6120
gcaggtgaca tatcggggcg gaagacttgg agattactgt tccggatcct cacattgagc 6180
ataggtgtgg tctgtcttgc catcaatatt gccacaattg caaaattgga tcacctggat 6240
aacatggctt cgaacacatg gacaacaact gaggctgacc gtgtgatatc tagcatcacg 6300
actccgctca aagtccctgt caaccagatt aatgacatgt ttcggattgt agcgcttgac 6360
ctacctctgc agatgacatc attacagaaa gaaatagcat cccaagtcgg gttcttggct 6420
gaaagtatca acaatgtttt atccaagaat ggatctgcag gcctggttct tgttaatgac 6480
cctgaatatg caggggggat cgctgtcagc ttgtaccaag gagatgcatc tgcaggccta 6540
aatttccagc ccatttcttt aatagaacat ccaagttttg tccctggtcc tactactgct 6600
aagggctgta taaggatccc gaccttccat atgggccctt cacattggtg ttactcacat 6660
aacatcattg catcaggttg ccaggatgcg agccactcca gtatgtatat ctctctgggg 6720
gtgctgaaag catcgcagac cgggtcgcct atcttcttga caacggccag ccagctcgtg 6780
gatgacaaca tcaaccggaa gtcatgcagc atcgtagcct caaaatacgg ttgtgatatc 6840
ctatgcagta ttgtgattga aacagaggat gaggattata ggtctgatcc ggctactagc 6900
atgattatag gtaggctgtt cttcaacggg tcatacacag agagcaagat taacacaggg 6960
tccatcttca gtctattctc tgctaactac cctgcggtgg ggtcgggtat tgtagtcggg 7020
gatgaagccg cattcccaat atatggtggg gtcaagcaga acacatggtt gttcaaccag 7080
ctcaaggatt ttggttactt cacccataat gatgtgtaca agtgcaatcg gactgatata 7140
cagcaaacta tcctggatgc atacaggcca cctaaaatct caggaaggtt atgggtacaa 7200
ggcatcctat tgtgcccagt ttcactgaga cctgatcctg gctgtcgctt aaaggtgttc 7260
aataccagca atgtgatgat gggggcagaa gcgaggttga tccaagtagg ctcaaccgtg 7320
tatctatacc aacgctcatc ctcatggtgg gtggtaggac tgacttacaa attagatgtg 7380
tcagaaataa cttcacagac aggtaacaca ctcaaccatg tagaccccat tgcccataca 7440
aagttcccaa gaccatcttt caggcgagat gcgtgtgcga ggccaaacat atgccctgct 7500
gtctgtgtct ccggagttta tcaggacatt tggccgatca gtacagccac caataacagc 7560
aacattgtgt gggttggaca gtacttagaa gcattctatt ccaggaaaga cccaagaata 7620
gggatagcaa cccagtatga gtggaaagtc accaaccagc tgttcaattc gaatactgag 7680
ggagggtact caaccacaac atgcttccgg aacaccaaac gggacaaggc atattgtgta 7740
gtgatatcag agtacgctga tggggtgttc ggatcataca ggatcgttcc tcagcttata 7800
gagattagaa caaccaccgg taaatctgag tgatgcatca atcctaaatt ggaatgacca 7860
atcaaaagct acgtagtgtc taacagcatt gcgaagcctg gtttaagaaa aaacttgggg 7920
gcgaatgccc atcaaccatg gatcaaactc aagctgacac tataatacaa cctgaagtcc 7980
atctgaattc accacttgtt cgcgcaaaat tggttcttct atggaaattg actgggttac 8040
ctttgccgtc tgatttgaga tcatttgtac taactacaca tgcagctgat gaccaaatcg 8100
caaaaaatga gactaggatc aaggccaaaa ttaattccct aatcgataac ttaatcaaac 8160
actgcaaggc aaggcaagtg gcactttcag ggttgacacc tgtcgtacat ccaacaactc 8220
tacagtggtt gctatccatc acatgtgaac gagcagacca ccttgcaaaa gtacgcgaga 8280
aatcagttaa gcaagcaatg tcagagaagc aacacgggtt tagacatctc ttttcggcag 8340
taagtcatca gttagttgga aacgccacac tgttctgtgc acaagactct agcaccgtga 8400
