CN107384958A - 基于反向遗传学构建的rsv反基因组质粒及其应用 - Google Patents

基于反向遗传学构建的rsv反基因组质粒及其应用 Download PDF

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CN107384958A
CN107384958A CN201710574632.2A CN201710574632A CN107384958A CN 107384958 A CN107384958 A CN 107384958A CN 201710574632 A CN201710574632 A CN 201710574632A CN 107384958 A CN107384958 A CN 107384958A
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何金生
彭向雷
许敏
付远辉
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Abstract

本发明公开基于反向遗传学构建的RSV反基因组质粒，所述RSV反基因组质粒为如序列表SEQ ID No.1所示的质粒pBRB‑RSV‑rLong/A2cp、如序列表SEQ ID No.2所示的质粒pBRB‑RSV‑rLong/A2cpts或如序列表SEQ ID No.3所示的质粒pBRB‑RSV‑rLong/A2cptsΔSH。本发明构建的重组质粒经拯救获得的重组RSV病毒具有明显的减毒效果，可用于制备RSV减毒活疫苗。

Description

基于反向遗传学构建的RSV反基因组质粒及其应用
技术领域
本发明涉及分子生物学和生物医学领域。更具体地，涉及一种基于反向遗传学构建的RSV反基因组重组质粒及其构建方法和应用。
背景技术
人呼吸道合胞病毒(Human Respiratory Syncytial Viru，RSV)是世界范围内引起婴幼儿和两岁以下儿童下呼吸道感染的最重要的病毒性病原，同时也在老年人和免疫缺陷人群引起严重感染。目前，利巴韦林和帕利珠单抗是仅有的用于RSV治疗和预防的药物，然而利巴韦林由于喷雾给药途径、药效不佳及存在致畸作用，阻碍了其在临床应用，帕利珠单抗主要用于2岁以下高危儿童且且价格昂贵，也未得到广泛使用。因而，开发新的RSV疫苗成为亟待解决的问题。
由于FI-RSV的失败，自20世纪60年代以来，获得在体内具有毒力减弱(attenuation,att)表型的RSV减毒活疫苗一直备受关注。现在认为，RSV减毒活疫苗的主要优势在于：减毒活疫苗能经滴鼻免疫诱导机体产生系统和局部免疫,因此可同时预防上、下呼吸道感染；减毒活疫苗能引起与自然感染相同的免疫应答，在嗣后的自然感染中不会产生疾病增强作用；在母传抗体存在的情况下，经鼻免疫的减毒活疫苗仍然具有在新生儿体内进行有效复制的能力。
RSV减毒活疫苗的研究主要经历了两个阶段，一是在20世纪近30年的时间里，主要采用传统的生物学方法获得RSV减毒活疫苗,包括宿主范围(host-range,hr)突变、冷传代(cold passage,cp)突变及化学诱变后的生物学筛选等，这些方法费时、费力，而且具有较大的偶然性；二是近10年来主要采用基因工程方法或反向遗传学(reverse genetics)获得重组RSV疫苗病毒，该方法首先利用分子生物学方法获得RSV反基因组感染性DNA克隆，在插入各种已知的突变或缺失病毒的非必需基因后，经拯救获得重组RSV疫苗病毒或RSV减毒活疫苗株。
反向遗传学不仅加深了人们对传统生物学方法获得的各种表型(att,ca,ts和hr)减毒活疫苗的遗传学机制的认识，同时大大加快了新型减毒活疫苗的研制步伐，突破了以传统生物学方法获得高度稳定的减毒株时存在的障碍，产生了第二代RSV减毒活疫苗或重组RSV，使RSV减毒活疫苗的研制工作发展到了一个崭新的阶段。
因此，通过反向遗传学技术进行构建和拯救RSV减毒疫苗病毒株是研发新的RSV疫苗的核心。
发明内容
本发明的第一个目的在于提供基于反向遗传学构建的RSV反基因组pBRB-RSV-rLong/A2cp、pBRB-RSV-rLong/A2cpts、pBRB-RSV-rLong/A2cptsΔSH质粒。
本发明的第二个目的在于提供一种上述质粒的构建方法。
本发明的第三个目的在于提供上述质粒在拯救重组RSV的应用。
为达到上述目的，本发明采用下述技术方案：
本发明一种基于反向遗传学构建的RSV反基因组质粒，所述质粒为如序列表SEQID No.1所示的质粒pBRB-RSV-rLong/A2cp、如序列表SEQ ID No.2所示的质粒pBRB-RSV-rLong/A2cpts或如序列表SEQ ID No.3所示的质粒pBRB-RSV-rLong/A2cptsΔSH。
进一步，所述质粒pBRB-RSV-rLong/A2cp在pBRB载体中装入重组序列获得的。
其中，所述的pBRB载体的序列如SEQ ID No.4所示；
所述重组序列的全长为15405nt：1-6nt为MluI克隆位点，7-26nt为T7启动子,27-67nt为锤头状核酶序列，68-15181nt为修饰后的RSV反基因组DNA序列，15182-15270nt为丁型肝炎核酶序列，15271-15399nt为T7终止子,15400-15405nt为NruI克隆位点；
所述修饰后的RSV反基因组DNA序列为在RSV Long株反基因组DNA序列的基础上删除了SH基因的非编码区112个nt(RSVLong株反基因组4497-4608nt)并在SH基因编码区末端引入4处同义突变：SH186C>T、SH189C>T、SH192A>T、SH194,195AG>GA，以增加RSVLong反基因组DNA结构的稳定性；在如下位置引入A2株上的五个冷传代(cp)突变：N 799G>A,aa267Val>Ile；F653,654AA>CG,aa218Glu>Ala；F1568,1569CA>TT,aa523Thr>Ile；L 956,957GT>AC,aa319Cys>Tyr；5068,5070C>T,T>C,aa1690His>Tyr。
进一步，所述质粒pBRB-RSV-rLong/A2cpts是在质粒pBRB-RSV-rLong/A2cp的基础上在如下位置再引入两个温度敏感型(ts)突变：M2 9T>C；L 2491,2492CA>TT,aa831Gln>Leu。
进一步，所述质粒pBRB-RSV-rLong/A2cptsΔSH是在质粒pBRB-RSV-rLong/A2cpts的基础上删除SH基因的GS-GE之间序列及部分SH-G基因间隔区序(RSVLong株反基因组4218-4496nt和4609-4666nt)。
进一步，上述对修饰后的的RSV反基因组DNA序列是对RSV Long株反基因组DNA序列(GenBank:AY911262.1)进行遗传学改造或修饰，即在保留了RSV Long株反基因组前导序列(Leader region/genomic promotor)、转录起始信号(Gene start signal，GS)、转录终止信号(Gene end signal，GE)和尾随序列(Trailer region/antigenomic promoter)等顺式作用元件及10个基因序列的基础上，在相应的位点引入五个cp突变(N 799G>A,aa267Val>Ile；F653,654AA>CG,aa218Glu>Ala；F1568,1569CA>TT,aa523Thr>Ile；L 956,957GT>AC,aa319Cys>Tyr；L 5068,5070C>T,T>C,aa1690His>Tyr)或两个ts突变(M2 9T>C；L 2491,2492CA>TT,aa831Gln>Leu)，删除SH基因的非编码区112个核苷酸(RSVLong株反基因组4497-4608nt)或SH基因的GS-GE之间序列及部分SH-G基因间隔区序(RSVLong株反基因组4218-4496nt和4609-4666nt)，并分别在反基因组两端添加了T7启动子终止子、锤头状核酶(hammerhead ribozyme，HHr)和丁型肝炎核酶(HDV ribozyme,HDVr)，获得了基于反向遗传学技术构建的RSV反基因组重组质粒。
本发明进一步提供了所述反基因组pBRB-RSV-rLong/A2cp质粒的构建方法，优选的，基于反向遗传学构建的RSV反基因组pBRB-RSV-rLong/A2cp质粒的构建方法包括以下步骤：
1)改造pBR322载体，将ClaI-NheI酶切位点之间序列替换为ClaI*-MluI-XbaI-HindIII-SacI-AsuII-NheI限制性内切酶多克隆位点，改造后载体命名为pBRB，序列如SEQID No.4所示；
2)按SEQ ID No.1所示序列，分段化学合成RSVLong株反基因组及附属表达原件，即分别合成DNA片段MluI-AsuII(A)、AsuII-NheI(B)、NheI-NruI(C)；所述A段含有T7启动子、锤头状核酶、RSVLong株反基因组1-5184nt，删除了SH基因的非编码区112个核苷酸(RSVLong株反基因组4497-4608nt)及A2cp突变N 799G>A,aa267Val>Ile；所述B段含有RSVLong株反基因组5179-11006nt及A2cp突变F653,654AA>CG,aa218Glu>Ala、F1568,1569CA>TT,aa523Thr>Ile、L 956,957GT>AC,aa319Cys>Tyr；所述C段含有RSVLong株反基因组11001-15226n、A2cp突变L5068,5070C>T,T>C,aa1690His>Tyr及丁型肝炎核酶序列、T7终止子序列；
3)按C、B、A顺序，依次将各片段按常规分子克隆方法连接到pBRB载体上，获得质粒pBRB-RSV-rLong/A2cp，序列如SEQ ID No.1所示。
所述质粒pBRB-RSV-rLong/A2cpts的构建方法与pBRB-RSV-rLong/A2cp大体相同，其区别在于：步骤2)合成B段基因除含有RSVLong株反基因组5179-11006nt及3个A2cp突变外，还含有两个ts突变(M2 9T>C；L 2491,2492CA>TT,aa831Gln>Leu)。
所述质粒pBRB-RSV-rLong/A2cptsΔSH的构建方法与质粒pBRB-RSV-rLong/A2cpts大体相同，其区别在于：步骤2)中合成的A片段除含有T7启动子、锤头状核酶、RSVLong株反基因组1-5184nt及A2cp突变N 799G>A,aa267Val>Ile外，还删除SH基因的GS-GE之间序列及部分SH-G基因间隔区序(RSVLong株反基因组4218-4496nt和4609-4666nt)。
在本发明中，所述步骤2)各个基因片段的合成方法包括但不限于全基因合成方法或RT-PCR扩增法，优选为全基因合成方法；
所述的基因片段的连接载体不限于以上pBRB载体，也可以连接在其他的载体上。
所述重组质粒中各个基因片段的连接顺序不限于C-B-A顺序，其他的连接顺序也在本发明的保护范围之内。
采用相似或相同的制备原理，RSV反基因组也可以连接至其他的表达载体中或修改的表达载体中，表达载体的修改方式不限于本申请例举的插入多克隆位点(MCS)方法。
本发明还提供了上述RSV反基因组质粒pBRB-RSV-rLong/A2cp、pBRB-RSV-rLong/A2cpts、pBRB-RSV-rLong/A2cptsΔSH经拯救获得的重组RSV，拯救的方法，包括以下步骤：
1)分别转染上述pBRB-RSV-rLong/A2cp、pBRB-RSV-rLong/A2cpts、pBRB-RSV-rLong/A2cptsΔSH质粒和四种辅助蛋白质粒(pCITE-N,P,L,M2-1)至宿主细胞(BHK T7/9)培养；
2)盲传至宿主细胞(Vero)继续培养至病毒空斑形成。
本发明进一步提供了所述重组RSV在制备抗RSV药物中的应用。
优选的，所述应用为所述重组RSV在制备RSV疫苗上的应用。
本发明利用实时定量PCR检测连续传代后重组病毒的基因组拷贝数、免疫酶法检测空斑形成单位(PFU/mL)来确定重组病毒的复制能力和病毒滴度，利用突变位点测序分析来检测连续传代后重组病毒的稳定性。
本发明的有益效果如下：
1.本发明构建的重组质粒以RSV Long株为母本，引入RSV A2株冷传代(cp)突变和温度敏感型(ts)突变，为后续RSV基因组改造或修饰奠定了基础。
2.本发明构建的重组质粒经拯救获得的重组RSV病毒具有明显的减毒效果，可用于制备RSV减毒活疫苗。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的说明。
图1示出载体pBRB的酶切鉴定电泳图，其中M代表分子量标记物。
图2示出RSV基因片段pBRB-RSV-A；pBRB-RSV-B；pBRB-RSV-C质粒的酶切鉴定电泳图，其中M代表分子量标记物，A：pBRB-RSV-A，B：pBRB-RSV-B，C：pBRB-RSV-C；。
图3示出重组pBRB-RSV-rLong/A2cp质粒的酶切鉴定电泳图，其中M代表分子量标记物；1：NruⅠ、NheⅠ；2：AsuⅡ、NheⅠ；3：AsuⅡ、SacⅠ；4：SacⅠ、MluⅠ；5：EcoRⅤ；6：SpeⅠ；7：AflⅡ。
图4示出质粒共转染BHK-T7/9细胞后于倒置显微镜下(NIKON ECLIPSE TE 2000-S×100)观察到的细胞融合。
图5示出盲传Vero细胞后于倒置显微镜下(NIKON ECLIPSE TE 2000-S×100)观察到的细胞融合。
图6示出免疫酶法检测重组RSV病毒连续传代后病毒滴度即空斑形成单位(PFU/mL)。
图7示出实时定量PCR检测重组病毒连续传代后病毒N基因的拷贝数。
图8示出第10、20、30代RSV-rLong/A2cp重组病毒突变位点的局部测序结果。
图9示出第10、20、30代RSV-rLong/A2cpts重组病毒突变位点的局部测序结果。
图10示出第10、20、30代RSV-rLong/A2cptsΔSH重组病毒突变位点的局部测序结果。
具体实施方式
为了更清楚地说明本发明，下面结合优选实施例和附图对本发明做进一步的说明。附图中相似的部件以相同的附图标记进行表示。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围。
实施例1：pBRB-RSV-rLong/A2cp质粒的构建
1)将载体pBR322改造为pBRB，即化学合成两条单链DNA；5’-CGACGCGTTACGTCTAGAGGTCAAGCTTGAGCGAGCTCGGCGTTCGAAGGCGG-3’、5’-CTAGCCGCCTTCGAACGCCGAGCTCGCTCAAGCTTGACCTCTAGACGTAACGCGT-3’，将其混合退火后连入到pBR322的ClaI/NheI位点间，替换原有片段，获得新的多克隆位点(MCS)ClaI*-MluI-XbaI-HindIII-SacI-AsuII-NheI，将改造后的载体pBRB用进行SacⅠ酶切鉴定，琼脂糖凝胶电泳可见4.2kb的目的条带(图1)，序列如SEQ ID No.4所示。
2)RSV-rLong/A2cp反基因组的基因片段合成
根据已知RSVLong株反基因组及表达附属元件序列，将其分成A(MluI-AsuII)、B(AsuII-NheI)、C(NheI-NruI)3段，分别化学合成，其中A段包括T7启动子、锤头状核酶、RSVLong株反基因组1-5184nt、A2cp突变：N 799G>A,aa267Val>Ile及删除了SH基因的非编码区的112nt(RSVLong株反基因组4497-4608nt)并在SH基因编码区末端引入4处同义突变，即SH186C>T、SH189C>T、SH192A>T、SH194,195AG>GA；B段包括RSVLong株反基因组5179-11006nt及A2cp突变：F653,654AA>CG,aa218Glu>Ala、F1568,1569CA>TT,aa523Thr>Ile、L956,957GT>AC,aa319Cys>Tyr；C段包括RSVLong株反基因组11001-15226n及L 5068,5070C>T,T>C,aa1690His>Tyr A2cp突变、丁型肝炎核酶序列、T7终止子序列。合成后的片段分别插入pBRB载体中，命名为pBRB-RSV-A(1-5135nt)、pBRB-RSV-B(5135-10956nt)、pBRB-RSV-C(10956-15405nt)质粒，将获得的质粒分别用MluI、AsuII(图2A)；AsuII、NheI(图2B)；NruI、NheI(图2C)进行酶切鉴定，分别于5143bp、5828bp、4456bp处得到目的条带(图2)，即基因片段。
3)pBRB-RSV-rLong/A2cp质粒构建
利用AsuⅡ和NheⅠ内切酶双酶切pBRB-RSV-C质粒和pBRB-RSV-B质粒，分别回收C质粒的7.9kb载体片段和B质粒的5.8kb插入片段，用T4DNA连接酶连接，转化感受态大肠杆菌DH5ɑ，经筛选后可获得pBRB-RSV-CB质粒；利用MluⅠ和AsuⅡ内切酶双酶切pBRB-RSV-CB和pBRB-RSV-A质粒，分别回收前者的13.7kb片段和后者的5.1kb片段，用T4DNA连接酶连接，转化感受态大肠杆菌DH5ɑ，经筛选后可获得pBRB-RSV-CBA质粒，即pBRB-RSV-rLong/A2cp质粒。将最终获得的重组质粒分别用NruI/NheI(1)、AsuII/NheI(2)、AsuII/SacI(3)、SacI/MluI(4)、EcoRV(5)、SpeI(6)、AflII(7)限制性内切酶进行酶切鉴定，分别在相应位置得到目的条带(图3)。经全基因测序后发现pBRB-RSV-rLong/A2cp质粒序列与预期相符，如SEQID No.1所示。
实施例2：pBRB-RSV-rLong/A2cpts质粒的构建
1)载体pBR322改造与实施例1相同，改造后的载体pBRB序列如SEQ ID No.4所示。
2)RSV-rLong/A2cpts反基因组的基因片段合成
RSV-rLong/A2cpts基因序列及分段合成与实施例1基本相同，只是B段基因在实施例1基础上增加了两个ts突变(M2 9T>C；L2491,2492CA>TT,aa831Gln>Leu)。合成后基因片段克隆及酶切鉴定同实施例1。
3)pBRB-RSV-rLong/A2cpts质粒构建
质粒构建过程同实施例1，获得的重组质粒pBRB-RSV-rLong/A2cpts经酶切及测序鉴定后与预期相符，质粒全基因序列如SEQ ID No.2所示。
实施例3：pBRB-RSV-rLong/A2cptsΔSH质粒的构建
1)载体pBR322改造与实施例1相同，改造后的载体pBRB序列如SEQ ID No.4所示。
2)RSV-rLong/A2cptsΔSH反基因组的基因片段合成
RSV-rLong/A2cptsΔSH基因序列及分段合成与实施例2基本相同，只是A段基因在实施例2基础上还删除SH基因的GS-GE之间序列及部分SH-G基因间隔区序(RSVLong株反基因组4218-4496nt和4609-4666nt)。合成后基因片段克隆及酶切鉴定同实施例2。
3)pBRB-RSV-rLong/A2cptsΔSH质粒构建
质粒构建过程同实施例2，获得的重组质粒pBRB-RSV-rLong/A2cptsΔSH经酶切及测序鉴定后与预期相符，质粒全基因序列如SEQ ID No.3所示。
实施例4重组病毒的拯救
将BHK T7/9细胞接种在6孔板，24h后分别转染所述pBRB-RSV-rLong/A2cp、pBRB-RSV-rLong/A2cpts、pBRB-RSV-rLong/A2cptsΔSH质粒和四种辅助蛋白质粒(pCITE-N,P,L,M2-1)至宿主细胞(BHK T7/9)于33℃下培养，其中实验组1转染pBRB-RSV-rLong/A2cp和pCITE-N,P,L,M2-1，实验组2转染pBRB-RSV-rLong/A2cpts和pCITE-N,P,L,M2-1，实验组3转染pBRB-RSV-rLong/A2cptsΔSH和pCITE-N,P,L,M2-1，mock组不转染质粒。转染96h后盲传至Vero细胞继续放置于33℃下培养，实验组在转染(图4)和盲传(图5)时出现细胞融合，初步判断成功拯救出重组病毒。
实施例5重组病毒的鉴定
利用免疫酶法和实时定量PCR(引物如序列表SEQ ID No.5H和SEQ ID No.6所示，表1)检测连续传代后重组病毒的滴度(图6，空斑形成单位)和复制能力(图7，N基因的拷贝数)，结果都显示拯救出的重组病毒与野生型RSV病毒相比复制能力明显降低(*P<0.05；**P<0.01)，表明重组病毒在体外明显减毒。设计引物RT-PCR检测第10、20、30代重组病毒突变位点，所用引物如表1所示，，测序结果显示pBRB-RSV-rLongA2cp(图8)、pBRB-RSV-rLong/A2cpts(图9)、pBRB-RSV-rLong/A2cptsΔSH(图10)突变位点没有变化，说明拯救出的重组病毒在体外连续传代的过程中是稳定的。
表1检测重组病毒突变位点的引物
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定，对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动，这里无法对所有的实施方式予以穷举，凡是属于本发明的技术方案所引伸出的显而易见的变化或变动仍处于本发明的保护范围之列。
SEQUENCE LISTING
<110> 北京交通大学
<120> 基于反向遗传学构建的RSV反基因组质粒及其应用
<130> JLC17I0280E
<160> 18
<170> PatentIn version 3.5
<210> 1
<211> 18815
<212> DNA
<213> 质粒pBRB-RSV-rLong/A2cp
<400> 1
acgcgttaat acgactcact atagggccgt ctgatgaggc cgaaaggccg aaactccgta 60
aggagtcacg ggaaaaaatg cgtacaacaa acttgcgtaa accaaaaaaa tggggcaaat 120
aagaatttga taagtaccac ttaaatttaa ctcccttggt tagagatggg cagcaattcg 180
ttgagtatga taaaagttag attacaaaat ttgtttgaca atgatgaagt agcattgtta 240
aaaataacat gctatactga caaattaata catttaacta atgctttggc taaggcagtg 300
atacatacaa tcaaattgaa tggcattgtg tttgtgcatg ttattacaag tagtgatatt 360
tgccctaata ataatattgt agtaaaatcc aatttcacaa caatgccagt gctacaaaat 420
ggaggttata tatgggaaat gatggaatta acacattgct ctcaacctaa tggtctaata 480
gatgacaatt gtgaaattaa attctccaaa aaactaagtg attcaacaat gaccaattat 540
atgaatcaat tatctgaatt acttggattt gatcttaatc cataaattat aattaatatc 600
