CN113881643B - 一种6a型丙肝病毒亚基因组复制子及应用 - Google Patents
一种6a型丙肝病毒亚基因组复制子及应用 Download PDFInfo
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Abstract
本发明涉及基因工程技术领域，具体公开了一种6a型丙肝病毒(HCV)亚基因组复制子及应用。所述复制子包括在NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白、NS5B蛋白上的适应性突变。本发明的6a型HCV毒株来自中国，其获得的6a型丙肝病毒亚基因组复制子模型RNA复制非常高效(RNA复制表达量达到109.8拷贝/μg RNA)，且有对应的高效全长感染性克隆。本发明还利用该复制子系统检测了对IFNα2b、Daclatasvir和Sofosbuvir药物的敏感性，证明了复制子是良好的药物筛选模型。另外，本发明还利用SGR‑CH6a系统研究了3’UTR区域的序列差异在复制环节的影响和作用。
Description
技术领域
本发明涉及基因工程技术领域，尤其是涉及一种6a型丙肝病毒亚基因组复制子及应用。
背景技术
HCV主要分为8种主要基因型(genotype，GT)和67种亚型基因。主要基因型之间序列差异约为30％，多数主要基因型又可分为多种亚型(用a、b、c等表示)，各亚型之间的序列差异为20-25％。不同基因型HCV具有一定的地区和人群分布，治疗反应和致病机制的差异性。除了遗传生物学差异外，基因型还是影响药物治疗效果的一个重要因素。其中，3a和6a型HCV越来越受到更多关注。
HCV亚基因组复制子是在HCV全基因组中将核心蛋白(Core)至非结构蛋白2(NS2)的一段基因位置替换为非病毒成分新霉素磷酸转移酶(neomycin phosphatetransferase，Neo)基因(G418抗性筛选)和脑心肌炎病毒(encephalomyocarditis virus，EMCV)核糖核酸病毒IRES的基因编码序列。因此HCV复制子克隆具有双顺反子，分别编码抗G418的Neo基因和HCV复制酶(NS3-NS5B)基因。将复制子克隆体外转录为RNA后转染人肝癌细胞系Huh7等，经G418抗性筛选可获得具有HCV RNA高水平复制的细胞克隆。
HCV亚基因组复制子体系可部分模拟HCV的真实复制周期但不产生感染性病毒颗粒，通过测定HCV正链和负链RNA及非结构蛋白水平，可评价HCV复制的水平。HCV复制子系统自建立以来，对DAAs的研究和发展起到非常重要的作用。复制子已经成为非结构蛋白的功能研究、筛选靶向抗病毒药物，DAAs研究、抗病毒宿主靶点和抗药性研究必不可少的工具。
2014年，Yu等人根据16个HCV 6a毒株合成一个共有的GT6a序列，加入Fluc、Neo、IRES元件筛选了高效复制的第一个6a型复制子。同年，Kinge等构建了第一个HCV GT5a毒株SA1的亚基因组复制子，并用该系统证实了S2205I依然有增进复制的作用。2018年，Camus等人继续用同样的方法筛选GSI6a-1(6a)的亚基因组复制子，证明S2208G突变对复制增强作用比S2208I更佳。
HCV亚基因组复制子体系可部分模拟HCV的真实复制周期但不产生感染性病毒颗粒，通过测定HCV正链和负链RNA及非结构蛋白水平，可评价HCV复制的水平。HCV复制子系统自建立以来，对DAAs的研究和发展起到非常重要的作用。复制子已经成为非结构蛋白的功能研究、筛选靶向抗病毒药物，DAAs研究、抗病毒宿主靶点和抗药性研究必不可少的工具。
目前已经两个6a型HCV毒株复制子模型，但是还未有针对我国6a型HCV毒株(参照本申请人申请过的中国专利CN110129341A)的复制子模型。
发明内容
本发明的目的在于克服上述现有技术的不足之处而提供一种6a型丙肝病毒亚基因组复制子及应用。
为实现上述目的，本发明采取的技术方案为：
第一目的，本发明提供了一种6a型丙肝病毒亚基因组复制子，所述复制子包括在NS3蛋白、NS4A蛋白、NS4B蛋白、NS5A蛋白、NS5B蛋白上的适应性突变；
所述适应性突变包括以下氨基酸残基的突变：
1)在NS3蛋白上的第1120位的谷氨酰胺突变为赖氨酸；
2)在NS3蛋白上的第1285位的苏氨酸突变为丙氨酸；
3)在NS3蛋白上的第1303位的赖氨酸突变为精氨酸；
4)在NS3蛋白上的第1370位的苏氨酸突变为异亮氨酸；
5)在NS3蛋白上的第1469位的苯丙氨酸突变为亮氨酸；
6)在NS3蛋白上的第1555位的缬氨酸突变为亮氨酸；
7)在NS3蛋白上的第1648位的赖氨酸突变为谷氨酰胺；
8)在NS4A蛋白上的第1677位的丙氨酸突变为丝氨酸；
9)在NS4A蛋白上的第1696位的赖氨酸突变为精氨酸；
10)在NS4B蛋白上的第1720位的异亮氨酸突变为苯丙氨酸；
11)在NS4B蛋白上的第1795位的亮氨酸突变为蛋氨酸；
12)在NS5A蛋白上的第2358位的苯丙氨酸突变为半胱氨酸；
13)在NS5A蛋白上的第2388位的天冬氨酸突变为丙氨酸；
14)在NS5A蛋白上的第2392位的谷氨酸突变为甘氨酸；
15)在NS5A蛋白上的第2423位的天冬氨酸突变为甘氨酸；
16)在NS5B蛋白上的第2805位的天冬酰胺突变为天冬氨酸；
17)在NS5B蛋白上的第2927位的丙氨酸突变为苏氨酸；
18)在NS5B蛋白上的第2941位的丙氨酸突变为缬氨酸；
19)在NS5B蛋白上的第2987位的天冬氨酸突变为甘氨酸；
20)在NS5B蛋白上的第2989位的酪氨酸突变为苯丙氨酸。
作为本发明所述6a型丙肝病毒亚基因组复制子的优选实施方式，复制子的核苷酸全长序列如SEQ ID NO:1所示。
第二目的，本发明提供了上述6a型丙肝病毒亚基因组复制子的构建方法，包括以下步骤：
