CN110684081A - 一种编码分泌型多肽氨基酸的重组溶瘤新城疫病毒及其制备方法 - Google Patents

一种编码分泌型多肽氨基酸的重组溶瘤新城疫病毒及其制备方法 Download PDF

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CN110684081A
CN110684081A CN201910822416.4A CN201910822416A CN110684081A CN 110684081 A CN110684081 A CN 110684081A CN 201910822416 A CN201910822416 A CN 201910822416A CN 110684081 A CN110684081 A CN 110684081A
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高恶斌
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Abstract

本发明提供了制备编码分泌型多肽氨基酸的重组溶瘤新城疫病毒，包含有小分子多肽pNaKtide，能够结合肉瘤病毒蛋白激酶(Src)，并抑制与Src相关的Na/K‑ATP酶/Src/ROS信号通路的活性；也可改善细胞核的氧化应激，显著降低细胞内ROS的水平，以减弱细胞衰老；实现pNaKtide多肽作为一种抗氧化剂在治疗肿瘤疾病的细胞和动物模型上展现出良好的治疗效果。同时，还提供相应的制备方法，该方法获得的病毒成功率高、效果稳定，具有较高的医疗价值。

Description

一种编码分泌型多肽氨基酸的重组溶瘤新城疫病毒及其制备 方法
技术领域
本发明属于基因生物领域，特别涉及一种编码分泌型多肽氨基酸的重组溶瘤新城疫病毒及其制备方法。
背景技术
新城疫病毒在病毒分类学中的位置属于分子负链RNA目、副黏病毒科、副黏病毒亚科、禽腮腺炎病毒或禽副黏病毒属。该病毒主要危害鸡、珠鸡和火鸡，在被侵袭的鸡群中迅速传播，强毒株可使鸡群全群毁灭。弱毒株仅引起鸡群呼吸道感染和产蛋量下降，但可迅速康复。人类可因接触病禽和活毒疫苗而引起结膜炎或淋巴腺炎，但很快便康复。近些年研究表明，新城疫病毒对抑制肿瘤细胞的再生并诱导肿瘤细胞的凋亡，有一定的效果，因此，如何操作简单获得质量优良的新城疫病毒成为一个重要的研究课题。
发明内容
为了获得质量优良的新城疫病毒，本发明提供了一种分泌型多肽氨基酸，其序列如 SEQ ID NO.1所示。
还提供了编码上述的分泌型多肽氨基酸的核苷酸，其序列如SEQ ID NO.2所示。
还提供了分泌型多肽氨基酸作为抗氧化剂中的应用。
还提供了分泌型多肽氨基酸在治疗肿瘤疾病药物中的应用。
还提供了编码分泌型多肽氨基酸的重组溶瘤新城疫病毒Lasota。
还提供了一种上述重组溶瘤新城疫病毒Lasota的制备方法，步骤为：
(1)重组溶瘤新城疫病毒Lasota全基因组cDNA序列转录载体pBR-rNDV-pNaK
将化学合成的多肽核苷酸序列SEQ ID NO.2以及新城疫编码的6个基因序列按照克隆策略通过PCR扩增、酶切、连接的方式连接到中间载体pBR322-M上，中间载体如SEQ IDNO.3所示，其顺序依次为核酸蛋白基因NP、pNaKtide多肽、磷酸蛋白基因P、基质基因M、融合蛋白基因F、血凝素蛋白基因HN和聚合酶蛋白基因L，其中在多肽核苷酸序列SEQ ID NO.2的5’端插入一个内部核糖体进入位点序列IRES。同时，在全基因组的头部和尾部分别连接具有自我剪切功能的拳头状核酶序列HamRz与丁肝核酶序列Rbz，得到重组载体pBR-rNDV-pNaK，如SEQ ID NO.4所示；
(2)辅助质粒构建
以步骤(1)中重组溶瘤新城疫病毒Lasota的cDNA为模板，采用高保真酶与核酸蛋白基因NP、磷酸蛋白基因P及聚合酶蛋白基因L分别对应的上游引物、下游引物进行 PCR扩增核酸蛋白基因NP、磷酸蛋白基因P及聚合酶蛋白基因L片段，产物经 (0.05-2.89)％琼脂糖凝胶电泳鉴定，切胶回收；核酸蛋白基因NP、磷酸蛋白基因P 的PCR产物插入pCI-neo载体的BmtI与MluI克隆酶切位点，聚合酶蛋白基因L的PCR 产物插入到pCI-neo载体的MluI与NotI克隆酶切位点，获得三个转录辅助质粒分别命名为pCI-NP、pCI-P、pCI-L，并测序验证；其中
NP基因上游引物：
5’-CCGGCTAGCATGTCCTCCGTATTTGATGAGTAC-3’；
NP基因下游引物：
5’-TAGAGGTACCACGCGTTCAATACCCCCAGTCGGTGTCGTTA-3’；
P基因上游引物：5’-CCGGCTAGCATGGCCACCTTTACAGATGCAGA-3’；
P基因下游引物：5’-TCGACGCGTTTAGCCATTTAGAGCAAGGCGC-3’；
L基因上游引物：5’-CGACGCGTATGGCGAGCTCCGGTCCTGA-3’；
L基因下游引物：
PCI-NotR:5’-GGAAAAAAGCGGCCGCTTAAGAGTCACAGTTACTGTAATATC-3’；
(3)细胞转染与病毒检测，获得病毒
将步骤(2)获得的三个转录辅助质粒pCI-NP、pCI-P、pCI-L及步骤(1)获得的重组溶瘤新城疫病毒Lasota全基因组cDNA序列转录载体pBR-rNDV-pNak，按照质量比为(2-6)：(1-3)：(1-3)：(0.2-1.5)，分别转化大肠杆菌感受态细胞DH5a，(30-42)℃， Amp抗性，大体积摇菌，利用QIAGEN质粒制备试剂盒抽提转染所用的质粒，并测定各质粒的浓度，采用脂质体试剂盒进行病毒检测，结果均为阳性的样品中含有拯救成功的重组溶瘤新城疫病毒。
有益效果：本发明提供的制备编码分泌型多肽氨基酸的重组溶瘤新城疫病毒，包含有小分子多肽pNaKtide，能够结合肉瘤病毒蛋白激酶(Src)，并抑制与Src相关的 Na/K-ATP酶/Src/ROS信号通路的活性；也可改善细胞核的氧化应激，显著降低细胞内 ROS的水平，以减弱细胞衰老；实现pNaKtide多肽作为一种抗氧化剂在治疗肿瘤疾病的细胞和动物模型上展现出良好的治疗效果。同时，本发明提供的制备方法，获得的病毒成功率高、效果稳定，具有较高的医疗价值。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例详细说明如后。
具体实施方式
下面结合实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
细胞转染与病毒检测试验
将含有三个转录辅助质粒pCI-NP、pCI-P、pCI-L及重组新城疫病毒全基因组cDNA序列转录载体pBR-rNDV-pNak按照质量比为(2-6)：(1-3)：(1-3)：(0.2-1.5)，分别转化大肠杆菌感受态细胞DH5a，在温度为(30-42)℃，Amp抗性，大体积摇菌，利用 QIAGEN质粒制备试剂盒抽提转染所用的质粒，并测定各质粒的浓度。
实施例1
采用Invitrogen公司的脂质体2000试剂盒，将制备好的三个转录辅助质粒pCI-NP、 pCI-P、pCI-L及重组新城疫病毒全基因组cDNA序列转录质粒pBR-rNDV-pNak按照质量比为4：2：2：1的比例，共转染BSR-T7/5细胞，转染60～72小时将细胞转移至-70℃反复冻融3次，然后将冻融的细胞及其上清按0.2ml/胚接种9～11日龄SPF鸡胚，弃去 24小时内死亡的鸡胚，收集24小时后死亡的鸡胚尿囊液并按照OIE标准对其进行血凝 (HA)试验和血凝抑制(HI)试验，结果均为阳性的样品含有拯救成功的重组溶瘤新城疫病毒，其中阳性的样品占比92％以上。
实施例2
采用Invitrogen公司的脂质体2000试剂盒，将制备好的三个转录辅助质粒pCI-NP、 pCI-P、pCI-L及重组新城疫病毒全基因组cDNA序列转录质粒pBR-rNDV-pNak按照质量比为2：1.5：3：0.5的比例，共转染BSR-T7/5细胞，转染60～70小时将细胞转移至 -85℃反复冻融2-5次，然后将冻融的细胞及其上清按(0.06-0.5)ml/胚接种7-13日龄 SPF鸡胚，弃去20-30小时内死亡的鸡胚，收集20-30小时后死亡的鸡胚尿囊液并按照 OIE标准对其进行血凝(HA)试验和血凝抑制(HI)试验，结果均为阳性的样品含有拯救成功的重组溶瘤新城疫病毒，其中阳性的样品占比90％以上。。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 天益健康科学研究院（镇江）有限公司
<120> 一种编码分泌型多肽氨基酸的重组溶瘤新城疫病毒及其制备方法
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 53
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro
20 25 30
Gln Ser Ala Thr Trp Leu Ala Leu Ser Arg Ile Ala Gly Leu Cys Asn
35 40 45
Arg Ala Val Phe Gln
50
<210> 2
<211> 162
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcg 60
gggcgtaaaa aacgccgtca gcgccgtcgt ccgccgcagt ctgctacctg gctggctctg 120
agccgtattg caggtctgtg caaccgcgca gttttccagt aa 162
<210> 3
<211> 4346
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 3
tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 60
aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 120
ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 180
aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat 240
tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc ttcaagaatt 300
ctcatgtttg acagcttatc atcgataagc ttctcgagat ggttccgcgg tttaaaccac 360
ctaggtatct agagtgacgc gtgtcctgca ggcgcgcctt aagttaatta agatctcggt 420
actcgttcga actcaaataa atgtcttaaa aaaaggttgc gcacaattat tcttgagtgt 480
agtctcgtca ttcaccaaat ctttgtttgg tgggtcggca tggcatctcc acctcctcgc 540
ggtccgacct gggcatccga aggaggacgc acgtccactc ggatggctaa gggagagcca 600
tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttggtc gacattagga 660
tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg 720
cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg 780
cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct 840
ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct 900
gcttcctaat gcaggagtcg cataagggag agcgtcgacc gatgcccttg agagccttca 960
acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg 1020
tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg 1080
aggaccgctt tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct 1140
tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc 1200
aggccattat cgccggcatg gcggccgacg cgctgggcta cgtcttgctg gcgttcgcga 1260
cgcgaggctg gatggccttc cccattatga ttcttctcgc ttccggcggc atcgggatgc 1320
ccgcgttgca ggccatgctg tccaggcagg tagatgacga ccatcaggga cagcttcaag 1380
gatcgctcgc ggctcttacc agcctaactt cgatcactgg accgctgatc gtcacggcga 1440
tttatgccgc ctcggcgagc acatggaacg ggttggcatg gattgtaggc gccgccctat 1500
accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa 1560
tggaagccgg cggcacctcg ctaacggatt caccactcca agaattggag ccaatcaatt 1620
cttgcggaga actgtgaatg cgcaaaccaa cccttggcag aacatatcca tcgcgtccgc 1680
catctccagc agccgcacgc ggcgcatctc gggcagcgtt gggtcctggc cacgggtgcg 1740
catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta 1800
gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc 1860
gacctgagca acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg 1920
gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc 1980
ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct 2040
ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc 2100
atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac 2160
ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc 2220
ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga 2280
gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct 2340
ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct 2400
cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg 2460
cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag 2520
cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat 2580
atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc 2640
gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 2700
cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 2760
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 2820
cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 2880
aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 2940
cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 3000
gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 3060
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 3120
cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 3180
aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 3240
tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 3300
ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 3360
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 3420
ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 3480
agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 3540
atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 3600
cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 3660
ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 3720
ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 3780
agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 3840
agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc 3900
gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 3960
cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 4020
gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 4080
tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 4140
tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat 4200
aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 4260
cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 4320
cccaactgat cttcagcatc ttttac 4346
<210> 4
<211> 20265
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 4
aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc 60
gtatcacgag gccctttcgt cttcaagaat tctcatgttt gacagcttat catcgataag 120
cttctcgaga tggttccgcg gtaatacgac tcactatagg ggttgttaac gcaacactga 180
tgaggccgaa aggccgaaac tccgtaagga gtcaccaaac agagaatccg tgagttacga 240
taaaaggcga aagagcaatt gaagtcacac gggtagaagg tgtgaatctc gagtgcgagc 300
ccgaagcaca aactcgagaa agccttctgc caacatgtcc tccgtatttg atgagtacga 360
acagctcctc gcggctcaga ctcgccccaa cggagctcat ggagggggag aaaaagggag 420
taccttaaaa gtagacgtcc cggtattcac tcttaacagt gatgacccag aagatagatg 480
gagctttgtg gtattctgcc tccggattgc tgttagcgaa gatgccaaca aaccactcag 540
gcaaggtgct ctcatatctc ttttatgctc ccactcacag gtaatgagga accatgttgc 600
ccttgcaggg aaacagaatg aagccacatt ggccgtgctt gagattgatg gctttgccaa 660
cggcacgccc cagttcaata ataggagtgg agtgtctgaa gagagagcac agagatttgc 720
gatgatagca ggatctctcc ctcgggcatg cagcaacgga accccgttcg tcacagccgg 780
ggccgaagat gatgcaccag aagacatcac cgataccctg gagaggatcc tctctatcca 840
ggctcaagta tgggtcacag tagcaaaagc cattactgcg tatgagactg cagatgagtc 900
ggaaacaagg cgaatcaata agtatatgca gcaaggcagg gtccaaaaga aatacatcct 960
ctaccccgta tgcaggagca caatccaact cacgatcaga cagtctcttg cagtccgcat 1020
ctttttggtt agcgagctca agagaggccg caacacggca ggtggtacct ctacttatta 1080
taacctggta ggggacgtag actcatacat caggaatacc gggcttactg cattcttctt 1140
gacactcaag tacggaatca acaccaagac atcagccctt gcacttagta gcctctcagg 1200
cgacatccag aagatgaagc agctcatgcg tttgtatcgg atgaaaggag ataatgcgcc 1260
gtacatgaca ttacttggtg atagtgacca gatgagcttt gcgcctgccg agtatgcaca 1320
actttactcc tttgccatgg gtatggcatc agtcctagat aaaggtactg ggaaatacca 1380
atttgccagg gactttatga gcacatcatt ctggagactt ggagtagagt acgctcaggc 1440
tcagggaagt agcattaacg aggatatggc tgccgagcta aagctaaccc cagcagcaag 1500
gaggggcctg gcagctgctg cccaacgggt ctccgaggag accagcagca tagacatgcc 1560
tactcaacaa gtcggagtcc tcactgggct tagcgagggg gggtcccaag ctctacaagg 1620
cggatcgaat agatcgcaag ggcaaccaga agccggggat ggggagaccc aattcctgga 1680
tcggatgaga gcggtagcaa atagcatgag ggaggcgcca aactctgcac agggcactcc 1740
ccaatcgggg cctcccccaa ctcctgggcc atcccaagat aacgacaccg actgggggta 1800
ttgaaccggt tctgcagtcg acggtaccgc gggcccggga tccgcccctc tccctccccc 1860
ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg 1920
ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc 1980
ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg 2040
aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg 2100
accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca 2160
cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata 2220
gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc 2280
cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt 2340
gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt 2400
tgaaaaacac gatgataata tggccacaac cttaattaat gtacaggatg caactcctgt 2460
cttgcattgc actaagtctt gcacttgtca cgaattcggg gcgtaaaaaa cgccgtcagc 2520
gccgtcgtcc gccgcagtct gctacctggc tggctctgag ccgtattgca ggtctgtgca 2580
accgcgcagt tttccagtaa cctggccggc caccggttac gagctctgga caaaacccag 2640
cctgcttcca caaaaacatc ccaatgccct cacccgtagt cgacccctcg atttgcggct 2700
ctatatgacc acaccctcaa acaaacatcc ccctctttcc tccctccccc tgctgtacaa 2760
ctccgcacgc cctagatacc acaggcacaa tgcggctcac taacaatcaa aacagagccg 2820
agggaattag aaaaaagtac gggtagaaga gggatattca gagatcaggg caagtctccc 2880
gagtctctgc tctctcctct acctgataga ccaggacaaa catggccacc tttacagatg 2940
cagagatcga cgagctattt gagacaagtg gaactgtcat tgacaacata attacagccc 3000
agggtaaacc agcagagact gttggaagga gtgcaatccc acaaggcaag accaaggtgc 3060
tgagcgcagc atgggagaag catgggagca tccagccacc ggccagtcaa gacaaccccg 3120
atcgacagga cagatctgac aaacaaccat ccacacccga gcaaacgacc ccgcatgaca 3180
gcccgccggc cacatccgcc gaccagcccc ccacccaggc cacagacgaa gccgtcgaca 3240
cacagctcag gaccggagca agcaactctc tgctgttgat gcttgacaag ctcagcaata 3300