aactagcaaa tcaatgtcac tagcaccatt agttaatata aaacttaaca gaagacaaaa 660
atggggcaaa taaatcaact cagccaaccc aaccatggac acaacccaca atgataccac 720
accacaaaga ctgatgatca cagacatgag accgttgtca cttgagacta caataacatc 780
actaaccaga gacatcataa cacacagatt tatatactta ataaatcatg aatgcatagt 840
gagaaaactt gatgaaagac aggccacatt tacattcctg gtcaactatg aaatgaaact 900
attgcacaaa gtaggaagca ctaaatataa aaaatatact gaatacaaca caaaatatgg 960
cactttccct atgccgatat tcatcaatca tgatgggttc ttagaatgca ttggcattaa 1020
gcctacaaag catactccca taatatacaa gtatgatctc aatccatgaa tttcaacaca 1080
agattcacac aatccaaaac aacaacttta tgcataacta cactccatag tccaaatgga 1140
gcctgaaaat tatagtaatt taaaattaag gagagacata agatagaaga tggggcaaat 1200
acaaagatgg ctcttagcaa agtcaagttg aatgatacac tcaacaaaga tcaacttctg 1260
tcatctagca aatacaccat ccaacggagc acaggagata gtattgatac tcctaattat 1320
gatgtgcaga aacacatcaa taagttatgt ggcatgttat taatcacaga agatgctaat 1380
cataaattca ctgggttaat aggtatgtta tatgctatgt ctaggttagg aagagaagac 1440
accataaaaa tactcagaga tgcgggatat catgtaaaag caaatggagt agatgtaaca 1500
acacatcgtc aagacatcaa tgggaaagaa atgaaatttg aagtgttaac attggcaagc 1560
ttaacaactg aaattcaaat caacattgag atagaatcta gaaaatccta caaaaaaatg 1620
ctaaaagaaa tgggagaggt agctccagaa tacaggcatg attctcctga ttgtgggatg 1680
ataatattat gtatagcagc attagtaata accaaattgg cagcagggga tagatctggt 1740
cttacagccg tgattaggag agctaataat gtcctaaaaa atgaaatgaa acgttacaaa 1800
ggcttactac ccaaggatat agccaacagc ttctatgaag tgtttgaaaa acatccccac 1860
tttatagatg tttttgttca ttttggtata gcacaatctt ccaccagagg tggcagtaga 1920
gttgaaggga tttttgcagg attgtttatg aatgcctatg gtgcagggca agtaatgcta 1980
cggtggggag tcttagcaaa atcaattaaa aatattatgt taggacatgc tagtgtgcaa 2040
gcagaaatgg aacaagttgt tgaggtttat gaatatgccc aaaaattggg tggagaagca 2100
ggattctacc atatattgaa caacccaaaa gcatcattat tatctttgac tcaatttcct 2160
cacttttcca gtgtagtatt aggcaatgct gctggcctag gcataatggg agagtacaga 2220
ggtacaccga ggaatcaaga tctatatgat gcagcaaagg catatgctga acaactcaaa 2280
gaaaatggtg tgattaacta cagtgtatta gacttgacag cagaagaact agaggctatc 2340
aaacatcagc ttaatccaaa agataatgat gtagagcttt gagttaataa aaaatggggc 2400
aaataaatca tcatggaaaa gtttgctcct gaattccatg gagaagatgc aaacaacagg 2460
gctactaaat tcctagaatc aataaagggc aaattcacat cacctaaaga tcccaagaaa 2520
aaagatagta tcatatctgt caactcaata gatatagaag taaccaaaga aagccctata 2580
acatcaaatt caaccattat taacccaaca aatgagacag atgataatgc agggaacaag 2640
cccaattatc aaagaaaacc tctagtaagt ttcaaagaag accctatacc aagtgataat 2700
cccttttcaa aactatacaa agaaaccata gagacatttg ataacaatga agaagaatct 2760
agctattcat atgaagaaat aaatgatcag acgaacgata atataactgc aagattagat 2820
aggattgatg aaaaattaag tgaaatacta ggaatgcttc acacattagt agtagcaagt 2880
gcaggaccta catctgctag ggatggtata agagatgcca tggttggttt aagagaagaa 2940
atgatagaaa aaatcagaac tgaagcatta atgaccaatg acagattaga agctatggca 3000
agactcagga atgaggaaag tgaaaagatg gcaaaagaca catcagatga agtgtctctc 3060
aatccaacat cagagaaatt gaacaacctg ttggaaggga atgatagtga caatgatcta 3120
tcacttgaag atttctgatt agttacaaat ctgcacttca acacacaaca ccaacagaag 3180
accaacaaac aaaccaaccc actcatccaa ccaaacatcc atccgccaat cagccaaaca 3240
gccaacaaaa caaccagcca atccaaaacc agccacctgg aaaaaatcga caatatagtt 3300
acaaaaaaag aaaagggtgg ggcaaatatg gaaacatacg tgaacaagct tcacgaaggc 3360
tccacataca cagctgctgt tcaatacaat gtcctagaaa aagacgatga ccctgcatca 3420
cttacaatat gggtgcccat gttccaatca tctatgccag cagatttact tataaaagaa 3480
ctagctaatg tcaacatact agtgaaacaa atatccacac ccaagggacc ttcactaaga 3540
gtcatgataa actcaagaag tgcattgctt gcacaaatgc ccagcaaatt taccatatgt 3600
gctaatgtgt ccttggatga aagaagcaaa ctggcatatg atgtaaccac accctgtgaa 3660
atcaaggcat gtagtctaac atgcctaaaa tcaaaaaata tgttaactac agttaaagat 3720
ctcactatga agacactcaa ccccacacat gatattattg ctttatgtga atttgaaaac 3780
atagtaacat caaaaaaagt cataatacca acatacctaa gatccatcag tgtcagaaat 3840
aaagatctga acacacttga aaatataaca accactgaat tcaaaaatgc catcacaaat 3900
gcaaaaatca tcccttactc aggattacta ttagtcatca cagtgactga caacaaagga 3960
gcattcaaat acataaagcc gcaaagtcaa ttcatagtag atcttggagc ttacctagaa 4020
aaagaaagta tatattatgt taccacaaat tggaagcaca cagctacacg atttgcaatc 4080
aaacccatgg aagattaacc tttttcctcc acatcagtga gtcaattcat acaaactttc 4140
tacctacatt cttcacttca ccattacaat cacaaacact ctgtggttca accaatcaaa 4200
caaaacttat ctgaagtctc agatcatccc aagtcattgt tcatcagatc tagtaatcaa 4260
ataagttaat aaaaatatac acatggggca aataatcatc ggaggaaatc caactaatca 4320
caatatctgt taacatagac aagtcaacac accagacaga atcaaccaat ggaaaataca 4380
tccataacaa tagaattctc aagcaaattc tggccttact ttacactaat acacatgatc 4440
acaacaataa tctctttgct aatcataatc tccatcatga ctgcaatact aaacaaactt 4500
tgtgaatata acgtattcca taacaaaacc tttgagttac caagagctcg agttaatact 4560
tgataaagta gttaattaaa aatagtcata acaatgaact aggatatcaa gactaacaat 4620
aacgttgggg caaatgcaaa catgtccaaa aacaaggacc aacgcaccgc taagacacta 4680
gaaaagacct gggacactct caatcattta ttattcatat catcgggctt atataagtta 4740
aatcttaaat ctatagcaca aatcacatta tccattctgg caatgataat ctcaacttca 4800
cttataatta cagccatcat attcatagcc tcggcaaacc acaaagtcac actaacaact 4860
gcaatcatac aagatgcaac aagccagatc aagaacacaa ccccaacata cctcactcag 4920
gaccctcagc ttggaatcag cttctccaat ctgtctgaaa ttacatcaca aaccaccacc 4980
atactagctt caacaacacc aggagtgaag tcaaacctgc aacccacaac agtcaagact 5040
aaaaacacaa caacaaccca aacacaaccc agcaagccca ctacaaaaca acgccaaaac 5100
aaaccaccaa acaaacccaa taatgatttt cacttcgaag tgtttaactt tgtaccctgc 5160
agcatatgca gcaacaatcc aacctgctgg gctatctgca aaagaatacc aaacaaaaaa 5220
ccaggaaaga aaaccaccac caagcctaca aaaaaaccaa ccttcaagac aaccaaaaaa 5280
gatctcaaac ctcaaaccac taaaccaaag gaagtaccca ccaccaagcc cacagaagag 5340
ccaaccatca acaccaccaa aacaaacatc acaactacac tgctcaccaa caacaccaca 5400
ggaaatccaa aactcacaag tcaaatggaa accttccact caacctcctc cgaaggcaat 5460
ctaagccctt ctcaagtctc cacaacatcc gagcacccat cacaaccctc atctccaccc 5520
aacacaacac gccagtagtt attaaaaaac atattatcac aaaaggcctt gaccaactca 5580
aacagaatca aaataaactc tggggcaaat aacaatggag ttgccaatcc tcaaagcaaa 5640
tgcaattacc acaatcctcg ctgcagtcac attttgcttt gcttctagtc aaaacatcac 5700
tgaagaattt tatcaatcaa catgcagtgc agttagcaaa ggctatctta gtgctctaag 5760
aactggttgg tatactagtg ttataactat agaattaagt aatatcaagg aaaataagtg 5820
taatggaaca gatgctaagg taaaattgat aaaccaagaa ttagataaat ataaaaatgc 5880
tgtaacagaa ttgcagttgc tcatgcaaag cacaacagca gcaaacaatc gagccagaag 5940
agaactacca aggtttatga attatacact caacaatacc aaaaaaacca atgtaacatt 6000
aagcaagaaa aggaaaagaa gatttcttgg ttttttgtta ggtgttggat ctgcaatcgc 6060
cagtggcatt gctgtatcta aggtcctgca cttagaagga gaagtgaaca agatcaaaag 6120
tgctctacta tccacaaaca aggccgtagt cagcttatca aatggagtta gtgtcttaac 6180
cagcaaagtg ttagacctca aaaactatat agataaacaa ttgttaccta ttgtgaataa 6240
gcaaagctgc agaatatcaa atatagcgac tgtgatagag ttccaacaaa agaacaacag 6300
actactagag attaccaggg aatttagtgt taatgcaggt gtaactacac ctgtaagcac 6360
ttacatgtta actaatagtg aattattgtc attaatcaat gatatgccta taacaaatga 6420
tcagaaaaag ttaatgtcca acaatgttca aatagttaga cagcaaagtt actctatcat 6480
gtccataata aaagaggaag tcttagcata tgtagtacaa ttaccactat atggtgtgat 6540
agatacacct tgttggaaat tacacacatc ccctctatgt acaaccaaca caaaagaagg 6600
gtcaaacatc tgtttaacaa gaactgacag aggatggtac tgtgacaatg caggatcagt 6660
atctttcttc ccacaagctg aaacatgtaa agttcaatcg aatcgagtat tttgtgacac 6720
aatgaacagt ttaacattac caagtgaagt aaatctctgc aatgttgaca tattcaatcc 6780
caaatatgat tgtaaaatta tgacttcaaa aacagatgta agcagctccg ttatcacatc 6840
tctaggagcc attgtgtcat gctatggcaa aactaaatgt acagcatcca ataaaaatcg 6900
tggaatcata aagacatttt ctaacgggtg tgattatgta tcaaataaag gggtggacac 6960
tgtgtctgta ggtaacacat tatattatgt aaataagcaa gaaggcaaaa gtctctatgt 7020
aaaaggtgaa ccaataataa atttctatga cccattagta ttcccctctg atgaatttga 7080
tgcatcaata tctcaagtca atgagaagat taaccagagt ttagcattta ttcgtaaatc 7140
cgatgaatta ttacatcatg taaatgctgg taaatcaacc attaatatca tgataactac 7200
tataattata gtgattatag taatattgtt atcattaatt gctgttggac tgctcctata 7260
ctgtaaggcc agaagcacac cagtcacact aagcaaggat caactgagtg gtataaataa 7320
tattgcattt agtaactgaa taaaaatagc acctaatcat gttcttacaa tggtttacta 7380
tctgctcata gacaacccat ctatcattgg attttcttaa aatctgaact tcatcgaaac 7440
tcttatctat aaaccatctc acttacacta tttaagtaga ttcctagttt atagttatat 7500
aaaacacaat tgaataccag attaacttac tatctgtaaa aatgagaact ggggcaaata 7560
tgtcacgaag gaatccttgc aaatttgaaa ttcgaggtca ttgcttgaat ggtaagagat 7620
gtcattttag tcataattat tttgaatggc caccccatgc actgctcgta agacaaaact 7680
ttatgttaaa cagaatactt aagtctatgg ataaaagtat agatacctta tcagaaataa 7740
gtggagctgc agagttggac agaacagaag agtatgctct tggtgtagtt ggagtgctag 7800
agagttatat aggatcaata aataatataa ctaaacaatc agcatgtgtt gccatgagca 7860
aactcctcac tgaactcaat agtgatgata tcaaaaaact gagagacaat gaagagctaa 7920
attcacccaa gataagagtg tacaatactg tcatatcata tattgaaagc aacaggaaaa 7980
acaataaaca aactatccat ctgttaaaaa gattgccagc agacgtattg aagaaaacca 8040
tcaaaaacac attggatatc cacaagagca taaccatcaa caacccaaaa gaattaactg 8100
ttagtgatac aaatgaccat gccaaaaata atgatactac ctgacaaata tccttgtagt 8160
ataacttcca tactaataac aagtagatgt agagtcacta tgtataatcg aaagaacaca 8220
ctatatttca atcaaaacaa cccaaataac catatgtact caccgaatca aacattcaat 8280
gaaatccatt ggacctcaca agacttgatt gacacaattc aaaattttct acagcatcta 8340
ggtgttattg aggatatata tacaatatat atattagtgt cataacactc aatcctaata 8400
ctgaccatat cgttgaatta ttaattcaaa taattcaagc tgtgggacaa aatggatccc 8460
attattaatg gaaattctgc taatgtttat ctaaccgata gttatttaaa aggtgttatc 8520
tctttctcag agtgtaatgc tttaggaagt tacatattca atggtcctta tctcaaaaat 8580
gattatacca acttaattag tagacaaaat ccattaatag aacacatgaa tctaaagaaa 8640
ctaaatataa cacagtcctt aatatctaag tatcataaag gtgaaataaa attagaagag 8700
cctacttatt ttcagtcatt acttatgaca tacaagagta tgacctcgtt ggaacagatt 8760
gctaccacta atttacttaa aaagataata agaagagcta tagaaataag tgatgtcaaa 8820
gtctatgcta tattgaataa actagggctt aaagaaaagg acaagattaa atccaacaat 8880
ggacaggatg aagacaactc agttattacg accataatca aagatgatat actttcagct 8940
gttaaggata atcaatctca tcttaaagca gacaaaaatc actctacaaa acaaaaagac 9000
acaatcaaaa caacactctt gaagaaatta atgtgttcaa tgcagcatcc tccatcatgg 9060
ttaatacatt ggtttaattt atacacaaaa ttaaacaaca tattaacaca gtatcgttca 9120
aatgaggtta aaaaccatgg gtttatattg atagataatc aaactcttag tggatttcaa 9180