将CH6a_ORF_m26基因组克隆的Core-NS2段基因替换为NPT II基因和EMCV的IRES序列，引入Q1120K、N2805D和A2927T三个适应性突变，获得CH6a毒株的亚基因组复制子SGR-CH6a_20m。
第三目的，本发明提供了一种多核苷酸，包括如上述的6a型丙肝病毒亚基因组复制子。
第四目的，本发明提供了一种表达载体，所述表达载体包括上述的多核苷酸。
第五目的，本发明提供了一种基因工程化的细胞，所述细胞包括上述的多核苷酸或表达载体，更优选地，细胞包括Huh7.5。
第六目的，本发明提供了上述6a型丙肝病毒亚基因组复制子在制备抗病毒药物上的应用。
第七目的，本发明提供了上述6a型丙肝病毒亚基因组复制子在筛选(HCV)HCV感染药物中的应用。
与现有技术相比，本发明具有以下有益效果：
本发明的6a型HCV毒株来自中国，其获得的6a型丙肝病毒亚基因组复制子模型RNA复制非常高效(RNA复制表达量达到109.8拷贝/μg RNA)，且有对应的高效全长感染性克隆。本发明还利用该复制子系统检测了其对IFNα2b、Daclatasvir和Sofosbuvir药物的敏感性，证明了复制子是良好的药物筛选模型。另外，本发明还利用SGR-CH6a系统研究了3’UTR区域的序列差异在复制环节的影响和作用。
附图说明
图1为6a型丙肝病毒亚基因组复制子的结构和突变位点图；
图2为不同嵌合3’UTR的SGR-CH6a复制子的活性图；
图3为6a型丙肝病毒亚基因组复制子在Huh7.5.1细胞内的表达水平图；
图4为IFNα2b对6a型丙肝病毒亚基因组复制子的抑制作用；
图5为6a型丙肝病毒亚基因组复制子SGR-CH6a(SGR-JFH1做对照)对抗病毒药物不同浓度的效应图。
具体实施方式
为更好的说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。
在以下实施例中，所使用的实验方法如无特殊说明，均为常规方法，所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明选用的6a型HCV毒株为中国专利CN110129341A中的CH6a毒株。
HCV 3′UTR分为可变区(varible region，VR)、polyU/UC区和3’-X区。
实施例1、复制子筛选
步骤：
1.与转染HCV病毒一样，提前24h在6孔板每孔铺3.5×105个细胞；
2.线性化HCV亚基因组复制子后，体外转录RNA后，RQ1去除DNA模板；
3.5μl Lipo 2000包裹5μg RNA转染Huh7.5.1-VISI细胞；
4.转染后48h将细胞转移至10cm皿加G418(800μg/mL)筛选，未转染细胞作对照，看细胞死亡情况，及时更换培养基；
5.对照组细胞死光后，G418浓度减半，大约筛选3周；
6.活细胞结晶紫染色；
7.免疫荧光检测非结构蛋白NS5A表达；
8.提取RNA扩增亚基因组检测核苷酸的突变。
将专利CN110129341A中获得的CH6a_ORF_m26基因组克隆的Core-NS2段基因替换为NPT II基因和EMCV的IRES序列，并引入16个HCV GT6a欧洲毒株合成的非结构蛋白共有序列GT6a亚基因组复制子的突变Q1120K和今年刚报道的HK6acc和HK2cc毒株的两个适应性突变N2805D(在NS5B蛋白上的第2805位的天冬酰胺突变为天冬氨酸)和A2927T(在NS5B蛋白上的第2927位的丙氨酸突变为苏氨酸)，构建了CH6a毒株的亚基因组复制子SGR-CH6a_20m(复制子的结构和突变位点如图1所示)。转染Huh7.5.1-VISI-GFP后，G418药物筛选2-3周，本发明获得了高效复制CH6a亚基因组的细胞克隆。
体外转录SGR-CH6a RNA(5μg)转染Huh7.5.1-VISI-mCherry细胞，G418(800μg/mL)筛选2-3周，用结晶紫染色稳定表达复制子的细胞。本发明设计克隆将SGR-CH6a_20m的JFH13’UTR的VR、polyUC和3’-X区域分别替换为CH6a毒株的序列，G418筛选2-3周后结晶紫染色，其不同嵌合3’UTR的SGR-CH6a复制子(序列如SEQ ID NO:1所示)的活性如图2所示。
6a型丙肝病毒亚基因组复制子在Huh7.5.1细胞内的表达水平如图3所示。
实施例2、6a型丙肝病毒亚基因组测序和拷贝数测定
设计CH6a亚基因组复制子序列扩增的特异引物(引物表1)。提取表达6a型丙肝病毒亚基因组复制子的细胞的RNA，反转录cDNA。分两段RT-PCR扩增亚基因组，检测非结构蛋白(NS3-NS5B)区域的突变；绝对定量PCR测RNA拷贝数。
1.提取细胞RNA后，用PolyA作引物反转，先用NS5B通用HCV扩增引物鉴定毒株是否为CH6a；
2.用SGR-CH6a特异引物(序列见引物表1)两段扩增SGR-CH6a的亚基因组；
3.测序分析复制子序列，并比对分析序列发现选取的5个细胞克隆只有1个克隆有一个突变位点S2361T(G7244C)，其它克隆无突变；
4.用qPCR绝对定量的方法检测细胞RNA反转录产物cDNA内SGR-CH6aRNA的拷贝数；
5.本发明6a型丙肝病毒亚基因组复制子的复制水平达到109.8拷贝/μg RNA。
表1 SGR-CH6a引物序列
实施例3、6a型丙肝病毒亚基因组复制子的干扰处理
本发明的复制子系统检测IFNα2b药物的敏感性：
(A)培养基加IFNα2b(10IU/mL)处理表达CH6a复制子的Huh7.5.1-VISI-GFP细胞2周，通过荧光显微镜观察细胞核的GFP，评估复制子的表达水平。(B)IFNα2b(10IU/mL)处理第4天复制子的表达水平，对照不作处理。
参考图4，IFNα2b处理4天后，细胞核表达的GFP明显下降，对照无变化。说明IFNα2b抑制CH6a复制子的复制，与报道的临床实验和治疗结果有很好的相关性。