aatcgtccaa tgctaaaaag ggcccatggt cgagccccca agaggggaat caccaacgtc 3360
cgactcaaca gcaggggagt caacccagtc gcggaaacag tcaggaaaga ccgcagaacc 3420
aagtcaaggc cgcccctgga aaccagggca cagacgtgaa cacagcatat catggacaat 3480
gggaggagtc acaactatca gctggtgcaa cccctcatgc tctccgatca aggcagagcc 3540
aagacaatac ccttgtatct gcggatcatg tccagccacc tgtagacttt gtgcaagcga 3600
tgatgtctat gatggaggcg atatcacaga gagtaagtaa ggtcgactat cagctagatc 3660
ttgtcttgaa acagacatcc tccatcccta tgatgcggtc cgaaatccaa cagctgaaaa 3720
catctgttgc agtcatggaa gccaacttgg gaatgatgaa gattctggat cccggttgtg 3780
ccaacatttc atctctgagt gatctacggg cagttgcccg atctcacccg gttttagttt 3840
caggccctgg agacccctct ccctatgtga cacaaggagg cgaaatggca cttaataaac 3900
tttcgcaacc agtgccacat ccatctgaat tgattaaacc cgccactgca tgcgggcctg 3960
atataggagt ggaaaaggac actgtccgtg cattgatcat gtcacgccca atgcacccga 4020
gttcttcagc caagctccta agcaagttag atgcagccgg gtcgatcgag gaaatcagga 4080
aaatcaagcg ccttgctcta aatggctaat tactactgcc acacgtagcg ggtccctgtc 4140
cactcggcat cacacggaat ctgcaccgag ttcccccccg cagacccaag gtccaactct 4200
ccaagcggca atcctctctc gcttcctcag ccccactgaa tgatcgcgta accgtaatta 4260
atctagctac atttaagatt aagaaaaaat acgggtagaa ttggagtgcc ccaattgtgc 4320
caagatggac tcatctagga caattgggct gtactttgat tctgcccatt cttctagcaa 4380
cctgttagca tttccgatcg tcctacaaga cacaggagat gggaagaagc aaatcgcccc 4440
gcaatatagg atccagcgcc ttgacttgtg gactgatagt aaggaggact cagtattcat 4500
caccacctat ggattcatct ttcaagttgg gaatgaagaa gccactgtcg gcatgatcga 4560
tgataaaccc aagcgcgagt tactttccgc tgcgatgctc tgcctaggaa gcgtcccaaa 4620
taccggagac cttattgagc tggcaagggc ctgtctcact atgatagtca catgcaagaa 4680
gagtgcaact aatactgaga gaatggtttt ctcagtagtg caggcacccc aagtgctgca 4740
aagctgtagg gttgtggcaa acaaatactc atcagtgaat gcagtcaagc acgtgaaagc 4800
gccagagaag attcccggga gtggaaccct agaatacaag gtgaactttg tctccttgac 4860
tgtggtaccg aagaaggatg tctacaagat ccctgctgca gtattgaagg tttctggctc 4920
gagtctgtac aatcttgcgc tcaatgtcac tattaatgtg gaggtagacc cgaggagtcc 4980
tttggttaaa tctctgtcta agtctgacag cggatactat gctaacctct tcttgcatat 5040
tggacttatg accaccgtag ataggaaggg gaagaaagtg acatttgaca agctggaaaa 5100
gaaaataagg agccttgatc tatctgtcgg gctcagtgat gtgctcgggc cttccgtgtt 5160
ggtaaaagca agaggtgcac ggactaagct tttggcacct ttcttctcta gcagtgggac 5220
agcctgctat cccatagcaa atgcttctcc tcaggtggcc aagatactct ggagtcaaac 5280
cgcgtgcctg cggagcgtta aaatcattat ccaagcaggt acccaacgcg ctgtcgcagt 5340
gaccgccgac cacgaggtta cctctactaa gctggagaag gggcacaccc ttgccaaata 5400
caatcctttt aagaaataag ctgcgtctct gagattgcgc tccgcccact cacccagatc 5460
atcatgacac aaaaaactaa tctgtcttga ttatttacag ttagtttacc tgtctatcaa 5520
gttagaaaaa acacgggtag aagattctgg atcccggttg gcgccctcca ggtgcaagat 5580
gggctccaga ccttctacca agaacccagc acctatgatg ctgactatcc gggttgcgct 5640
ggtactgagt tgcatctgtc cggcaaactc cattgatggc aggcctcttg cagctgcagg 5700
aattgtggtt acaggagaca aagccgtcaa catatacacc tcatcccaga caggatcaat 5760
catagttaag ctcctcccga atctgcccaa ggataaggag gcatgtgcga aagccccctt 5820
ggatgcatac aacaggacat tgaccacttt gctcaccccc cttggtgact ctatccgtag 5880
gatacaagag tctgtgacta catctggagg ggggagacag gggcgcctta taggtgccat 5940
tattggcggt gtggctcttg gggttgcaac tgccgcacaa ataacagcgg ccgcagctct 6000
gatacaagcc aaacaaaatg ctgccaacat cctccgactt aaagagagca ttgccgcaac 6060
caatgaggct gtgcatgagg tcactgacgg attatcgcaa ctagcagtgg cagttgggaa 6120
gatgcagcag tttgttaatg accaatttaa taaaacagct caggaattag actgcatcaa 6180
aattgcacag caagttggtg tagagctcaa cctgtaccta accgaattga ctacagtatt 6240
cggaccacaa atcacttcac ccgctttaaa caagctgact attcaggcac tttacaatct 6300
agctggtgga aatatggatt acttattgac taagttaggt gtagggaaca atcaactcag 6360
ctcattaatc ggtagcggct taatcaccgg taaccctatt ctatacgact cacagactca 6420
actcttgggt atacaggtaa ctctaccttc agtcgggaac ctaaataata tgcgtgccac 6480
ctacttggaa accttatccg taagcacaac caggggattt gcctcggcac ttgtcccaaa 6540
agtggtgaca caggtcggtt ctgtgataga agaacttgac acctcatact gtatagaaac 6600
tgacttagat ttatattgta caagaatagt aacgttccct atgtcccctg gtatttattc 6660
ctgcttgagc ggcaatacgt cggcctgtat gtactcaaag accgaaggcg cacttactac 6720
accatacatg actatcaaag gttcagtcat cgccaactgc aagatgacaa catgtagatg 6780
tgtaaacccc ccgggtatca tatcgcaaaa ctatggagaa gccgtgtctc taatagataa 6840
acaatcatgc aatgttttat ccttaggcgg gataacttta aggctcagtg gggaattcga 6900