tttattttga atcaatatgg ttgtatagtt tatcataagg aactcaaaag aattactgtg 9240
acaacctata atcaattctt gacatggaaa gatattagcc ttagtagatt aaatgtttgt 9300
ttaattacat ggattagtaa ctgcttgaac acattaaata aaagcttagg cttaagatgc 9360
ggattcaata atgttatctt gacacaacta ttcctttatg gtgattacat actaaagcta 9420
tttcacaatg aggggttcta cataataaaa gaggtagagg gatttattat gtctctaatt 9480
ttaaatataa cagaagaaga tcaattcaga aaacgatttt ataatagtat gctcaacaac 9540
atcacagatg ctgctaataa agctcagaaa aatctgctat caagagtatg tcatacatta 9600
ttagataaga cagtatccga taatataata aatggcagat ggataattct attaagtaag 9660
ttccttaaat taattaagct tgcaggtgac aataacctta acaatctgag tgaactatat 9720
tttttgttca gaatatttgg acacccaatg gtagatgaaa gacaagccat ggatgctgtt 9780
aaagttaatt gcaatgagac caaattttac ttgttaagca gtttgagtat gttaagaggt 9840
gcctttatat atagaattat aaaagggttt gtaaataatt acaacagatg gcctacttta 9900
agaaatgcta ttgttttacc cttaagatgg ttaacttact ataaactaaa cacttatcct 9960
tctttgttgg aacttacaga aagagatttg attgtgttat caggactacg tttctatcgt 10020
gagtttcggt tgcctaaaaa agtggatctt gaaatgatta taaatgataa agctatatca 10080
ccccctaaaa atttgatatg gactagtttc cctagaaatt atatgccgtc acacatacaa 10140
aactatatag aacatgaaaa attaaaattt tccgagagtg ataaatcaag aagagtatta 10200
gagtattatt taagagataa caaattcaat gaatgtgatt tatacaactg tgtagttaat 10260
caaagttatc tcaacaaccc taatcatgtg gtatcattga caggcaaaga aagagaactc 10320
agtgtaggta gaatgtttgc aatgcaaccg ggaatgttca gacaggttca aatattggca 10380
gagaaaatga tagctgaaaa cattttacaa ttctttcctg aaagtcttac aagatatggt 10440
gatctagaac tacaaaaaat attagaattg aaagcaggaa taagtaacaa atcaaatcgc 10500
tacaatgata attacaacaa ttacattagt aagtgctcta tcatcacaga tctcagcaaa 10560
ttcaatcaag catttcgata tgaaacgtca tgtatttgta gtgatgtgct ggatgaactg 10620
catggtgtac aatctctatt ttcctggtta catttaacta ttcctcatgt cacaataata 10680
tgcacatata ggcatgcacc cccctatata agagatcata ttgtagatct taacaatgta 10740
gatgaacaaa gtggattata tagatatcac atgggtggta ttgaagggtg gtgtcaaaaa 10800
ctatggacca tagaagctat atcactattg gatctaatat ctctcaaagg gaaattctca 10860
attactgctt taattaatgg tgacaatcaa tcaatagata taagcaaacc agtcagactc 10920
atggaaggtc aaactcatgc tcaagcagat tatttgctag cattaaatag ccttaaatta 10980
ctgtataaag agtatgcagg cataggtcac aaattaaaag gaactgagac ttatatatca 11040
cgagatatgc aatttatgag taaaacaatt caacataacg gtgtatatta ccctgctagt 11100
ataaagaaag tcctaagagt gggaccgtgg ataaacacta tacttgatga tttcaaagtg 11160
agtctagaat ctataggtag tttgacacaa gaattagaat atagaggtga aagtctatta 11220
tgcagtttaa tatttagaaa tgtatggtta tataatcaaa ttgctctaca attaaaaaat 11280
catgcgttat gtaacaataa attatatttg gacatattaa aggttctgaa acacttaaaa 11340
acctttttta atcttgataa tattgataca gcattaacat tgtatatgaa tttacccatg 11400
ttatttggtg gtggtgatcc caacttgtta tatcgaagtt tctatagaag aactcctgat 11460
ttcctcacag aggctatagt tcactctgtg ttcatactta gttattatac aaaccatgac 11520
ttaaaagata aacttcaaga tttgtcagat gatagattga ataagttctt aacatgcata 11580
atcacgtttg acaaaaaccc taatgctgaa ttcgtaacat tgatgagaga tcctcaagct 11640
ttagggtctg agagacaagc taaaattact agtgaaatca atagactggc agttacagag 11700
gttttgagta cagctccaaa caaaatattc tccaaaagtg cacaacatta taccactaca 11760
gagatagatc taaatgatat tatgcaaaat atagaaccta catatcctca cgggctaaga 11820
gttgtttatg aaagtttacc cttttataaa gcagagaaaa tagtaaatct tatatcaggt 11880
acaaaatcta taactaacat actggaaaag acttctgcca tagacttaac agatattgat 11940
agagccactg agatgatgag gaaaaacata actttgctta taaggatact tccattggat 12000
tgtaacagag ataaaagaga aatattgagt atggaaaacc taagtattac tgaattaagc 12060
aaatatgtta gggaaagatc ttggtcttta tccaatatag ttggtgttac atcacccagt 12120
atcatgtata caatggacat caaatataca acaagcacta tagctagtgg cataattata 12180
gagaaatata atgttaacag tttaacacgt ggtgagagag gaccaactaa accatgggtt 12240
ggttcatcta cacaagagaa aaaaacaatg ccagtttata atagacaagt tttaaccaaa 12300
aaacaaagag atcaaataga tctattagca aaattggatt gggtgtatgc atctatagat 12360
aacaaggatg aattcatgga agaactcagc ataggaaccc ttgggttaac atatgaaaag 12420
gccaaaaaat tatttccaca atatttaagt gtcaactatt tgcatcgcct tacagtcagt 12480
agtagaccat gtgaattccc tgcatcaata ccagcttata gaacaacaaa ttatcacttt 12540
gacactagcc ctattaatcg catattaaca gaaaagtatg gtgatgaaga tattgacata 12600
gtattccaaa actgtataag ctttggcctt agcttaatgt cagtagtaga acaatttact 12660
aatgtatgtc ctaacagaat tattctcata cctaagctta atgagataca tttgatgaaa 12720
cctcccatat tcacaggtga tgttgatatt cacaagttaa aacaagtgat acaaaaacag 12780
catatgtttt taccagacaa aataagtttg actcaatatg tggaattatt cttaagtaac 12840
aaaacactca aatctggatc tcatgttaat tctaatttaa tattggcaca taaaatatct 12900
gactattttc ataatactta cattttaagt actaatttag ctggacattg gattctaatt 12960
atacaactta tgaaagattc taaaggtatt tttgaaaaag attggggaga gggatatata 13020
actgatcata tgtttattaa tttgaaagtt ttcttcaatg cttataagac ctatctcttg 13080
tgttttcata aaggttatgg caaagcaaaa ctggagtgtg atatgaacac ttcagatctt 13140
ctatgtgtat tggaattaat agacagtagt tattggaagt ctatgtctaa ggtattttta 13200
gaacaaaaag ttatcaaata cattcttagc caagatgcaa gtttacatag agtaaaagga 13260
tgtcatagct tcaaattatg gtttcttaaa cgtcttaatg tagcagaatt tacagtttgc 13320
ccttgggttg ttaacataga ttatcatcca acacatatga aagcaatatt aacttatata 13380
gatcttgtta gaatgggatt gataaatata gatagaatac acattaaaaa taaacacaaa 13440
ttcaatgatg aattttatac ttctaatctc ttttacatta attataactt ctcagataat 13500
actcatctat taactaaata cataaggatt gctaattcag aattagaaaa taattacaac 13560
aaattatatc atcctacacc agaaacccta gagaatatac tagccaatcc gattaaaagt 13620
aatgacaaaa agacactgaa cgactattgt ataggtaaaa atgttgactc aataatgtta 13680
ccattgttat ctaataagaa gcttgttaaa tcgtctgcaa tgattagaac caattacagc 13740
aaacaagacc tgtacaatct attccctacg gttgtgatcg atagaattat agatcattca 13800
ggtaatacag ccaaatccaa ccaactttac actactactt cccatcaaat atctttagtg 13860
cacaatagca catcacttta ttgcatgctt ccttggcatc atattaatag attcaatttt 13920
gtatttagtt ctacaggttg taaaattagt atagagtata ttttaaaaga ccttaaaatt 13980
aaagatccta attgtatagc attcataggt gaaggagcag ggaatttatt attgcgtaca 14040
gtggtggaac ttcatcctga cataagatat atttacagaa gtctgaaaga ttgcaatgat 14100
catagtttac ctattgagtt tttaaggcta tacaatggac atatcaacat tgattatggt 14160
gaaaatttga ccattcctgc tacagatgca accaacaaca ttcattggtc ttatttacat 14220
ataaagtttg ctgaacctat cagtcttttt gtatgtgatg ccgaattgcc tgtaacagtc 14280
aactggagta aaattataat agaatggagc aagcatgtaa gaaaatgcaa gtactgttcc 14340
tcagttaata aatgtacgtt aatagtaaaa tatcatgctc aagatgatat tgatttcaaa 14400
ttagacaata taactatatt aaaaacttat gtatgcttag gcagtaagtt aaagggatcg 14460
gaggtttact tagtccttac aataggtcct gcaaatatat ttccagtatt taatgtagta 14520
caaaatgcta aattgatact atcaagaacc aaaaatttca tcatgcctaa gaaagctgat 14580
aaagagtcta ttgatgcaaa tattaaaagt ttgataccct ttctttgtta ccctataaca 14640
aaaaaaggaa ttaatactgc attgtcaaaa ctaaagagtg ttgttagtgg agatatacta 14700
tcatattcta tagctggacg gaatgaagtt ttcagcaata aacttataaa tcataagcat 14760
atgaacatct taaagtggtt caatcatgtt ttaaatttca gatcaacaga actaaactat 14820
aaccatttat atatggtaga atctacatat ccttacctaa gtgaattgtt aaacagcttg 14880
acaactaatg aacttaaaaa actgattaaa atcacaggta gtctgttata caactttcat 14940
aatgaataat gaataaagat cttataataa aaattcctat agctatacac tagcactgta 15000
ttcaattata gttattaaaa aattaaaaat catataattt tttataaaaa taacttttag 15060
tgaactaatc ctaaagttat cattttgatc taggaggaat aaatttaaat cccaatctaa 15120
ttggtttata tgtgtattaa ctaaactacg agatattagt ttttgacact ttttttctcg 15180
tgggtcggca tggcatctcc acctcctcgc ggtccgacct gggcatccga aggaggacgc 15240
acgtccactc ggatggctaa gggagagcca ctgctaacaa agcccgaaag gaagctgagt 15300
tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct 15360
tgaggggttt tttgctgaaa ggaggaacta tatccggatt cgcgacgcga ggctggatgg 15420
ccttccccat tatgattctt ctcgcttccg gcggcatcgg gatgcccgcg ttgcaggcca 15480
tgctgtccag gcaggtagat gacgaccatc agggacagct tcaaggatcg ctcgcggctc 15540
ttaccagcct aacttcgatc actggaccgc tgatcgtcac ggcgatttat gccgcctcgg 15600
cgagcacatg gaacgggttg gcatggattg taggcgccgc cctatacctt gtctgcctcc 15660
ccgcgttgcg tcgcggtgca tggagccggg ccacctcgac ctgaatggaa gccggcggca 15720
cctcgctaac ggattcacca ctccaagaat tggagccaat caattcttgc ggagaactgt 15780
gaatgcgcaa accaaccctt ggcagaacat atccatcgcg tccgccatct ccagcagccg 15840
cacgcggcgc atctcgggca gcgttgggtc ctggccacgg gtgcgcatga tcgtgctcct 15900
gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc 15960
gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac 16020
atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg 16080
caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac 16140
atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat 16200
ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt 16260
aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa 16320
tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg 16380
ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga 16440
acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct 16500
cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac 16560
agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt 16620
tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg 16680
cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata 16740
ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact 16800
gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 16860
atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 16920
caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 16980
cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 17040
taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 17100
ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 17160
tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 17220
gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 17280
ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 17340
aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 17400
aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 17460
agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 17520
cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 17580
gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 17640
atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 17700
gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 17760
tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 17820
gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 17880
ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 17940
actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 18000
ccagttaata gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg 18060
tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 18120
cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 18180
ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 18240
ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 18300
tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat 18360
agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 18420
atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 18480
gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 18540
aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 18600
tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 18660
aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa 18720
gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt 18780
cttcaagaat tctcatgttt gacagcttat catcg 18815
<210> 2
<211> 18815
<212> DNA
<213> 质粒pBRB-RSV-rLong/A2cpts
<400> 2
acgcgttaat acgactcact atagggccgt ctgatgaggc cgaaaggccg aaactccgta 60
aggagtcacg ggaaaaaatg cgtacaacaa acttgcgtaa accaaaaaaa tggggcaaat 120
aagaatttga taagtaccac ttaaatttaa ctcccttggt tagagatggg cagcaattcg 180
ttgagtatga taaaagttag attacaaaat ttgtttgaca atgatgaagt agcattgtta 240
aaaataacat gctatactga caaattaata catttaacta atgctttggc taaggcagtg 300
atacatacaa tcaaattgaa tggcattgtg tttgtgcatg ttattacaag tagtgatatt 360
tgccctaata ataatattgt agtaaaatcc aatttcacaa caatgccagt gctacaaaat 420
ggaggttata tatgggaaat gatggaatta acacattgct ctcaacctaa tggtctaata 480
gatgacaatt gtgaaattaa attctccaaa aaactaagtg attcaacaat gaccaattat 540
atgaatcaat tatctgaatt acttggattt gatcttaatc cataaattat aattaatatc 600
aactagcaaa tcaatgtcac tagcaccatt agttaatata aaacttaaca gaagacaaaa 660
atggggcaaa taaatcaact cagccaaccc aaccatggac acaacccaca atgataccac 720
accacaaaga ctgatgatca cagacatgag accgttgtca cttgagacta caataacatc 780
actaaccaga gacatcataa cacacagatt tatatactta ataaatcatg aatgcatagt 840
gagaaaactt gatgaaagac aggccacatt tacattcctg gtcaactatg aaatgaaact 900
attgcacaaa gtaggaagca ctaaatataa aaaatatact gaatacaaca caaaatatgg 960
cactttccct atgccgatat tcatcaatca tgatgggttc ttagaatgca ttggcattaa 1020
gcctacaaag catactccca taatatacaa gtatgatctc aatccatgaa tttcaacaca 1080
agattcacac aatccaaaac aacaacttta tgcataacta cactccatag tccaaatgga 1140
gcctgaaaat tatagtaatt taaaattaag gagagacata agatagaaga tggggcaaat 1200
acaaagatgg ctcttagcaa agtcaagttg aatgatacac tcaacaaaga tcaacttctg 1260
tcatctagca aatacaccat ccaacggagc acaggagata gtattgatac tcctaattat 1320
gatgtgcaga aacacatcaa taagttatgt ggcatgttat taatcacaga agatgctaat 1380
cataaattca ctgggttaat aggtatgtta tatgctatgt ctaggttagg aagagaagac 1440
accataaaaa tactcagaga tgcgggatat catgtaaaag caaatggagt agatgtaaca 1500
acacatcgtc aagacatcaa tgggaaagaa atgaaatttg aagtgttaac attggcaagc 1560
ttaacaactg aaattcaaat caacattgag atagaatcta gaaaatccta caaaaaaatg 1620
ctaaaagaaa tgggagaggt agctccagaa tacaggcatg attctcctga ttgtgggatg 1680
ataatattat gtatagcagc attagtaata accaaattgg cagcagggga tagatctggt 1740
cttacagccg tgattaggag agctaataat gtcctaaaaa atgaaatgaa acgttacaaa 1800
ggcttactac ccaaggatat agccaacagc ttctatgaag tgtttgaaaa acatccccac 1860
tttatagatg tttttgttca ttttggtata gcacaatctt ccaccagagg tggcagtaga 1920
gttgaaggga tttttgcagg attgtttatg aatgcctatg gtgcagggca agtaatgcta 1980
cggtggggag tcttagcaaa atcaattaaa aatattatgt taggacatgc tagtgtgcaa 2040
gcagaaatgg aacaagttgt tgaggtttat gaatatgccc aaaaattggg tggagaagca 2100
ggattctacc atatattgaa caacccaaaa gcatcattat tatctttgac tcaatttcct 2160
cacttttcca gtgtagtatt aggcaatgct gctggcctag gcataatggg agagtacaga 2220
ggtacaccga ggaatcaaga tctatatgat gcagcaaagg catatgctga acaactcaaa 2280
gaaaatggtg tgattaacta cagtgtatta gacttgacag cagaagaact agaggctatc 2340
aaacatcagc ttaatccaaa agataatgat gtagagcttt gagttaataa aaaatggggc 2400
aaataaatca tcatggaaaa gtttgctcct gaattccatg gagaagatgc aaacaacagg 2460
gctactaaat tcctagaatc aataaagggc aaattcacat cacctaaaga tcccaagaaa 2520
aaagatagta tcatatctgt caactcaata gatatagaag taaccaaaga aagccctata 2580
acatcaaatt caaccattat taacccaaca aatgagacag atgataatgc agggaacaag 2640
cccaattatc aaagaaaacc tctagtaagt ttcaaagaag accctatacc aagtgataat 2700
cccttttcaa aactatacaa agaaaccata gagacatttg ataacaatga agaagaatct 2760
agctattcat atgaagaaat aaatgatcag acgaacgata atataactgc aagattagat 2820
aggattgatg aaaaattaag tgaaatacta ggaatgcttc acacattagt agtagcaagt 2880
gcaggaccta catctgctag ggatggtata agagatgcca tggttggttt aagagaagaa 2940
atgatagaaa aaatcagaac tgaagcatta atgaccaatg acagattaga agctatggca 3000
agactcagga atgaggaaag tgaaaagatg gcaaaagaca catcagatga agtgtctctc 3060
aatccaacat cagagaaatt gaacaacctg ttggaaggga atgatagtga caatgatcta 3120
tcacttgaag atttctgatt agttacaaat ctgcacttca acacacaaca ccaacagaag 3180
accaacaaac aaaccaaccc actcatccaa ccaaacatcc atccgccaat cagccaaaca 3240
gccaacaaaa caaccagcca atccaaaacc agccacctgg aaaaaatcga caatatagtt 3300
acaaaaaaag aaaagggtgg ggcaaatatg gaaacatacg tgaacaagct tcacgaaggc 3360
tccacataca cagctgctgt tcaatacaat gtcctagaaa aagacgatga ccctgcatca 3420
cttacaatat gggtgcccat gttccaatca tctatgccag cagatttact tataaaagaa 3480
ctagctaatg tcaacatact agtgaaacaa atatccacac ccaagggacc ttcactaaga 3540
gtcatgataa actcaagaag tgcattgctt gcacaaatgc ccagcaaatt taccatatgt 3600
gctaatgtgt ccttggatga aagaagcaaa ctggcatatg atgtaaccac accctgtgaa 3660
atcaaggcat gtagtctaac atgcctaaaa tcaaaaaata tgttaactac agttaaagat 3720
ctcactatga agacactcaa ccccacacat gatattattg ctttatgtga atttgaaaac 3780
atagtaacat caaaaaaagt cataatacca acatacctaa gatccatcag tgtcagaaat 3840
aaagatctga acacacttga aaatataaca accactgaat tcaaaaatgc catcacaaat 3900
gcaaaaatca tcccttactc aggattacta ttagtcatca cagtgactga caacaaagga 3960
gcattcaaat acataaagcc gcaaagtcaa ttcatagtag atcttggagc ttacctagaa 4020
aaagaaagta tatattatgt taccacaaat tggaagcaca cagctacacg atttgcaatc 4080
aaacccatgg aagattaacc tttttcctcc acatcagtga gtcaattcat acaaactttc 4140
tacctacatt cttcacttca ccattacaat cacaaacact ctgtggttca accaatcaaa 4200
caaaacttat ctgaagtctc agatcatccc aagtcattgt tcatcagatc tagtaatcaa 4260
ataagttaat aaaaatatac acatggggca aataatcatc ggaggaaatc caactaatca 4320
caatatctgt taacatagac aagtcaacac accagacaga atcaaccaat ggaaaataca 4380
tccataacaa tagaattctc aagcaaattc tggccttact ttacactaat acacatgatc 4440
acaacaataa tctctttgct aatcataatc tccatcatga ctgcaatact aaacaaactt 4500
tgtgaatata acgtattcca taacaaaacc tttgagttac caagagctcg agttaatact 4560
tgataaagta gttaattaaa aatagtcata acaatgaact aggatatcaa gactaacaat 4620
aacgttgggg caaatgcaaa catgtccaaa aacaaggacc aacgcaccgc taagacacta 4680
gaaaagacct gggacactct caatcattta ttattcatat catcgggctt atataagtta 4740
aatcttaaat ctatagcaca aatcacatta tccattctgg caatgataat ctcaacttca 4800
cttataatta cagccatcat attcatagcc tcggcaaacc acaaagtcac actaacaact 4860
gcaatcatac aagatgcaac aagccagatc aagaacacaa ccccaacata cctcactcag 4920
gaccctcagc ttggaatcag cttctccaat ctgtctgaaa ttacatcaca aaccaccacc 4980
atactagctt caacaacacc aggagtgaag tcaaacctgc aacccacaac agtcaagact 5040
aaaaacacaa caacaaccca aacacaaccc agcaagccca ctacaaaaca acgccaaaac 5100
aaaccaccaa acaaacccaa taatgatttt cacttcgaag tgtttaactt tgtaccctgc 5160
agcatatgca gcaacaatcc aacctgctgg gctatctgca aaagaatacc aaacaaaaaa 5220
ccaggaaaga aaaccaccac caagcctaca aaaaaaccaa ccttcaagac aaccaaaaaa 5280
gatctcaaac ctcaaaccac taaaccaaag gaagtaccca ccaccaagcc cacagaagag 5340
ccaaccatca acaccaccaa aacaaacatc acaactacac tgctcaccaa caacaccaca 5400
ggaaatccaa aactcacaag tcaaatggaa accttccact caacctcctc cgaaggcaat 5460
ctaagccctt ctcaagtctc cacaacatcc gagcacccat cacaaccctc atctccaccc 5520
aacacaacac gccagtagtt attaaaaaac atattatcac aaaaggcctt gaccaactca 5580
aacagaatca aaataaactc tggggcaaat aacaatggag ttgccaatcc tcaaagcaaa 5640
tgcaattacc acaatcctcg ctgcagtcac attttgcttt gcttctagtc aaaacatcac 5700
tgaagaattt tatcaatcaa catgcagtgc agttagcaaa ggctatctta gtgctctaag 5760
aactggttgg tatactagtg ttataactat agaattaagt aatatcaagg aaaataagtg 5820
taatggaaca gatgctaagg taaaattgat aaaccaagaa ttagataaat ataaaaatgc 5880
tgtaacagaa ttgcagttgc tcatgcaaag cacaacagca gcaaacaatc gagccagaag 5940
agaactacca aggtttatga attatacact caacaatacc aaaaaaacca atgtaacatt 6000
aagcaagaaa aggaaaagaa gatttcttgg ttttttgtta ggtgttggat ctgcaatcgc 6060
cagtggcatt gctgtatcta aggtcctgca cttagaagga gaagtgaaca agatcaaaag 6120
tgctctacta tccacaaaca aggccgtagt cagcttatca aatggagtta gtgtcttaac 6180
cagcaaagtg ttagacctca aaaactatat agataaacaa ttgttaccta ttgtgaataa 6240
gcaaagctgc agaatatcaa atatagcgac tgtgatagag ttccaacaaa agaacaacag 6300
actactagag attaccaggg aatttagtgt taatgcaggt gtaactacac ctgtaagcac 6360
ttacatgtta actaatagtg aattattgtc attaatcaat gatatgccta taacaaatga 6420
tcagaaaaag ttaatgtcca acaatgttca aatagttaga cagcaaagtt actctatcat 6480
gtccataata aaagaggaag tcttagcata tgtagtacaa ttaccactat atggtgtgat 6540
agatacacct tgttggaaat tacacacatc ccctctatgt acaaccaaca caaaagaagg 6600
gtcaaacatc tgtttaacaa gaactgacag aggatggtac tgtgacaatg caggatcagt 6660
atctttcttc ccacaagctg aaacatgtaa agttcaatcg aatcgagtat tttgtgacac 6720
aatgaacagt ttaacattac caagtgaagt aaatctctgc aatgttgaca tattcaatcc 6780
caaatatgat tgtaaaatta tgacttcaaa aacagatgta agcagctccg ttatcacatc 6840
tctaggagcc attgtgtcat gctatggcaa aactaaatgt acagcatcca ataaaaatcg 6900
tggaatcata aagacatttt ctaacgggtg tgattatgta tcaaataaag gggtggacac 6960
tgtgtctgta ggtaacacat tatattatgt aaataagcaa gaaggcaaaa gtctctatgt 7020
aaaaggtgaa ccaataataa atttctatga cccattagta ttcccctctg atgaatttga 7080
tgcatcaata tctcaagtca atgagaagat taaccagagt ttagcattta ttcgtaaatc 7140
cgatgaatta ttacatcatg taaatgctgg taaatcaacc attaatatca tgataactac 7200
tataattata gtgattatag taatattgtt atcattaatt gctgttggac tgctcctata 7260
ctgtaaggcc agaagcacac cagtcacact aagcaaggat caactgagtg gtataaataa 7320
tattgcattt agtaactgaa taaaaatagc acctaatcat gttcttacaa tggtttacta 7380
tctgctcata gacaacccat ctatcattgg attttcttaa aatctgaact tcatcgaaac 7440
tcttatctat aaaccatctc acttacacta tttaagtaga ttcctagttt atagttatat 7500
aaaacacaat tgaataccag attaacttac tatctgtaaa aatgagaact ggggcaaaca 7560
tgtcacgaag gaatccttgc aaatttgaaa ttcgaggtca ttgcttgaat ggtaagagat 7620
gtcattttag tcataattat tttgaatggc caccccatgc actgctcgta agacaaaact 7680
ttatgttaaa cagaatactt aagtctatgg ataaaagtat agatacctta tcagaaataa 7740