本发明的复制子系统检测Daclatasvir和Sofosbuvir药物的敏感性：
96孔板每孔种9000个G418筛选的稳定表达复制子的细胞，24h后改不同梯度浓度药物Daclatasvir(A)和Sofosbuvir(B)处理，72h后NS5A蛋白的抗体做免疫荧光检测复制子的表达水平(±SEM)。
参考图5，Daclatasvir和Sofosbuvir药物的效应浓度EC50(nM，95％CI)分别为0.010(0.0068-0.016)和338.2(282.2-405.3)，和CH6acc系统相当。说明该亚基因组复制子系统是筛选药物的有效模型。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
SEQUENCE LISTING
<110> 中山大学
<120> 一种6a型丙肝病毒亚基因组复制子及应用
<130> 2021.09.29
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 8021
<212> DNA
<213> 人工合成
<400> 1
gccagcccct taacggggcg acactccgcc attatcactc ccctgtgagg aactactgtc 60
ttcacgcaga aagcgtctag ccatggcgtt agtatgagtg tcgtgcagcc tccaggcccc 120
cccctcccgg gagagccata gtggtctgcg gaaccggtga gtacaccgga attgccagga 180
tgaccgggtc ctttccattg gatcaaaccc gctcaatgcc tggagatttg ggcgtgcccc 240
cgcaagactg ctagccgagt agcgttgggt tgcgaaaggc cttgtggtac tgcctgatag 300
ggtgcttgcg agtgccccgg gaggtctcgt agaccgtgca tcatgagcac acttccaaaa 360
ccccaaagaa aaaccaaaag aaacaccaac ggcgcgccaa tgattgaaca agatggattg 420
cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag 480
acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt 540
tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta 600
tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg 660
ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt 720
gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat 780
ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg 840
atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca 900
gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc 960
catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc 1020
gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat 1080
attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc 1140
gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgaggccgg 1200
ccgcggccgc aacagaccac aacggtttcc ctctagcggg atcaattccg cccccccccc 1260
ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat 1320
tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct 1380
tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg 1440
tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc 1500
tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg 1560
tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg 1620
tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga 1680
aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt 1740
agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa 1800
aaacacgata cgcgtatggc tcccatcact gcgtatgcac agcagacgag gggcctagtc 1860