tgtaacttat cagaagaata tctcaataca agattctcaa gtaataataa caggcaatct 6960
tgatatctca actgagcttg ggaatgtcaa caactcgatc agtaatgctt tgaataagtt 7020
agaggaaagc aacagaaaac tagacaaagt caatgtcaaa ctgactagca catctgccct 7080
cattacctat atcgttttga ctatcatatc tcttgttttt ggtatactta gcctgattct 7140
agcatgctac ctaatgtaca agcaaaaggc gcaacaaaag accttattat ggcttgggaa 7200
taatactcta gatcagatga gagccactac aaaaatgtga acacagatga ggaacgaagg 7260
tttccctaat agtaatttgt gtgaaagttc tggtagtctg tcagttcaga gagttaagaa 7320
aaaactaccg gttgtagatg accaaaggac gatatacggg tagaacggta agagaggccg 7380
cccctcaatt gcgagccagg cttcacaacc tccgttctac cgcttcaccg acaacagtcc 7440
tcaatcatgg accgcgccgt tagccaagtt gcgttagaga atgatgaaag agaggcaaaa 7500
aatacatggc gcttgatatt ccggattgca atcttattct taacagtagt gaccttggct 7560
atatctgtag cctccctttt atatagcatg ggggctagca cacctagcga tcttgtaggc 7620
ataccgacta ggatttccag ggcagaagaa aagattacat ctacacttgg ttccaatcaa 7680
gatgtagtag ataggatata taagcaagtg gcccttgagt ctccgttggc attgttaaaa 7740
actgagacca caattatgaa cgcaataaca tctctctctt atcagattaa tggagctgca 7800
aacaacagtg ggtggggggc acctatccat gacccagatt atataggggg gataggcaaa 7860
gaactcattg tagatgatgc tagtgatgtc acatcattct atccctctgc atttcaagaa 7920
catctgaatt ttatcccggc gcctactaca ggatcaggtt gcactcgaat accctcattt 7980
gacatgagtg ctacccatta ctgctacacc cataatgtaa tattgtctgg atgcagagat 8040
cactcacatt catatcagta tttagcactt ggtgtgctcc ggacatctgc aacagggagg 8100
gtattctttt ctactctgcg ttccatcaac ctggacgaca cccaaaatcg gaagtcttgc 8160
agtgtgagtg caactcccct gggttgtgat atgctgtgct cgaaagtcac ggagacagag 8220
gaagaagatt ataactcagc tgtccctacg cggatggtac atgggaggtt agggttcgac 8280
ggccagtacc acgaaaagga cctagatgtc acaacattat tcggggactg ggtggccaac 8340
tacccaggag tagggggtgg atcttttatt gacagccgcg tatggttctc agtctacgga 8400
gggttaaaac ccaattcacc cagtgacact gtacaggaag ggaaatatgt gatatacaag 8460
cgatacaatg acacatgccc agatgagcaa gactaccaga ttcgaatggc caagtcttcg 8520
tataagcctg gacggtttgg tgggaaacgc atacagcagg ctatcttatc tatcaaggtg 8580
tcaacatcct taggcgaaga cccggtactg actgtaccgc ccaacacagt cacactcatg 8640
ggggccgaag gcagaattct cacagtaggg acatctcatt tcttgtatca acgagggtca 8700
tcatacttct ctcccgcgtt attatatcct atgacagtca gcaacaaaac agccactctt 8760
catagtcctt atacattcaa tgccttcact cggccaggta gtatcccttg ccaggcttca 8820
gcaagatgcc ccaacccgtg tgttactgga gtctatacag atccatatcc cctaatcttc 8880
tatagaaacc acaccttgcg aggggtattc gggacaatgc ttgatggtgt acaagcaaga 8940
cttaaccctg cgtctgcagt attcgatagc acatcccgca gtcgcattac tcgagtgagt 9000
tcaagcagta ccaaagcagc atacacaaca tcaacttgtt ttaaagtggt caagactaat 9060
aagacctatt gtctcagcat tgctgaaata tctaatactc tcttcggaga attcagaatc 9120
gtcccgttac tagttgagat cctcaaagat gacggggtta gagaagccag gtctggctag 9180
ttgagtcaat tataaaggag ttggaaagat ggcattgtat cacctatctt ccacgacatc 9240
aagaatcaaa gcgatcgccg acgcgtgctc gaattccatg ttgccagttg accacaatca 9300
gccagtgctc atgcgatcag attaagcctt gtcaatagtc tcttgattaa gaaaaaatgt 9360
aagtggcaat gagatacaag gcaaaacagc tcatggtaaa taatacgggt aggacatggc 9420
gagctccggt cctgaaaggg cagagcatca gattatccta ccagagtcac acctgtcttc 9480
accattggtc aagcacaaac tactctatta ctggaaatta actgggctac cgcttcctga 9540
tgaatgtgac ttcgaccacc tcattctcag tcgacaatgg aaaaaaatac ttgaatcggc 9600
ctctcctgat actgagagaa tgataaaact cggaagggca gtacaccaaa ctcttaacca 9660
caattccaga ataaccggag tgctccaccc caggtgttta gaagaactgg ctaatattga 9720
ggtcccagat tcaaccaaca aatttcggaa gattgagaag aagatccaaa ttcacaacac 9780
gagatatgga gaactgttca caaggctgtg tacgcatata gagaagaaac tgctggggtc 9840
atcttggtct aacaatgtcc cccggtcaga ggagttcagc agcattcgta cggatccggc 9900
attctggttt cactcaaaat ggtccacagc caagtttgca tggctccata taaaacagat 9960
ccagaggcat ctgatggtgg cagctaggac aaggtctgcg gccaacaaat tggtgatgct 10020
aacccataag gtaggccaag tctttgtcac tcctgaactt gtcgttgtga cgcatacgaa 10080
tgagaacaag ttcacatgtc ttacccagga acttgtattg atgtatgcag atatgatgga 10140
gggcagagat atggtcaaca taatatcaac cacggcggtg catctcagaa gcttatcaga 10200
gaaaattgat gacattttgc ggttaataga cgctctggca aaagacttgg gtaatcaagt 10260
ctacgatgtt gtatcactaa tggagggatt tgcatacgga gctgtccagc tactcgagcc 10320
gtcaggtaca tttgcaggag atttcttcgc attcaacctg caggagctta aagacattct 10380
aattggcctc ctccccaatg atatagcaga atccgtgact catgcaatcg ctactgtatt 10440