gtggagctgc agagttggac agaacagaag agtatgctct tggtgtagtt ggagtgctag 7800
agagttatat aggatcaata aataatataa ctaaacaatc agcatgtgtt gccatgagca 7860
aactcctcac tgaactcaat agtgatgata tcaaaaaact gagagacaat gaagagctaa 7920
attcacccaa gataagagtg tacaatactg tcatatcata tattgaaagc aacaggaaaa 7980
acaataaaca aactatccat ctgttaaaaa gattgccagc agacgtattg aagaaaacca 8040
tcaaaaacac attggatatc cacaagagca taaccatcaa caacccaaaa gaattaactg 8100
ttagtgatac aaatgaccat gccaaaaata atgatactac ctgacaaata tccttgtagt 8160
ataacttcca tactaataac aagtagatgt agagtcacta tgtataatcg aaagaacaca 8220
ctatatttca atcaaaacaa cccaaataac catatgtact caccgaatca aacattcaat 8280
gaaatccatt ggacctcaca agacttgatt gacacaattc aaaattttct acagcatcta 8340
ggtgttattg aggatatata tacaatatat atattagtgt cataacactc aatcctaata 8400
ctgaccatat cgttgaatta ttaattcaaa taattcaagc tgtgggacaa aatggatccc 8460
attattaatg gaaattctgc taatgtttat ctaaccgata gttatttaaa aggtgttatc 8520
tctttctcag agtgtaatgc tttaggaagt tacatattca atggtcctta tctcaaaaat 8580
gattatacca acttaattag tagacaaaat ccattaatag aacacatgaa tctaaagaaa 8640
ctaaatataa cacagtcctt aatatctaag tatcataaag gtgaaataaa attagaagag 8700
cctacttatt ttcagtcatt acttatgaca tacaagagta tgacctcgtt ggaacagatt 8760
gctaccacta atttacttaa aaagataata agaagagcta tagaaataag tgatgtcaaa 8820
gtctatgcta tattgaataa actagggctt aaagaaaagg acaagattaa atccaacaat 8880
ggacaggatg aagacaactc agttattacg accataatca aagatgatat actttcagct 8940
gttaaggata atcaatctca tcttaaagca gacaaaaatc actctacaaa acaaaaagac 9000
acaatcaaaa caacactctt gaagaaatta atgtgttcaa tgcagcatcc tccatcatgg 9060
ttaatacatt ggtttaattt atacacaaaa ttaaacaaca tattaacaca gtatcgttca 9120
aatgaggtta aaaaccatgg gtttatattg atagataatc aaactcttag tggatttcaa 9180
tttattttga atcaatatgg ttgtatagtt tatcataagg aactcaaaag aattactgtg 9240
acaacctata atcaattctt gacatggaaa gatattagcc ttagtagatt aaatgtttgt 9300
ttaattacat ggattagtaa ctgcttgaac acattaaata aaagcttagg cttaagatgc 9360
ggattcaata atgttatctt gacacaacta ttcctttatg gtgattacat actaaagcta 9420
tttcacaatg aggggttcta cataataaaa gaggtagagg gatttattat gtctctaatt 9480
ttaaatataa cagaagaaga tcaattcaga aaacgatttt ataatagtat gctcaacaac 9540
atcacagatg ctgctaataa agctcagaaa aatctgctat caagagtatg tcatacatta 9600
ttagataaga cagtatccga taatataata aatggcagat ggataattct attaagtaag 9660
ttccttaaat taattaagct tgcaggtgac aataacctta acaatctgag tgaactatat 9720
tttttgttca gaatatttgg acacccaatg gtagatgaaa gacaagccat ggatgctgtt 9780
aaagttaatt gcaatgagac caaattttac ttgttaagca gtttgagtat gttaagaggt 9840
gcctttatat atagaattat aaaagggttt gtaaataatt acaacagatg gcctacttta 9900
agaaatgcta ttgttttacc cttaagatgg ttaacttact ataaactaaa cacttatcct 9960
tctttgttgg aacttacaga aagagatttg attgtgttat caggactacg tttctatcgt 10020
gagtttcggt tgcctaaaaa agtggatctt gaaatgatta taaatgataa agctatatca 10080
ccccctaaaa atttgatatg gactagtttc cctagaaatt atatgccgtc acacatacaa 10140
aactatatag aacatgaaaa attaaaattt tccgagagtg ataaatcaag aagagtatta 10200
gagtattatt taagagataa caaattcaat gaatgtgatt tatacaactg tgtagttaat 10260
caaagttatc tcaacaaccc taatcatgtg gtatcattga caggcaaaga aagagaactc 10320
agtgtaggta gaatgtttgc aatgcaaccg ggaatgttca gacaggttca aatattggca 10380
gagaaaatga tagctgaaaa cattttacaa ttctttcctg aaagtcttac aagatatggt 10440
gatctagaac tacaaaaaat attagaattg aaagcaggaa taagtaacaa atcaaatcgc 10500
tacaatgata attacaacaa ttacattagt aagtgctcta tcatcacaga tctcagcaaa 10560
ttcaatcaag catttcgata tgaaacgtca tgtatttgta gtgatgtgct ggatgaactg 10620
catggtgtac aatctctatt ttcctggtta catttaacta ttcctcatgt cacaataata 10680
tgcacatata ggcatgcacc cccctatata agagatcata ttgtagatct taacaatgta 10740
gatgaacaaa gtggattata tagatatcac atgggtggta ttgaagggtg gtgtcaaaaa 10800
ctatggacca tagaagctat atcactattg gatctaatat ctctcaaagg gaaattctca 10860
attactgctt taattaatgg tgacaatcaa tcaatagata taagcaaacc agtcagactc 10920
atggaaggtc aaactcatgc tttagcagat tatttgctag cattaaatag ccttaaatta 10980
ctgtataaag agtatgcagg cataggtcac aaattaaaag gaactgagac ttatatatca 11040
cgagatatgc aatttatgag taaaacaatt caacataacg gtgtatatta ccctgctagt 11100
ataaagaaag tcctaagagt gggaccgtgg ataaacacta tacttgatga tttcaaagtg 11160
agtctagaat ctataggtag tttgacacaa gaattagaat atagaggtga aagtctatta 11220
tgcagtttaa tatttagaaa tgtatggtta tataatcaaa ttgctctaca attaaaaaat 11280
catgcgttat gtaacaataa attatatttg gacatattaa aggttctgaa acacttaaaa 11340
acctttttta atcttgataa tattgataca gcattaacat tgtatatgaa tttacccatg 11400
ttatttggtg gtggtgatcc caacttgtta tatcgaagtt tctatagaag aactcctgat 11460
ttcctcacag aggctatagt tcactctgtg ttcatactta gttattatac aaaccatgac 11520
ttaaaagata aacttcaaga tttgtcagat gatagattga ataagttctt aacatgcata 11580
atcacgtttg acaaaaaccc taatgctgaa ttcgtaacat tgatgagaga tcctcaagct 11640
ttagggtctg agagacaagc taaaattact agtgaaatca atagactggc agttacagag 11700
gttttgagta cagctccaaa caaaatattc tccaaaagtg cacaacatta taccactaca 11760
gagatagatc taaatgatat tatgcaaaat atagaaccta catatcctca cgggctaaga 11820
gttgtttatg aaagtttacc cttttataaa gcagagaaaa tagtaaatct tatatcaggt 11880
acaaaatcta taactaacat actggaaaag acttctgcca tagacttaac agatattgat 11940
agagccactg agatgatgag gaaaaacata actttgctta taaggatact tccattggat 12000
tgtaacagag ataaaagaga aatattgagt atggaaaacc taagtattac tgaattaagc 12060
aaatatgtta gggaaagatc ttggtcttta tccaatatag ttggtgttac atcacccagt 12120
atcatgtata caatggacat caaatataca acaagcacta tagctagtgg cataattata 12180
gagaaatata atgttaacag tttaacacgt ggtgagagag gaccaactaa accatgggtt 12240
ggttcatcta cacaagagaa aaaaacaatg ccagtttata atagacaagt tttaaccaaa 12300
aaacaaagag atcaaataga tctattagca aaattggatt gggtgtatgc atctatagat 12360
aacaaggatg aattcatgga agaactcagc ataggaaccc ttgggttaac atatgaaaag 12420
gccaaaaaat tatttccaca atatttaagt gtcaactatt tgcatcgcct tacagtcagt 12480
agtagaccat gtgaattccc tgcatcaata ccagcttata gaacaacaaa ttatcacttt 12540
gacactagcc ctattaatcg catattaaca gaaaagtatg gtgatgaaga tattgacata 12600
gtattccaaa actgtataag ctttggcctt agcttaatgt cagtagtaga acaatttact 12660
aatgtatgtc ctaacagaat tattctcata cctaagctta atgagataca tttgatgaaa 12720
cctcccatat tcacaggtga tgttgatatt cacaagttaa aacaagtgat acaaaaacag 12780
catatgtttt taccagacaa aataagtttg actcaatatg tggaattatt cttaagtaac 12840
aaaacactca aatctggatc tcatgttaat tctaatttaa tattggcaca taaaatatct 12900
gactattttc ataatactta cattttaagt actaatttag ctggacattg gattctaatt 12960
atacaactta tgaaagattc taaaggtatt tttgaaaaag attggggaga gggatatata 13020
actgatcata tgtttattaa tttgaaagtt ttcttcaatg cttataagac ctatctcttg 13080
tgttttcata aaggttatgg caaagcaaaa ctggagtgtg atatgaacac ttcagatctt 13140
ctatgtgtat tggaattaat agacagtagt tattggaagt ctatgtctaa ggtattttta 13200
gaacaaaaag ttatcaaata cattcttagc caagatgcaa gtttacatag agtaaaagga 13260
tgtcatagct tcaaattatg gtttcttaaa cgtcttaatg tagcagaatt tacagtttgc 13320
ccttgggttg ttaacataga ttatcatcca acacatatga aagcaatatt aacttatata 13380
gatcttgtta gaatgggatt gataaatata gatagaatac acattaaaaa taaacacaaa 13440
ttcaatgatg aattttatac ttctaatctc ttttacatta attataactt ctcagataat 13500
actcatctat taactaaata cataaggatt gctaattcag aattagaaaa taattacaac 13560
aaattatatc atcctacacc agaaacccta gagaatatac tagccaatcc gattaaaagt 13620
aatgacaaaa agacactgaa cgactattgt ataggtaaaa atgttgactc aataatgtta 13680
ccattgttat ctaataagaa gcttgttaaa tcgtctgcaa tgattagaac caattacagc 13740
aaacaagacc tgtacaatct attccctacg gttgtgatcg atagaattat agatcattca 13800
ggtaatacag ccaaatccaa ccaactttac actactactt cccatcaaat atctttagtg 13860
cacaatagca catcacttta ttgcatgctt ccttggcatc atattaatag attcaatttt 13920
gtatttagtt ctacaggttg taaaattagt atagagtata ttttaaaaga ccttaaaatt 13980
aaagatccta attgtatagc attcataggt gaaggagcag ggaatttatt attgcgtaca 14040
gtggtggaac ttcatcctga cataagatat atttacagaa gtctgaaaga ttgcaatgat 14100
catagtttac ctattgagtt tttaaggcta tacaatggac atatcaacat tgattatggt 14160
gaaaatttga ccattcctgc tacagatgca accaacaaca ttcattggtc ttatttacat 14220
ataaagtttg ctgaacctat cagtcttttt gtatgtgatg ccgaattgcc tgtaacagtc 14280
aactggagta aaattataat agaatggagc aagcatgtaa gaaaatgcaa gtactgttcc 14340
tcagttaata aatgtacgtt aatagtaaaa tatcatgctc aagatgatat tgatttcaaa 14400
ttagacaata taactatatt aaaaacttat gtatgcttag gcagtaagtt aaagggatcg 14460
gaggtttact tagtccttac aataggtcct gcaaatatat ttccagtatt taatgtagta 14520
caaaatgcta aattgatact atcaagaacc aaaaatttca tcatgcctaa gaaagctgat 14580
aaagagtcta ttgatgcaaa tattaaaagt ttgataccct ttctttgtta ccctataaca 14640
aaaaaaggaa ttaatactgc attgtcaaaa ctaaagagtg ttgttagtgg agatatacta 14700
tcatattcta tagctggacg gaatgaagtt ttcagcaata aacttataaa tcataagcat 14760
atgaacatct taaagtggtt caatcatgtt ttaaatttca gatcaacaga actaaactat 14820
aaccatttat atatggtaga atctacatat ccttacctaa gtgaattgtt aaacagcttg 14880
acaactaatg aacttaaaaa actgattaaa atcacaggta gtctgttata caactttcat 14940
aatgaataat gaataaagat cttataataa aaattcctat agctatacac tagcactgta 15000
ttcaattata gttattaaaa aattaaaaat catataattt tttataaaaa taacttttag 15060
tgaactaatc ctaaagttat cattttgatc taggaggaat aaatttaaat cccaatctaa 15120
ttggtttata tgtgtattaa ctaaactacg agatattagt ttttgacact ttttttctcg 15180
tgggtcggca tggcatctcc acctcctcgc ggtccgacct gggcatccga aggaggacgc 15240
acgtccactc ggatggctaa gggagagcca ctgctaacaa agcccgaaag gaagctgagt 15300
tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct 15360
tgaggggttt tttgctgaaa ggaggaacta tatccggatt cgcgacgcga ggctggatgg 15420
ccttccccat tatgattctt ctcgcttccg gcggcatcgg gatgcccgcg ttgcaggcca 15480
tgctgtccag gcaggtagat gacgaccatc agggacagct tcaaggatcg ctcgcggctc 15540
ttaccagcct aacttcgatc actggaccgc tgatcgtcac ggcgatttat gccgcctcgg 15600
cgagcacatg gaacgggttg gcatggattg taggcgccgc cctatacctt gtctgcctcc 15660
ccgcgttgcg tcgcggtgca tggagccggg ccacctcgac ctgaatggaa gccggcggca 15720
cctcgctaac ggattcacca ctccaagaat tggagccaat caattcttgc ggagaactgt 15780
gaatgcgcaa accaaccctt ggcagaacat atccatcgcg tccgccatct ccagcagccg 15840
cacgcggcgc atctcgggca gcgttgggtc ctggccacgg gtgcgcatga tcgtgctcct 15900
gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc 15960
gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac 16020
atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg 16080
caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac 16140
atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat 16200
ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt 16260
aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa 16320
tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg 16380
ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga 16440
acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct 16500
cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac 16560
agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt 16620
tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg 16680
cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata 16740
ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact 16800
gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 16860
atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 16920
caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 16980
cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 17040
taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 17100
ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 17160
tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 17220
gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 17280
ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 17340
aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 17400
aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 17460
agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 17520
cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 17580
gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 17640
atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 17700
gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 17760
tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 17820
gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 17880
ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 17940
actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 18000
ccagttaata gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg 18060
tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 18120
cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 18180
ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 18240
ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 18300
tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat 18360
agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 18420
atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 18480
gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 18540
aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 18600
tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 18660
aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa 18720
gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt 18780
cttcaagaat tctcatgttt gacagcttat catcg 18815
<210> 3
<211> 18485
<212> DNA
<213> 质粒pBRB-RSV-rLong/A2cpts∆SH
<400> 3
acgcgttaat acgactcact atagggccgt ctgatgaggc cgaaaggccg aaactccgta 60
aggagtcacg ggaaaaaatg cgtacaacaa acttgcgtaa accaaaaaaa tggggcaaat 120
aagaatttga taagtaccac ttaaatttaa ctcccttggt tagagatggg cagcaattcg 180
ttgagtatga taaaagttag attacaaaat ttgtttgaca atgatgaagt agcattgtta 240
aaaataacat gctatactga caaattaata catttaacta atgctttggc taaggcagtg 300
atacatacaa tcaaattgaa tggcattgtg tttgtgcatg ttattacaag tagtgatatt 360
tgccctaata ataatattgt agtaaaatcc aatttcacaa caatgccagt gctacaaaat 420
ggaggttata tatgggaaat gatggaatta acacattgct ctcaacctaa tggtctaata 480
gatgacaatt gtgaaattaa attctccaaa aaactaagtg attcaacaat gaccaattat 540
atgaatcaat tatctgaatt acttggattt gatcttaatc cataaattat aattaatatc 600
aactagcaaa tcaatgtcac tagcaccatt agttaatata aaacttaaca gaagacaaaa 660
atggggcaaa taaatcaact cagccaaccc aaccatggac acaacccaca atgataccac 720
accacaaaga ctgatgatca cagacatgag accgttgtca cttgagacta caataacatc 780
actaaccaga gacatcataa cacacagatt tatatactta ataaatcatg aatgcatagt 840
gagaaaactt gatgaaagac aggccacatt tacattcctg gtcaactatg aaatgaaact 900
attgcacaaa gtaggaagca ctaaatataa aaaatatact gaatacaaca caaaatatgg 960
cactttccct atgccgatat tcatcaatca tgatgggttc ttagaatgca ttggcattaa 1020
gcctacaaag catactccca taatatacaa gtatgatctc aatccatgaa tttcaacaca 1080
agattcacac aatccaaaac aacaacttta tgcataacta cactccatag tccaaatgga 1140
gcctgaaaat tatagtaatt taaaattaag gagagacata agatagaaga tggggcaaat 1200
acaaagatgg ctcttagcaa agtcaagttg aatgatacac tcaacaaaga tcaacttctg 1260
tcatctagca aatacaccat ccaacggagc acaggagata gtattgatac tcctaattat 1320
gatgtgcaga aacacatcaa taagttatgt ggcatgttat taatcacaga agatgctaat 1380
cataaattca ctgggttaat aggtatgtta tatgctatgt ctaggttagg aagagaagac 1440
accataaaaa tactcagaga tgcgggatat catgtaaaag caaatggagt agatgtaaca 1500
acacatcgtc aagacatcaa tgggaaagaa atgaaatttg aagtgttaac attggcaagc 1560
ttaacaactg aaattcaaat caacattgag atagaatcta gaaaatccta caaaaaaatg 1620
ctaaaagaaa tgggagaggt agctccagaa tacaggcatg attctcctga ttgtgggatg 1680
ataatattat gtatagcagc attagtaata accaaattgg cagcagggga tagatctggt 1740
cttacagccg tgattaggag agctaataat gtcctaaaaa atgaaatgaa acgttacaaa 1800
ggcttactac ccaaggatat agccaacagc ttctatgaag tgtttgaaaa acatccccac 1860
tttatagatg tttttgttca ttttggtata gcacaatctt ccaccagagg tggcagtaga 1920
gttgaaggga tttttgcagg attgtttatg aatgcctatg gtgcagggca agtaatgcta 1980
cggtggggag tcttagcaaa atcaattaaa aatattatgt taggacatgc tagtgtgcaa 2040
gcagaaatgg aacaagttgt tgaggtttat gaatatgccc aaaaattggg tggagaagca 2100
ggattctacc atatattgaa caacccaaaa gcatcattat tatctttgac tcaatttcct 2160
cacttttcca gtgtagtatt aggcaatgct gctggcctag gcataatggg agagtacaga 2220
ggtacaccga ggaatcaaga tctatatgat gcagcaaagg catatgctga acaactcaaa 2280
gaaaatggtg tgattaacta cagtgtatta gacttgacag cagaagaact agaggctatc 2340
aaacatcagc ttaatccaaa agataatgat gtagagcttt gagttaataa aaaatggggc 2400
aaataaatca tcatggaaaa gtttgctcct gaattccatg gagaagatgc aaacaacagg 2460
gctactaaat tcctagaatc aataaagggc aaattcacat cacctaaaga tcccaagaaa 2520
aaagatagta tcatatctgt caactcaata gatatagaag taaccaaaga aagccctata 2580
acatcaaatt caaccattat taacccaaca aatgagacag atgataatgc agggaacaag 2640
cccaattatc aaagaaaacc tctagtaagt ttcaaagaag accctatacc aagtgataat 2700
cccttttcaa aactatacaa agaaaccata gagacatttg ataacaatga agaagaatct 2760
agctattcat atgaagaaat aaatgatcag acgaacgata atataactgc aagattagat 2820
aggattgatg aaaaattaag tgaaatacta ggaatgcttc acacattagt agtagcaagt 2880
gcaggaccta catctgctag ggatggtata agagatgcca tggttggttt aagagaagaa 2940
atgatagaaa aaatcagaac tgaagcatta atgaccaatg acagattaga agctatggca 3000
agactcagga atgaggaaag tgaaaagatg gcaaaagaca catcagatga agtgtctctc 3060
aatccaacat cagagaaatt gaacaacctg ttggaaggga atgatagtga caatgatcta 3120
tcacttgaag atttctgatt agttacaaat ctgcacttca acacacaaca ccaacagaag 3180
accaacaaac aaaccaaccc actcatccaa ccaaacatcc atccgccaat cagccaaaca 3240
gccaacaaaa caaccagcca atccaaaacc agccacctgg aaaaaatcga caatatagtt 3300
acaaaaaaag aaaagggtgg ggcaaatatg gaaacatacg tgaacaagct tcacgaaggc 3360
tccacataca cagctgctgt tcaatacaat gtcctagaaa aagacgatga ccctgcatca 3420
cttacaatat gggtgcccat gttccaatca tctatgccag cagatttact tataaaagaa 3480
ctagctaatg tcaacatact agtgaaacaa atatccacac ccaagggacc ttcactaaga 3540
gtcatgataa actcaagaag tgcattgctt gcacaaatgc ccagcaaatt taccatatgt 3600
gctaatgtgt ccttggatga aagaagcaaa ctggcatatg atgtaaccac accctgtgaa 3660
atcaaggcat gtagtctaac atgcctaaaa tcaaaaaata tgttaactac agttaaagat 3720
ctcactatga agacactcaa ccccacacat gatattattg ctttatgtga atttgaaaac 3780
atagtaacat caaaaaaagt cataatacca acatacctaa gatccatcag tgtcagaaat 3840
aaagatctga acacacttga aaatataaca accactgaat tcaaaaatgc catcacaaat 3900
gcaaaaatca tcccttactc aggattacta ttagtcatca cagtgactga caacaaagga 3960
gcattcaaat acataaagcc gcaaagtcaa ttcatagtag atcttggagc ttacctagaa 4020
aaagaaagta tatattatgt taccacaaat tggaagcaca cagctacacg atttgcaatc 4080
aaacccatgg aagattaacc tttttcctcc acatcagtga gtcaattcat acaaactttc 4140
tacctacatt cttcacttca ccattacaat cacaaacact ctgtggttca accaatcaaa 4200
caaaacttat ctgaagtctc agatcatccc aagtcattgt tcatcagatc tagtaatcaa 4260
ataagttaat aaaaatatac acatgagctc aacgttgggg caaatgcaaa catgtccaaa 4320
aacaaggacc aacgcaccgc taagacacta gaaaagacct gggacactct caatcattta 4380
ttattcatat catcgggctt atataagtta aatcttaaat ctatagcaca aatcacatta 4440
tccattctgg caatgataat ctcaacttca cttataatta cagccatcat attcatagcc 4500
tcggcaaacc acaaagtcac actaacaact gcaatcatac aagatgcaac aagccagatc 4560
aagaacacaa ccccaacata cctcactcag gaccctcagc ttggaatcag cttctccaat 4620
ctgtctgaaa ttacatcaca aaccaccacc atactagctt caacaacacc aggagtgaag 4680
tcaaacctgc aacccacaac agtcaagact aaaaacacaa caacaaccca aacacaaccc 4740
agcaagccca ctacaaaaca acgccaaaac aaaccaccaa acaaacccaa taatgatttt 4800
cacttcgaag tgtttaactt tgtaccctgc agcatatgca gcaacaatcc aacctgctgg 4860
gctatctgca aaagaatacc aaacaaaaaa ccaggaaaga aaaccaccac caagcctaca 4920
aaaaaaccaa ccttcaagac aaccaaaaaa gatctcaaac ctcaaaccac taaaccaaag 4980
gaagtaccca ccaccaagcc cacagaagag ccaaccatca acaccaccaa aacaaacatc 5040
acaactacac tgctcaccaa caacaccaca ggaaatccaa aactcacaag tcaaatggaa 5100
accttccact caacctcctc cgaaggcaat ctaagccctt ctcaagtctc cacaacatcc 5160
gagcacccat cacaaccctc atctccaccc aacacaacac gccagtagtt attaaaaaac 5220
atattatcac aaaaggcctt gaccaactca aacagaatca aaataaactc tggggcaaat 5280
aacaatggag ttgccaatcc tcaaagcaaa tgcaattacc acaatcctcg ctgcagtcac 5340
attttgcttt gcttctagtc aaaacatcac tgaagaattt tatcaatcaa catgcagtgc 5400
agttagcaaa ggctatctta gtgctctaag aactggttgg tatactagtg ttataactat 5460
agaattaagt aatatcaagg aaaataagtg taatggaaca gatgctaagg taaaattgat 5520
aaaccaagaa ttagataaat ataaaaatgc tgtaacagaa ttgcagttgc tcatgcaaag 5580
cacaacagca gcaaacaatc gagccagaag agaactacca aggtttatga attatacact 5640
caacaatacc aaaaaaacca atgtaacatt aagcaagaaa aggaaaagaa gatttcttgg 5700
ttttttgtta ggtgttggat ctgcaatcgc cagtggcatt gctgtatcta aggtcctgca 5760
cttagaagga gaagtgaaca agatcaaaag tgctctacta tccacaaaca aggccgtagt 5820
cagcttatca aatggagtta gtgtcttaac cagcaaagtg ttagacctca aaaactatat 5880
agataaacaa ttgttaccta ttgtgaataa gcaaagctgc agaatatcaa atatagcgac 5940
tgtgatagag ttccaacaaa agaacaacag actactagag attaccaggg aatttagtgt 6000
taatgcaggt gtaactacac ctgtaagcac ttacatgtta actaatagtg aattattgtc 6060
attaatcaat gatatgccta taacaaatga tcagaaaaag ttaatgtcca acaatgttca 6120
aatagttaga cagcaaagtt actctatcat gtccataata aaagaggaag tcttagcata 6180
tgtagtacaa ttaccactat atggtgtgat agatacacct tgttggaaat tacacacatc 6240
ccctctatgt acaaccaaca caaaagaagg gtcaaacatc tgtttaacaa gaactgacag 6300