atgtcgactc tccttgctca tcaggttgtg agaggctgat aatagactct attggagcct 8460
tacaaacacg atggacaaga tgtaggtggg cttggcttca cattaaacag gtaatgagat 8520
accaggtgct tcagagtcgc ctacacgctc atgccaattc tgttagcaca tggtctgagg 8580
cgtgggggtt cattgggatc acaccagata tagtccttat tgtagactat aagagcaaaa 8640
tgtttactat cctgaccttc gaaatgatgc tgatgtattc agatgtcata gagggtcgtg 8700
ataatgtggt agctgtagga agtatgtcac caaacctaca gcctgtggtg gagaggattg 8760
aggtgctgtt tgatgtagtg gacaccttgg cgaggaggat tcatgatcct atttatgatc 8820
tggttgctgc cttagaaagc atggcatacg ctgccgtcca attgcacgat gctagtgaga 8880
cacacgcagg ggaattcttt tcgttcaatt tgacagaaat agagtccact cttgccccct 8940
tgctggatcc tggccaagtc ctatcggtga tgaggactat cagttattgt tacagtgggc 9000
tatcgcctga ccaagctgca gagttgctct gtgtgatgcg cttatttgga caccctctgc 9060
tctccgcaca acaagcagca aaaaaagtcc gggagtctat gtgtgcccct aaactgttag 9120
agcatgatgc aatactgcaa actctatctt tcttcaaggg aatcataatc aatggttaca 9180
ggaaaagtca ttctggagta tggcctgcaa ttgacccaga ttctatagtg gacgatgacc 9240
ttagacagct gtattacgag tcggcagaaa tttcacatgc tttcatgctt aagaaatatc 9300
ggtaccttag tatgattgag ttccgcaaga gcatagagtt tgacttaaat gatgacctga 9360
gcacattcct taaagacaaa gcaatctgca ggccaaaaga tcaatgggca cgcatcttcc 9420
ggaaatcatt gttcccttgc aaaacgaacc ttggcactag tatagatgtt aaaagtaatc 9480
gactgttgat agattttttg gagtcacatg acttcaatcc tgaggaagaa atgaagtatg 9540
tgactacgct agcatacctg gcagataatc aattctcagc atcatattca ctgaaggaga 9600
aagagatcaa gactactggc cggatcttcg ccaaaatgac caggaaaatg aggagctgtc 9660
aagtaatatt ggaatcacta ttgtccagtc acgtctgcaa attctttaag gagaacggtg 9720
tgtcaatgga acaactgtct ttgacaaaga gcttgcttgc aatgtcacag ttagcaccca 9780
ggatatcttc agttcgccag gcgacagcac gtagacagga cccaggactc agccactcta 9840
atggttgtaa tcacattgta ggagacttag gcccacacca gcaggacaga ccggcccgga 9900
agagtgtagt cgcaaccttc cttacaacag atcttcaaaa atattgcttg aattggcgat 9960
atgggagtat caagcttttc gcccaagcct taaaccagct attcggaatc gagcatgggt 10020
ttgaatggat acacctgaga ctgatgaata gcaccctgtt tgtcggggac ccattctcgc 10080
ctcctgaaag caaagtgctg agtgatcttg atgatgcgcc caattcagac atatttatcg 10140
tgtccgccag aggggggatt gaagggttat gccagaagct gtggaccatg atttcaataa 10200
gcataatcca ttgcgtggct gagaagatag gagcaagggt tgcggcgatg gttcagggag 10260
ataatcaggt aattgcaatc acgagagagc tgtataaggg agagacttac acgcagattc 10320
agccggagtt agatcgatta ggcaatgcat tttttgctga attcaaaaga cacaactatg 10380
caatgggaca taatctgaag cccaaagaga caatccaaag tcaatcattc tttgtgtatt 10440
cgaaacggat tttctgggaa gggagaattc ttagtcaagc actgaagaat gctaccaaac 10500
tatgcttcat tgcagatcac ctcggggata atactgtctc atcatgcagc aatctagcct 10560
ctacgataac ccgcttggtt gagaatgggt atgaaaagga cacagcattc attctgaata 10620