ctctggttta gaacagaatc aagcagctga gatgttgtgt ctgttgcgtc tgtggggtca 10500
cccactgctt gagtcccgta ttgcagcaaa ggcagtcagg agccaaatgt gcgcaccgaa 10560
aatggtagac tttgatatga tccttcaggt actgtctttc ttcaagggaa caatcatcaa 10620
cgggtacaga aagaagaatg caggtgtgtg gccgcgagtc aaagtggata caatatatgg 10680
gaaggtcatt gggcaactac atgcagattc agcagagatt tcacacgata tcatgttgag 10740
agagtataag agtttatctg cacttgaatt tgagccatgt atagaatatg accctgtcac 10800
caacctgagc atgttcctaa aagacaaggc aatcgcacac cccaacgata attggcttgc 10860
ctcgtttagg cggaaccttc tctccgaaga ccagaagaaa catgtaaaag aagcaacttc 10920
gactaatcgc ctcttgatag agtttttaga gtcaaatgat tttgatccat ataaagagat 10980
ggaatatctg acgacccttg agtaccttag agatgacaat gtggcagtat catactcgct 11040
caaggagaag gaagtgaaag ttaatggacg gatcttcgct aagctgacaa agaagttaag 11100
gaactgtcag gtgatggcgg aagggatcct agccgatcag attgcacctt tctttcaggg 11160
aaatggagtc attcaggata gcatatcctt gaccaagagt atgctagcga tgagtcaact 11220
gtcttttaac agcaataaga aacgtatcac tgactgtaaa gaaagagtat cttcaaaccg 11280
caatcatgat ccgaaaagca agaaccgtcg gagagttgca accttcataa caactgacct 11340
gcaaaagtac tgtcttaatt ggagatatca gacaatcaaa ttgttcgctc atgccatcaa 11400
tcagttgatg ggcctacctc acttcttcga atggattcac ctaagactga tggacactac 11460
gatgttcgta ggagaccctt tcaatcctcc aagtgaccct actgactgtg acctctcaag 11520
agtccctaat gatgacatat atattgtcag tgccagaggg ggtatcgaag gattatgcca 11580
gaagctatgg acaatgatct caattgctgc aatccaactt gctgcagcta gatcgcattg 11640
tcgtgttgcc tgtatggtac agggtgataa tcaagtaata gcagtaacga gagaggtaag 11700
atcagacgac tctccggaga tggtgttgac acagttgcat caagccagtg ataatttctt 11760
caaggaatta attcatgtca atcatttgat tggccataat ttgaaggatc gtgaaaccat 11820
caggtcagac acattcttca tatacagcaa acgaatcttc aaagatggag caatcctcag 11880
tcaagtcctc aaaaattcat ctaaattagt gctagtgtca ggtgatctca gtgaaaacac 11940
cgtaatgtcc tgtgccaaca ttgcctctac tgtagcacgg ctatgcgaga acgggcttcc 12000
caaagacttc tgttactatt taaactatat aatgagttgt gtgcagacat actttgactc 12060
tgagttctcc atcaccaaca attcgcaccc cgatcttaat cagtcgtgga ttgaagacat 12120
ctcttttgtg cactcatatg ttctgactcc tgcccaatta gggggactga gtaaccttca 12180
atactcaagg ctctacacta gaaatatcgg tgacccgggg actactgctt ttgcagagat 12240
caagcgacta gaagcagtgg gattactgag tcctaacatt atgactaata tcttaactag 12300
gccgcctggg aatggagatt gggccagtct gtgcaacgac ccatactctt tcaattttga 12360
gactgttgca agcccaaata ttgttcttaa gaaacatacg caaagagtcc tatttgaaac 12420
ttgttcaaat cccttattgt ctggagtgca cacagaggat aatgaggcag aagagaaggc 12480
attggctgaa ttcttgctta atcaagaggt gattcatccc cgcgttgcgc atgccatcat 12540
ggaggcaagc tctgtaggta ggagaaagca aattcaaggg cttgttgaca caacaaacac 12600
cgtaattaag attgcgctta ctaggaggcc attaggcatc aagaggctga tgcggatagt 12660
caattattct agcatgcatg caatgctgtt tagagacgat gttttttcct ccagtagatc 12720
caaccacccc ttagtctctt ctaatatgtg ttctctgaca ctggcagact atgcacggaa 12780
tagaagctgg tcacctttga cgggaggcag gaaaatactg ggtgtatcta atcctgatac 12840
gatagaactc gtagagggtg agattcttag tgtaagcgga gggtgtacaa gatgtgacag 12900
cggagatgaa caatttactt ggttccatct tccaagcaat atagaattga ccgatgacac 12960
cagcaagaat cctccgatga gggtaccata tctcgggtca aagacacagg agaggagagc 13020
tgcctcactt gcaaaaatag ctcatatgtc gccacatgta aaggctgccc taagggcatc 13080
atccgtgttg atctgggctt atggggataa tgaagtaaat tggactgctg ctcttacgat 13140
tgcaaaatct cggtgtaatg taaacttaga gtatcttcgg ttactgtccc ctttacccac 13200
ggctgggaat cttcaacata gactagatga tggtataact cagatgacat tcacccctgc 13260
atctctctac agggtgtcac cttacattca catatccaat gattctcaaa ggctgttcac 13320
tgaagaagga gtcaaagagg ggaatgtggt ttaccaacag atcatgctct tgggtttatc 13380
tctaatcgaa tcgatctttc caatgacaac aaccaggaca tatgatgaga tcacactgca 13440
cctacatagt aaatttagtt gctgtatcag agaagcacct gttgcggttc ctttcgagct 13500
acttggggtg gtaccggaac tgaggacagt gacctcaaat aagtttatgt atgatcctag 13560
ccctgtatcg gagggagact ttgcgagact tgacttagct atcttcaaga gttatgagct 13620
caatctggag tcatatccca cgatagagct aatgaacatt ctttcaatat ccagcgggaa 13680
gttgattggc cagtctgtgg tttcttatga tgaagatacc tccataaaga atgacgccat 13740
aatagtgtat gacaataccc gaaattggat cagtgaagct cagaattcag atgtggtccg 13800
cctatttgaa tatgcagcac ttgaagtgct cctcgactgt tcttaccaac tctattacct 13860
gagagtaaga ggcctagaca atattgtctt atatatgggt gatttataca agaatatgcc 13920
aggaattcta ctttccaaca ttgcagctac aatatctcat cccgtcattc attcaaggtt 13980