aggatggtac tgtgacaatg caggatcagt atctttcttc ccacaagctg aaacatgtaa 6360
agttcaatcg aatcgagtat tttgtgacac aatgaacagt ttaacattac caagtgaagt 6420
aaatctctgc aatgttgaca tattcaatcc caaatatgat tgtaaaatta tgacttcaaa 6480
aacagatgta agcagctccg ttatcacatc tctaggagcc attgtgtcat gctatggcaa 6540
aactaaatgt acagcatcca ataaaaatcg tggaatcata aagacatttt ctaacgggtg 6600
tgattatgta tcaaataaag gggtggacac tgtgtctgta ggtaacacat tatattatgt 6660
aaataagcaa gaaggcaaaa gtctctatgt aaaaggtgaa ccaataataa atttctatga 6720
cccattagta ttcccctctg atgaatttga tgcatcaata tctcaagtca atgagaagat 6780
taaccagagt ttagcattta ttcgtaaatc cgatgaatta ttacatcatg taaatgctgg 6840
taaatcaacc attaatatca tgataactac tataattata gtgattatag taatattgtt 6900
atcattaatt gctgttggac tgctcctata ctgtaaggcc agaagcacac cagtcacact 6960
aagcaaggat caactgagtg gtataaataa tattgcattt agtaactgaa taaaaatagc 7020
acctaatcat gttcttacaa tggtttacta tctgctcata gacaacccat ctatcattgg 7080
attttcttaa aatctgaact tcatcgaaac tcttatctat aaaccatctc acttacacta 7140
tttaagtaga ttcctagttt atagttatat aaaacacaat tgaataccag attaacttac 7200
tatctgtaaa aatgagaact ggggcaaaca tgtcacgaag gaatccttgc aaatttgaaa 7260
ttcgaggtca ttgcttgaat ggtaagagat gtcattttag tcataattat tttgaatggc 7320
caccccatgc actgctcgta agacaaaact ttatgttaaa cagaatactt aagtctatgg 7380
ataaaagtat agatacctta tcagaaataa gtggagctgc agagttggac agaacagaag 7440
agtatgctct tggtgtagtt ggagtgctag agagttatat aggatcaata aataatataa 7500
ctaaacaatc agcatgtgtt gccatgagca aactcctcac tgaactcaat agtgatgata 7560
tcaaaaaact gagagacaat gaagagctaa attcacccaa gataagagtg tacaatactg 7620
tcatatcata tattgaaagc aacaggaaaa acaataaaca aactatccat ctgttaaaaa 7680
gattgccagc agacgtattg aagaaaacca tcaaaaacac attggatatc cacaagagca 7740
taaccatcaa caacccaaaa gaattaactg ttagtgatac aaatgaccat gccaaaaata 7800
atgatactac ctgacaaata tccttgtagt ataacttcca tactaataac aagtagatgt 7860
agagtcacta tgtataatcg aaagaacaca ctatatttca atcaaaacaa cccaaataac 7920
catatgtact caccgaatca aacattcaat gaaatccatt ggacctcaca agacttgatt 7980
gacacaattc aaaattttct acagcatcta ggtgttattg aggatatata tacaatatat 8040
atattagtgt cataacactc aatcctaata ctgaccatat cgttgaatta ttaattcaaa 8100
taattcaagc tgtgggacaa aatggatccc attattaatg gaaattctgc taatgtttat 8160
ctaaccgata gttatttaaa aggtgttatc tctttctcag agtgtaatgc tttaggaagt 8220
tacatattca atggtcctta tctcaaaaat gattatacca acttaattag tagacaaaat 8280
ccattaatag aacacatgaa tctaaagaaa ctaaatataa cacagtcctt aatatctaag 8340
tatcataaag gtgaaataaa attagaagag cctacttatt ttcagtcatt acttatgaca 8400
tacaagagta tgacctcgtt ggaacagatt gctaccacta atttacttaa aaagataata 8460
agaagagcta tagaaataag tgatgtcaaa gtctatgcta tattgaataa actagggctt 8520
aaagaaaagg acaagattaa atccaacaat ggacaggatg aagacaactc agttattacg 8580
accataatca aagatgatat actttcagct gttaaggata atcaatctca tcttaaagca 8640
gacaaaaatc actctacaaa acaaaaagac acaatcaaaa caacactctt gaagaaatta 8700
atgtgttcaa tgcagcatcc tccatcatgg ttaatacatt ggtttaattt atacacaaaa 8760
ttaaacaaca tattaacaca gtatcgttca aatgaggtta aaaaccatgg gtttatattg 8820
atagataatc aaactcttag tggatttcaa tttattttga atcaatatgg ttgtatagtt 8880
tatcataagg aactcaaaag aattactgtg acaacctata atcaattctt gacatggaaa 8940
gatattagcc ttagtagatt aaatgtttgt ttaattacat ggattagtaa ctgcttgaac 9000
acattaaata aaagcttagg cttaagatgc ggattcaata atgttatctt gacacaacta 9060
ttcctttatg gtgattacat actaaagcta tttcacaatg aggggttcta cataataaaa 9120
gaggtagagg gatttattat gtctctaatt ttaaatataa cagaagaaga tcaattcaga 9180
aaacgatttt ataatagtat gctcaacaac atcacagatg ctgctaataa agctcagaaa 9240
aatctgctat caagagtatg tcatacatta ttagataaga cagtatccga taatataata 9300
aatggcagat ggataattct attaagtaag ttccttaaat taattaagct tgcaggtgac 9360
aataacctta acaatctgag tgaactatat tttttgttca gaatatttgg acacccaatg 9420
gtagatgaaa gacaagccat ggatgctgtt aaagttaatt gcaatgagac caaattttac 9480
ttgttaagca gtttgagtat gttaagaggt gcctttatat atagaattat aaaagggttt 9540
gtaaataatt acaacagatg gcctacttta agaaatgcta ttgttttacc cttaagatgg 9600
ttaacttact ataaactaaa cacttatcct tctttgttgg aacttacaga aagagatttg 9660
attgtgttat caggactacg tttctatcgt gagtttcggt tgcctaaaaa agtggatctt 9720
gaaatgatta taaatgataa agctatatca ccccctaaaa atttgatatg gactagtttc 9780
cctagaaatt atatgccgtc acacatacaa aactatatag aacatgaaaa attaaaattt 9840
tccgagagtg ataaatcaag aagagtatta gagtattatt taagagataa caaattcaat 9900
gaatgtgatt tatacaactg tgtagttaat caaagttatc tcaacaaccc taatcatgtg 9960
gtatcattga caggcaaaga aagagaactc agtgtaggta gaatgtttgc aatgcaaccg 10020
ggaatgttca gacaggttca aatattggca gagaaaatga tagctgaaaa cattttacaa 10080
ttctttcctg aaagtcttac aagatatggt gatctagaac tacaaaaaat attagaattg 10140
aaagcaggaa taagtaacaa atcaaatcgc tacaatgata attacaacaa ttacattagt 10200
aagtgctcta tcatcacaga tctcagcaaa ttcaatcaag catttcgata tgaaacgtca 10260
tgtatttgta gtgatgtgct ggatgaactg catggtgtac aatctctatt ttcctggtta 10320
catttaacta ttcctcatgt cacaataata tgcacatata ggcatgcacc cccctatata 10380
agagatcata ttgtagatct taacaatgta gatgaacaaa gtggattata tagatatcac 10440
atgggtggta ttgaagggtg gtgtcaaaaa ctatggacca tagaagctat atcactattg 10500
gatctaatat ctctcaaagg gaaattctca attactgctt taattaatgg tgacaatcaa 10560
tcaatagata taagcaaacc agtcagactc atggaaggtc aaactcatgc tttagcagat 10620
tatttgctag cattaaatag ccttaaatta ctgtataaag agtatgcagg cataggtcac 10680
aaattaaaag gaactgagac ttatatatca cgagatatgc aatttatgag taaaacaatt 10740
caacataacg gtgtatatta ccctgctagt ataaagaaag tcctaagagt gggaccgtgg 10800
ataaacacta tacttgatga tttcaaagtg agtctagaat ctataggtag tttgacacaa 10860
gaattagaat atagaggtga aagtctatta tgcagtttaa tatttagaaa tgtatggtta 10920
tataatcaaa ttgctctaca attaaaaaat catgcgttat gtaacaataa attatatttg 10980
gacatattaa aggttctgaa acacttaaaa acctttttta atcttgataa tattgataca 11040
gcattaacat tgtatatgaa tttacccatg ttatttggtg gtggtgatcc caacttgtta 11100
tatcgaagtt tctatagaag aactcctgat ttcctcacag aggctatagt tcactctgtg 11160
ttcatactta gttattatac aaaccatgac ttaaaagata aacttcaaga tttgtcagat 11220
gatagattga ataagttctt aacatgcata atcacgtttg acaaaaaccc taatgctgaa 11280
ttcgtaacat tgatgagaga tcctcaagct ttagggtctg agagacaagc taaaattact 11340
agtgaaatca atagactggc agttacagag gttttgagta cagctccaaa caaaatattc 11400
tccaaaagtg cacaacatta taccactaca gagatagatc taaatgatat tatgcaaaat 11460
atagaaccta catatcctca cgggctaaga gttgtttatg aaagtttacc cttttataaa 11520
gcagagaaaa tagtaaatct tatatcaggt acaaaatcta taactaacat actggaaaag 11580
acttctgcca tagacttaac agatattgat agagccactg agatgatgag gaaaaacata 11640
actttgctta taaggatact tccattggat tgtaacagag ataaaagaga aatattgagt 11700
atggaaaacc taagtattac tgaattaagc aaatatgtta gggaaagatc ttggtcttta 11760
tccaatatag ttggtgttac atcacccagt atcatgtata caatggacat caaatataca 11820
acaagcacta tagctagtgg cataattata gagaaatata atgttaacag tttaacacgt 11880
ggtgagagag gaccaactaa accatgggtt ggttcatcta cacaagagaa aaaaacaatg 11940
ccagtttata atagacaagt tttaaccaaa aaacaaagag atcaaataga tctattagca 12000
aaattggatt gggtgtatgc atctatagat aacaaggatg aattcatgga agaactcagc 12060
ataggaaccc ttgggttaac atatgaaaag gccaaaaaat tatttccaca atatttaagt 12120
gtcaactatt tgcatcgcct tacagtcagt agtagaccat gtgaattccc tgcatcaata 12180
ccagcttata gaacaacaaa ttatcacttt gacactagcc ctattaatcg catattaaca 12240
gaaaagtatg gtgatgaaga tattgacata gtattccaaa actgtataag ctttggcctt 12300
agcttaatgt cagtagtaga acaatttact aatgtatgtc ctaacagaat tattctcata 12360
cctaagctta atgagataca tttgatgaaa cctcccatat tcacaggtga tgttgatatt 12420
cacaagttaa aacaagtgat acaaaaacag catatgtttt taccagacaa aataagtttg 12480
actcaatatg tggaattatt cttaagtaac aaaacactca aatctggatc tcatgttaat 12540
tctaatttaa tattggcaca taaaatatct gactattttc ataatactta cattttaagt 12600
actaatttag ctggacattg gattctaatt atacaactta tgaaagattc taaaggtatt 12660
tttgaaaaag attggggaga gggatatata actgatcata tgtttattaa tttgaaagtt 12720
ttcttcaatg cttataagac ctatctcttg tgttttcata aaggttatgg caaagcaaaa 12780
ctggagtgtg atatgaacac ttcagatctt ctatgtgtat tggaattaat agacagtagt 12840
tattggaagt ctatgtctaa ggtattttta gaacaaaaag ttatcaaata cattcttagc 12900
caagatgcaa gtttacatag agtaaaagga tgtcatagct tcaaattatg gtttcttaaa 12960
cgtcttaatg tagcagaatt tacagtttgc ccttgggttg ttaacataga ttatcatcca 13020
acacatatga aagcaatatt aacttatata gatcttgtta gaatgggatt gataaatata 13080
gatagaatac acattaaaaa taaacacaaa ttcaatgatg aattttatac ttctaatctc 13140
ttttacatta attataactt ctcagataat actcatctat taactaaata cataaggatt 13200
gctaattcag aattagaaaa taattacaac aaattatatc atcctacacc agaaacccta 13260
gagaatatac tagccaatcc gattaaaagt aatgacaaaa agacactgaa cgactattgt 13320
ataggtaaaa atgttgactc aataatgtta ccattgttat ctaataagaa gcttgttaaa 13380
tcgtctgcaa tgattagaac caattacagc aaacaagacc tgtacaatct attccctacg 13440
gttgtgatcg atagaattat agatcattca ggtaatacag ccaaatccaa ccaactttac 13500
actactactt cccatcaaat atctttagtg cacaatagca catcacttta ttgcatgctt 13560
ccttggcatc atattaatag attcaatttt gtatttagtt ctacaggttg taaaattagt 13620
atagagtata ttttaaaaga ccttaaaatt aaagatccta attgtatagc attcataggt 13680
gaaggagcag ggaatttatt attgcgtaca gtggtggaac ttcatcctga cataagatat 13740
atttacagaa gtctgaaaga ttgcaatgat catagtttac ctattgagtt tttaaggcta 13800
tacaatggac atatcaacat tgattatggt gaaaatttga ccattcctgc tacagatgca 13860
accaacaaca ttcattggtc ttatttacat ataaagtttg ctgaacctat cagtcttttt 13920
gtatgtgatg ccgaattgcc tgtaacagtc aactggagta aaattataat agaatggagc 13980
aagcatgtaa gaaaatgcaa gtactgttcc tcagttaata aatgtacgtt aatagtaaaa 14040
tatcatgctc aagatgatat tgatttcaaa ttagacaata taactatatt aaaaacttat 14100
gtatgcttag gcagtaagtt aaagggatcg gaggtttact tagtccttac aataggtcct 14160
gcaaatatat ttccagtatt taatgtagta caaaatgcta aattgatact atcaagaacc 14220
aaaaatttca tcatgcctaa gaaagctgat aaagagtcta ttgatgcaaa tattaaaagt 14280
ttgataccct ttctttgtta ccctataaca aaaaaaggaa ttaatactgc attgtcaaaa 14340
ctaaagagtg ttgttagtgg agatatacta tcatattcta tagctggacg gaatgaagtt 14400
ttcagcaata aacttataaa tcataagcat atgaacatct taaagtggtt caatcatgtt 14460
ttaaatttca gatcaacaga actaaactat aaccatttat atatggtaga atctacatat 14520
ccttacctaa gtgaattgtt aaacagcttg acaactaatg aacttaaaaa actgattaaa 14580
atcacaggta gtctgttata caactttcat aatgaataat gaataaagat cttataataa 14640
aaattcctat agctatacac tagcactgta ttcaattata gttattaaaa aattaaaaat 14700
catataattt tttataaaaa taacttttag tgaactaatc ctaaagttat cattttgatc 14760
taggaggaat aaatttaaat cccaatctaa ttggtttata tgtgtattaa ctaaactacg 14820