ggcaccattg tgaccagcct aaccggacgt gacaaaaatg aggtcgaagg ggaggtacag 1920
gtggtctcca cggctaccca atccttcctg gcgacctcca ttaacggtgt catgtggacc 1980
gtttatcatg gggccggttc aaagactctc gccggaccga aaggaccagt gtgtcaaatg 2040
tacaccaacg tggacaagga cctagtaggg tggccatctc cccagggagc aagatccctc 2100
accccatgta cttgtggctc tagtgacctc tatttggtca cgagggaggc cgacgtcatc 2160
cccgcaagac gcaggggtga cagccgtgct gccctcctct ctcctaggcc cataagcacc 2220
ttgaaaggct cttcgggagg ccccattatg tgtccctcgg ggcacgttgt gggactcttc 2280
cgagcagccg tatgcacaag gggtgtagca aaatccttag attttatccc agtggaaaac 2340
atggagacga ctatgcgctc tccctcattc acagacaatt ccacaccccc tgcggtgccc 2400
cagacctatc aggtggggta cctgcacgca ccaacaggca gcggaaagag cacccgtgtc 2460
ccggcggcgt acgctagcca gggctacaag gtgttggtct tgaacccatc ggtggcggcg 2520
acgcttagct ttggctcgta catgaggcaa gcttacggcg tggagccgaa tgtccggact 2580
ggggtcagga ctataactac agggggcgcc atcacatact ccacatatgg gaaattcttg 2640
gccgatgggg gatgttccgg aggggcgtat gacatcatca tctgcgatga gtgccactct 2700
acagacccta cgacggtgtt gggcattggc acggttctcg accaggctga gaccgccggg 2760
gttcgtctta ccgtgcttgc aacagcaacg ccgccgggtt ctgtcaccgt accccatcct 2820
aacataacag agacagccct cccaactacg ggagaaatac cattttatgg aaagggcatc 2880
cctcttgagt acatcaaagg gggaagacac ctcatattct gtcactcgaa gaagaagtgc 2940
gacgagctgg ccgggaaact gaagtcactc ggcttaaacg ccgtcgcatt ctacagaggt 3000
gtcgacgtgt ccgttatccc cacttcgggc gacgtcgtcg tctgcgcgac agacgccctt 3060
atgaccggct acacaggcga tttcgattcc gtcatcgact gtaacgtagc cgtgacacag 3120
gtggtggatt tcagcttgga cccgacattt tccatagaga ctaccaccgt ccctcaggat 3180
gcggtatcac ggagccaacg acgaggccgc acggggcggg gtaaaccggg ggtgtacagg 3240
tttgtctccc agggggagag gccttcgggt atgttcgaca ccgtcgtcct gtgtgaggct 3300
tatgacacgg gatgcgcgtg gtacgaacta accccttctg aaacaactgt caggctgagg 3360
gcctatctga acactcctgg ccttcccgta tgccaagacc acctggaatt ttgggaaggc 3420
gtgtttactg gcttgaccca catagacgcc cactttctgt ctcagacgaa gcaggggggt 3480
gagaacttcg cgtacctcgt ggcataccag gctacagtat gcgccagggc caaagccccc 3540
ccgccttctt gggatacgat gtggaagtgt ctcatcagac ttaaacccac ccttaccggc 3600
cccactccac ttttgtatcg gctgggggcc gtacaaaatg aggtaataac aacccacccc 3660
ataaccaaat acatcatgac ctgcatgtct gcggatttgg aggtcatcac cagcacgtgg 3720
gtccttgtgg gtggagtcct agccgcgctc gcagcctact gcttgtcagt gggttgtgtt 3780
gtcatctgtg gcaggataac cttgactggc aagcctgctg ttgtccctga tcgtgagatc 3840
ttgtaccagc aatttgacga gatggaagag tgctctaggc acatccccta cctcgccgag 3900
ggccagcaga tcgccgagca attcagacaa aaggtgttgg gactccttca agcgagcgcc 3960