tcatctcagc aatgactcag ttgctgattg atgagcaata ttccctacaa ggagactact 10680
cagctgtgag aaaactgatt gggtcatcaa attaccgtaa tctcttagtg gcgtcgctca 10740
tacctggtca ggttggcggc tataatttct tgaatatcag tcgcctattc acacgcaata 10800
ttggtgatcc agtaacatgc gccatagcag atctgaagtg gttcattagg agcgggttaa 10860
tcccagagtt catcctgaag aatatattac tacgagatcc cggagacggg gggtggagta 10920
ctctatgtgc tgacccttac gcattaaata tcccctacac tcagctaccc acaacatacc 10980
tgaagaagca tactcagagg gcattactat ccgattctaa taatccgctt cttgcagggg 11040
tgcaattgga caatcaatac attgaagagg aggagtttgc acgattcctt ttggatcggg 11100
aatccgtgat gcctcgagtg gcacacacaa tcatggagtc aagtatacta gggaagagaa 11160
agaacatcca gggtttaatc gacactaccc ctacaatcat taagactgca ctcatgaggc 11220
agcccatatc tcgtagaaag tgtgataaaa tagttaatta ctcgattaac tacctgactg 11280
agtgccacga ttcattattg tcctgtagga cattcgagcc aaggaaggaa ataatatggg 11340
agtcagctat gatctcagta gaaacttgca gtgtcacaat tgcggagttc ctgcgcgcca 11400
ccagctggtc caacatcctg aacggtagga ctatttcggg tgtaacatct ccagacacta 11460
tagagctgct caaggggtca ttaattggag agaatgccca ttgtattctt tgtgagcagg 11520
gagacgagac attcacgtgg atgcacttag ccgggcccat ctatatacca gacccggggg 11580
tgaccgcatc caagatgaga gtgccgtatc ttgggtcaaa gacagaggaa aggcgtacgg 11640
catccatggc caccattaag ggcatgtctc accacctaaa ggccgctttg cgaggagcct 11700
ctgtgttggt gtgggccttt ggtgatactg aagaaagttg ggaacatgcc tgccttgtgg 11760
ccaatacaag gtgcaagatt aatcttccgc agctacgcct gctgaccccg acaccaagca 11820
gctctaacat ccaacatcga ctaaatgatg gtatcagcgt gcaaaaattt acacctgcta 11880
gcttatcccg agtggcgtca tttgttcaca tttgcaacga tttccaaaag ctagagagag 11940
atggatcttc cgtagactct aacttgatat atcagcaaat catgctgact ggtctaagta 12000
ttatggagac acttcatcct atgcacgtct catgggtata caacaatcag acaattcact 12060
tacataccgg aacatcgtgt tgtcctaggg aaatagagac aagcattgtt aatcccgcta 12120
ggggagaatt cccaacaata actctcacaa ctaacaatca gtttctgttt gattgtaatc 12180
ccatacatga tgaggcactt acaaaactgt cagtaagtga gttcaagttc caggagctta 12240
atatagactc aatgcagggt tacagtgctg tgaacctgct gagcagatgt gtggctaagc 12300
tgatagggga atgcattctg gaagacggta tcggatcgtc aatcaagaat gaagcaatga 12360
tatcatttga taactctatc aactggattt ctgaagcact caatagtgac ctgcgtttgg 12420
tattcctcca gctggggcaa gaactacttt gtgacctggc gtaccaaatg tactatctga 12480
gggtcatcgg ctatcattcc atcgtggcat atctgcagaa tactctagaa agaattcctg 12540
ttatccaact cgcaaacatg gcactcacca tatcccaccc agaagtatgg aggagagtga 12600
cagtgagcgg attcaaccaa ggttaccgga gtccctatct ggccactgtc gactttatcg 12660
ccgcatgtcg tgatatcatt gtgcaaggtg cccagcatta tatggctgat ttgttgtcag 12720
gagtagagtg ccaatataca ttctttaatg ttcaagacgg cgatctgaca ccgaagatgg 12780
aacaattttt agcccggcgc atgtgcttgt ttgtattgtt aactgggacg atccgaccac 12840