acatgcagtg ggcctggtca accatgacgg atcacaccaa cttgcagata cggattttat 14040
cgaaatgtct gcaaaactat tagtatcttg cacccgacgt gtgatctccg gcttatattc 14100
aggaaataag tatgatctgc tgttcccatc tgtcttagat gataacctga atgagaagat 14160
gcttcagctg atatcccggt tatgctgtct gtacacggta ctctttgcta caacaagaga 14220
aatcccgaaa ataagaggct taactgcaga agagaaatgt tcaatactca ctgagtattt 14280
actgtcggat gctgtgaaac cattacttag ccccgatcaa gtgagctcta tcatgtctcc 14340
taacataatt acattcccag ctaatctgta ctacatgtct cggaagagcc tcaatttgat 14400
cagggaaagg gaggacaggg atactatcct ggcgttgttg ttcccccaag agccattatt 14460
agagttccct tctgtgcaag atattggtgc tcgagtgaaa gatccattca cccgacaacc 14520
tgcggcattt ttgcaagagt tagatttgag tgctccagca aggtatgacg cattcacact 14580
tagtcagatt catcctgaac tcacatctcc aaatccggag gaagaccact tagtacgata 14640
cttgttcaga gggataggga ctgcatcttc ctcttggtat aaggcatctc atctcctttc 14700
tgtacccgag gtaagatgtg caagacacgg gaactcctta tacttagctg aagggagcgg 14760
agccatcatg agtcttctcg aactgcatgt accacatgaa actatctatt acaatacgct 14820
cttttcaaat gagatgaacc ccccgcaacg acatttcggg ccgaccccaa ctcagttttt 14880
gaattcggtt gtttatagga atctacaggc ggaggtaaca tgcaaagatg gatttgtcca 14940
agagttccgt ccattatgga gagaaaatac agaggaaagt gacctgacct cagataaagc 15000
agtggggtat attacatctg cagtgcccta cagatctgta tcattgctgc attgtgacat 15060
tgaaattcct ccagggtcca atcaaagctt actagatcaa ctagctatca atttatctct 15120
gattgccatg cattctgtaa gggagggcgg ggtagtaatc atcaaagtgt tgtatgcaat 15180
gggatactac tttcatctac tcatgaactt gtttgctccg tgttccacaa aaggatatat 15240
tctctctaat ggttatgcat gtcgaggaga tatggagtgt tacctggtat ttgtcatggg 15300
ttacctgggc gggcctacat ttgtacatga ggtggtgagg atggcaaaaa ctctggtgca 15360
gcggcacggt acgcttttgt ctaaatcaga tgagatcaca ctgaccaggt tattcacctc 15420
acagcggcag cgtgtgacag acatcctatc cagtccttta ccaagattaa taaagtactt 15480
gaggaagaat attgacactg cgctgattga agccggggga cagcccgtcc gtccattctg 15540
tgcggagagt ctggtgagca cgctagcgaa cataactcag ataacccaga tcatcgctag 15600
ccacattgac acagttatcc ggtctgtgat atatatggaa gctgagggtg atctcgctga 15660
cacagtattt ctatttaccc cttacaatct ctctactgac gggaaaaaga ggacatcact 15720
taaacagtgc acgagacaga tcctagaggt tacaatacta ggtcttagag tcgaaaatct 15780
caataaaata ggcgatataa tcagcctagt gcttaaaggc atgatctcca tggaggacct 15840
tatcccacta aggacatact tgaagcatag tacctgccct aaatatttga aggctgtcct 15900
aggtattacc aaactcaaag aaatgtttac agacacttct gtactgtact tgactcgtgc 15960
tcaacaaaaa ttctacatga aaactatagg caatgcagtc aaaggatatt acagtaactg 16020
tgactcttaa cgaaaatcac atattaatag gctccttttt tggccaattg tattcttgtt 16080
gatttaatca tattatgtta gaaaaaagtt gaaccctgac tccttaggac tcgaattcga 16140
actcaaataa atgtcttaaa aaaaggttgc gcacaattat tcttgagtgt agtctcgtca 16200
ttcaccaaat ctttgtttgg tgggtcggca tggcatctcc acctcctcgc ggtccgacct 16260
gggcatccga aggaggacgc acgtccactc ggatggctaa gggagagcca tagcataacc 16320
ccttggggcc tctaaacggg tcttgagggg ttttttggtc gacattagga tcctctacgc 16380
cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc 16440
cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg 16500
cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc 16560
accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat 16620
gcaggagtcg cataagggag agcgtcgacc gatgcccttg agagccttca acccagtcag 16680
ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat 16740
catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt 16800
tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct 16860
cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat 16920
cgccggcatg gcggccgacg cgctgggcta cgtcttgctg gcgttcgcga cgcgaggctg 16980
gatggccttc cccattatga ttcttctcgc ttccggcggc atcgggatgc ccgcgttgca 17040
ggccatgctg tccaggcagg tagatgacga ccatcaggga cagcttcaag gatcgctcgc 17100
ggctcttacc agcctaactt cgatcactgg accgctgatc gtcacggcga tttatgccgc 17160
ctcggcgagc acatggaacg ggttggcatg gattgtaggc gccgccctat accttgtctg 17220
cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa tggaagccgg 17280
cggcacctcg ctaacggatt caccactcca agaattggag ccaatcaatt cttgcggaga 17340
actgtgaatg cgcaaaccaa cccttggcag aacatatcca tcgcgtccgc catctccagc 17400
agccgcacgc ggcgcatctc gggcagcgtt gggtcctggc cacgggtgcg catgatcgtg 17460
ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa 17520
tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca 17580
acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg 17640
ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca 17700
cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc 17760
cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca 17820
tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac 17880
agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg 17940
gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg 18000
gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc 18060
tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg 18120
gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg 18180
ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat 18240
actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg 18300
aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc 18360
tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 18420
cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 18480
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 18540
gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 18600
gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 18660
ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 18720
atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 18780
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 18840
ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 18900
gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 18960
ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 19020
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 19080
agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 19140
ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 19200
aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 19260
tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 19320
cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 19380
tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 19440
cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 19500
ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 19560
gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac 19620
gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 19680
gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 19740
gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 19800
tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 19860
aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc 19920
cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 19980
caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 20040
cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 20100
ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 20160
aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 20220
tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaa 20265

Claims (6)

1.一种分泌型多肽氨基酸，其序列如SEQ ID NO.1所示。
2.编码权利要求1所述的分泌型多肽氨基酸的核苷酸，其序列如SEQ ID NO.2所示。
3.权利要求1所述的分泌型多肽氨基酸作为抗氧化剂的应用。
4.权利要求1所述的分泌型多肽氨基酸在制备抗肿瘤药物中的应用。
5.编码权利要求1所述的分泌型多肽氨基酸的重组溶瘤新城疫病毒Lasota。
6.一种根据权利要求5所述的重组溶瘤新城疫病毒Lasota的制备方法，其特征在于：步骤为：
(1)重组溶瘤新城疫病毒Lasota全基因组cDNA序列转录载体pBR-rNDV-pNaK将化学合成的多肽核苷酸序列SEQ ID NO.2以及新城疫编码的6个基因序列按照克隆策略通过PCR扩增、酶切、连接的方式连接到中间载体pBR322-M上，中间载体如SEQ ID NO.3所示，其顺序依次为核酸蛋白基因NP、pNaKtide多肽、磷酸蛋白基因P、基质基因M、融合蛋白基因F、血凝素蛋白基因HN和聚合酶蛋白基因L，其中在多肽核苷酸序列SEQ ID NO.2的5’端插入一个内部核糖体进入位点序列IRES；同时，在全基因组的头部和尾部分别连接具有自我剪切功能的拳头状核酶序列HamRz与丁肝核酶序列Rbz，得到重组载体pBR-rNDV-pNaK，如SEQ ID NO.4所示；
(2)辅助质粒构建
以步骤(1)中重组溶瘤新城疫病毒Lasota的cDNA为模板，采用高保真酶与核酸蛋白基因NP、磷酸蛋白基因P及聚合酶蛋白基因L分别对应的上游引物、下游引物进行PCR扩增核酸蛋白基因NP、磷酸蛋白基因P及聚合酶蛋白基因L片段，产物经(0.05-2.89)％琼脂糖凝胶电泳鉴定，切胶回收；核酸蛋白基因NP、磷酸蛋白基因P的PCR产物插入pCI-neo载体的BmtI与MluI克隆酶切位点，聚合酶蛋白基因L的PCR产物插入到pCI-neo载体的MluI与NotI克隆酶切位点，获得三个转录辅助质粒分别命名为pCI-NP、pCI-P、pCI-L，并测序验证；其中
NP基因上游引物：
5’-CCGGCTAGCATGTCCTCCGTATTTGATGAGTAC-3’，
NP基因下游引物：
5’-TAGAGGTACCACGCGTTCAATACCCCCAGTCGGTGTCGTTA-3’
P基因上游引物：5’-CCGGCTAGCATGGCCACCTTTACAGATGCAGA-3’
P基因下游引物：5’-TCGACGCGTTTAGCCATTTAGAGCAAGGCGC-3’
L基因上游引物：5’-CGACGCGTATGGCGAGCTCCGGTCCTGA-3’
L基因下游引物：
PCI-NotR:5’-GGAAAAAAGCGGCCGCTTAAGAGTCACAGTTACTGTAATATC-3’；
(3)细胞转染与病毒检测，获得病毒
将步骤(2)获得的三个转录辅助质粒pCI-NP、pCI-P、pCI-L及步骤(1)获得的重组溶瘤新城疫病毒Lasota全基因组cDNA序列转录载体pBR-rNDV-pNak，按照质量比为(2-6)：(1-3)：(1-3)：(0.2-1.5)，分别转化大肠杆菌感受态细胞DH5a，(30-42)℃，Amp抗性，大体积摇菌，利用QIAGEN质粒制备试剂盒抽提转染所用的质粒，并测定各质粒的浓度，采用脂质体试剂盒进行病毒检测，结果均为阳性的样品中含有拯救成功的重组溶瘤新城疫病毒。
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