agatattagt ttttgacact ttttttctcg tgggtcggca tggcatctcc acctcctcgc 14880
ggtccgacct gggcatccga aggaggacgc acgtccactc ggatggctaa gggagagcca 14940
ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag 15000
cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta 15060
tatccggatt cgcgacgcga ggctggatgg ccttccccat tatgattctt ctcgcttccg 15120
gcggcatcgg gatgcccgcg ttgcaggcca tgctgtccag gcaggtagat gacgaccatc 15180
agggacagct tcaaggatcg ctcgcggctc ttaccagcct aacttcgatc actggaccgc 15240
tgatcgtcac ggcgatttat gccgcctcgg cgagcacatg gaacgggttg gcatggattg 15300
taggcgccgc cctatacctt gtctgcctcc ccgcgttgcg tcgcggtgca tggagccggg 15360
ccacctcgac ctgaatggaa gccggcggca cctcgctaac ggattcacca ctccaagaat 15420
tggagccaat caattcttgc ggagaactgt gaatgcgcaa accaaccctt ggcagaacat 15480
atccatcgcg tccgccatct ccagcagccg cacgcggcgc atctcgggca gcgttgggtc 15540
ctggccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg 15600
ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg 15660
ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta 15720
aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg 15780
atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc 15840
ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg 15900
ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt 15960
catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa 16020
acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat taacgcttct 16080
ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga 16140
ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct 16200
ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag 16260
acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca 16320
gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta 16380
ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc 16440
atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 16500
cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 16560
gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 16620
ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 16680
agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 16740
tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 16800
ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 16860
gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 16920
ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 16980
gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 17040
aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 17100
aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 17160
ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 17220
gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 17280
gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 17340
tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 17400
ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 17460
ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 17520
atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 17580
ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 17640
tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 17700
attgctgcag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 17760
tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 17820
ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 17880
gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 17940
gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 18000
gcgtcaacac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 18060
aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 18120
taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 18180
tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 18240
tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 18300
atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 18360
tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat 18420
aaaaataggc gtatcacgag gccctttcgt cttcaagaat tctcatgttt gacagcttat 18480
catcg 18485
<210> 4
<211> 4209
<212> DNA
<213> pBRB载体
<400> 4
gaattctcat gtttgacagc ttatcatcga cgcgttacgt ctagaggtca agcttgagcg 60
agctcggcgt tcgaaggcgg ctagcgctat atgcgttgat gcaatttcta tgcgcacccg 120
ttctcggagc actgtccgac cgctttggcc gccgcccagt cctgctcgct tcgctacttg 180
gagccactat cgactacgcg atcatggcga ccacacccgt cctgtggatc ctctacgccg 240
gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg 300
acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg 360
tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac 420
cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc 480
aggagtcgca taagggagag cgtcgaccga tgcccttgag agccttcaac ccagtcagct 540
ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca 600
tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag gaccgctttc 660
gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg 720
ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag gccattatcg 780
ccggcatggc ggccgacgcg ctgggctacg tcttgctggc gttcgcgacg cgaggctgga 840
tggccttccc cattatgatt cttctcgctt ccggcggcat cgggatgccc gcgttgcagg 900
ccatgctgtc caggcaggta gatgacgacc atcagggaca gcttcaagga tcgctcgcgg 960
ctcttaccag cctaacttcg atcactggac cgctgatcgt cacggcgatt tatgccgcct 1020
cggcgagcac atggaacggg ttggcatgga ttgtaggcgc cgccctatac cttgtctgcc 1080
tccccgcgtt gcgtcgcggt gcatggagcc gggccacctc gacctgaatg gaagccggcg 1140
gcacctcgct aacggattca ccactccaag aattggagcc aatcaattct tgcggagaac 1200
tgtgaatgcg caaaccaacc cttggcagaa catatccatc gcgtccgcca tctccagcag 1260
ccgcacgcgg cgcatctcgg gcagcgttgg gtcctggcca cgggtgcgca tgatcgtgct 1320
cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc 1380
accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac 1440
aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc 1500
ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc 1560
tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg 1620
catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc 1680
agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag 1740
aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc 1800
ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga 1860
tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcagctg 1920
cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt 1980
cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg 2040
tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac 2100
tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa 2160
ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc 2220
actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 2280
gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 2340
cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 2400
ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 2460
ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 2520
ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 2580
agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 2640
cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 2700
aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 2760
gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 2820
agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 2880
ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 2940
cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 3000
tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 3060
aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 3120
tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 3180
atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 3240
cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 3300
gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 3360
gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 3420
tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc 3480
tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 3540
tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 3600
aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 3660
atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 3720
tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca 3780
catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 3840
aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 3900
tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 3960
gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 4020
tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 4080
tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 4140
taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt 4200
cgtcttcaa 4209
<210> 5
<211> 24
<212> DNA
<213> 实时定量PCR引物 F
<400> 5
agatcaactt ctgtcatcca gcaa 24
<210> 6
<211> 25
<212> DNA
<213> 实时定量PCR引物 R
<400> 6
gcacatcata attaggagta tcaat 25
<210> 7
<211> 21
<212> DNA
<213> N799G/A引物 F
<400> 7
gatctggtct tacagccgtg a 21
<210> 8
<211> 20
<212> DNA
<213> N799G/A引物 R
<400> 8
tgagttgttc agcatatgcc 20
<210> 9
<211> 23
<212> DNA
<213> F653,654AA/ CG引物 F
<400> 9
gcacaacagc agcaaacaat cga 23
<210> 10
<211> 22
<212> DNA
<213> F653,654AA/ CG引物 R
<400> 10
tcacagtacc atcctctgtc ag 22
<210> 11
<211> 22
<212> DNA
<213> F1568,1569CA/TT引物 F
<400> 11
gtggacactg tgtctgtagg ta 22
<210> 12
<211> 22
<212> DNA
<213> F1568,1569CA/TT引物 R
<400> 12
ttgcaaggat tccttcgtga ca 22
<210> 13
<211> 21
<212> DNA
<213> L 956,957GT/AC引物 F
<400> 13
tgcagcatcc tccatcatgg t 21
<210> 14
<211> 22
<212> DNA
<213> L 956,957GT/AC引物 R
<400> 14
gcatctgtga tgttgttgag ca 22
<210> 15
<211> 20
<212> DNA
<213> L5068,5070 C/T,T/C引物 F
<400> 15
agccaagatg caagtttaca 20
<210> 16
<211> 22
<212> DNA
<213> L5068,5070 C/T,T/C引物 R
<400> 16
ttgctcatgg caacacatgc tg 22
<210> 17
<211> 22
<212> DNA
<213> L2491,2492CA/TT引物 F
<400> 17
gtagtgatgt gctggatgaa ct 22
<210> 18
<211> 22
<212> DNA
<213> L2491,2492CA/TT引物 R
<400> 18
aacaagttgg gatcaccacc ac 22

Claims (5)

1.一种基于反向遗传学构建的RSV反基因组质粒，其特征在于：所述质粒为如序列表SEQ ID No.1所示的质粒pBRB-RSV-rLong/A2cp。
2.一种基于反向遗传学构建的RSV反基因组质粒，其特征在于：所述质粒为如序列表SEQ ID No.2所示的质粒pBRB-RSV-rLong/A2cpts。
3.一种基于反向遗传学构建的RSV反基因组质粒，其特征在于：所述质粒为如序列表SEQ ID No.3所示的质粒pBRB-RSV-rLong/A2cptsΔSH。
4.一种由权利要求1、2或3所述的RSV反基因组质粒经拯救获得的重组RSV。
5.权利要求4所述的重组RSV在制备RSV减毒活疫苗中的应用。
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CN114990079A (zh) * 2022-04-29 2022-09-02 广州医科大学附属第一医院（广州呼吸中心） 治疗性缺陷干扰颗粒及其在制备用于防治rsv病毒感染的产品中的应用

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