tcccaatcat acgatccctt aatgcgattg agaaatgtgc aattctcact cagttcttgt 12900
attacctacc gtcagtcgac atggcagtag cagacaaggc tcgtgtgtta tatcaactgt 12960
caataaatcc gaaaatagat gctttagtct ccaaccttta tttcaccaca aggaggttgc 13020
tttcaaatat caggggagat tcttcttcac gagcgcaaat tgcattcctc tacgaggagg 13080
aagtgatcgt tgatgtgcct gcatctaatc aatttgatca gtaccatcgt gaccccatcc 13140
taagaggagg tctatttttc tctctctcct taaaaatgga aaggatgtct ctgaaccgat 13200
ttgcagtaca gaccctgcca acccaggggt ctaactcgca gggttcacga cagaccttgt 13260
ggcgtgcctc accgttagca cactgcctta aatcagtagg gcaggtaagt accagctggt 13320
acaagtatgc tgtagtgggg gcgtctgtag agaaagtcca accaacaaga tcaacaagcc 13380
tctacatcgg ggagggcagt gggagtgtca tgacattatt ggagtatctg gaccctgcta 13440
caattatctt ctacaactcg ctattcagca atagcatgaa ccctccacaa aggaatttcg 13500
gactgatgcc cacacagttt caggactcag tcgtgtataa aaacatatca gcaggagttg 13560
actgcaagta cgggtttaag caagtctttc aaccattatg gcgtgatgta gatcaagaaa 13620
caaatgtggt agagacggcg ttcctaaact atgtgatgga agtagtgcca gtccactctt 13680
cgaagcgtgt cgtatgtgaa gttgagtttg acagggggat gcctgacgag atagtaataa 13740
cagggtacat acacgtgctg atggtgaccg catacagtct gcatcgagga gggcgtctaa 13800
taatcaaggt ctatcgtcac tccgaggctg tattccaatt cgtactctct gcgatagtca 13860
tgatgtttgg ggggcttgat atacaccgga actcgtacat gtcaactaac aaagaggagt 13920
acatcatcat agctgcggcg ccggaggcat taaactattc ctctgtacca gcaatattgc 13980
agagggtgaa gtctgttatt gaccagcagc ttacattaat ctctcctata gatctagaaa 14040
gattgcgcca tgagactgag tctctccgtg agaaggagaa taatctagta atatctctga 14100
cgagagggaa gtatcaactc cggccgacac agactgatat gcttctatca tacctaggtg 14160
ggagattcat caccctattc ggacagtctg ctagggattt gatggccact gatgttgctg 14220
accttgatgc taggaagatt gcattagttg atctactgat ggtggaatcc aacattattt 14280
taagtgagag cacagacttg gaccttgcac tgttgctgag cccgtttaac ttagacaaag 14340
ggcggaagat agttacccta gcaaaggcta ctacccgcca attgctgccc gtgtatatag 14400
catcagagat aatgtgcaat cggcaggcat tcacacacct gacatcaatt atacagcgtg 14460
gtgtcataag aatagaaaac atgcttgcta caacggaatt tgtccgacag tcagttcgcc 14520
cccagttcat aaaggaggtg ataactatag cccaagtcaa ccaccttttt tcagatctat 14580
ccaaactcgt gctttctcga tctgaagtca agcaagcact taaatttgtc ggttgctgta 14640
tgaagttcag aaatgcaagc aattaaacag gattgttatt gtcaaatcac cggttactat 14700
agtcaaatta atatgtaaag ttccctcttt caagagtgat taagaaaaaa cgcgtcaaag 14760
gtggcggttt cactgatttg ctcttggaag ttgggcatcc tccagccaat atatcggtgc 14820
cgaaatcgaa agtctgacag ctgatttgga atataagcac tgcataatca ctgagttacg 14880
ttgctttgct attccatgtc tggt 14904

Claims (2)

1.一种基因VII型新城疫标记疫苗株副粘病毒II型rY2-HB株，该毒株保藏于中国典型培养物保藏中心，保藏地址是，中国，武汉，武汉大学，保藏编号为CCTCC NO：V202052。
2.如权利要求1所述的基因VII型新城疫标记疫苗株副粘病毒II型rY2-HB株在制备基因VII型新城疫标记疫苗方面的应用。
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