aagcaggcag aagaactgaa gcctgctgtc cattccgcgt ggcctagggt ggaggagttt 4020
tggaggaagc acatgtggaa ctttgtcagc gggatccagt atttggcggg tttatccacc 4080
ctgcccggca acccagccgt ggcatcattg atgtcattta cagcgtcgct gaccagtcct 4140
ctgaggactt ctcagaccct gctcctcaac atactcggcg gctggatagc cacccaagtg 4200
gctccccccc ccgcgtccac agcttttgtc gtgagcggtc tagcaggagc cacggttgga 4260
agcatcgggc tcgggagggt gttggttgat gtgcttgccg gatacggggc cggtgtgtcg 4320
ggtgctctag tcgctttcaa gatcatgagc ggcgagtgcc caaccaccga agacatggtc 4380
aatctgctac ccgcgctgtt gtcgccaggg gctctcgtgg tgggagtcgt gtgtgctgcc 4440
atcttaagac gccacgtagg ccctgcggag ggtgccaatc agtggatgaa caggctaata 4500
gcctttgcat caagaggcaa ccatgtgtcc ccgacgcact acgtgcctga gactgacgcg 4560
tcaaaaaatg tgactcagat actcacttct cttaccatca ccagcctact ccgcaggtta 4620
catcagtggg tcaatgagga cacggccacc ccttgcgcta cctcatggtt acgcgacgtg 4680
tgggactggg tgtgcacagt gctatctgac tttaaagtat ggttgcaagc caaacttttc 4740
cctcgcctgc cggggatccc cttcctctcg tgccaaacgg gatatagggg agtctgggca 4800
ggggacgggg tgtgccacac cacttgtacc tgtggggccg tgatagctgg acacgtcaaa 4860
aatggctcca tgaaaatcac aggacccaag acatgcagca acacttggca cgggactttc 4920
ccaatcaacg ccaccactac cggccccagc acaccacgac cagccctcaa ctatcagcgc 4980
gctctttggc gggtgtccgc cgaggactac gttgaggtgc ggaggttggg cgactgccac 5040
tacgtggtag gggtcactgc tgacgggttg aaatgccctt gccaggtacc tgcacctgaa 5100
ttcttcactg aggtcgatgg cgtgaggata caccgttacg cgccaccttg caagcccttg 5160
ctcagggacg aagtgacttt ctctgtgggt ctttcaaact atgtcatagg gtctcagctc 5220
ccttgcgagc cagagcctga cgtgaccgtg gtcacctcaa tgctcacaga ccccgtgcac 5280
atcaccgcag agacggcagc acggcggttg aagagggggt cccccccctc tttagccagc 5340
tcatcggcca gccagctgtc tgcaccgtcc ctcaaggcta cttgtacaac ctccagagac 5400
cacccggaca tggagctcat cgaggccaac ctcctctgga ggcaggagat gggaggcaac 5460
atcactcgag tcgagtcaga gaacaaagtc gtagtccttg actcctttga gcctctaacc 5520
gctgagtatg acgagaggga aatctcagtg tcagctgagt gccacaagcc acccaggccc 5580
aaattccctc cagctctccc agtatgggcc aggcccgact acaatccacc tctcctacaa 5640
gcatggcaaa tgcccggata cgagcctcca gtcgtgtctg gatgtgccgt cgctccacct 5700
aaaccggcac caattcctcc gccgaggcgg aagaggctag tgcacttgga tgagtccacg 5760
gtctcgcacg ccttggcgca gcttgccgac aaggtatttg tggagagtgg tagtgacccg 5820
ggacctagtt cagactcggg actatcaata gccagtcctg tcccgcctgt ctcaacaaca 5880
ccggatgacg cctgctcaga agcagagtcc tatagctcaa tgccccctct tgagggagag 5940
cctggtgacc ctgacctgag ctcaggctct tggtccactg tgagcgatca ggatgatgtc 6000
gtgtgttgtt ccatgtccta ttcctggacg ggggctctaa taacaccatg cgctgcggag 6060
gaggagaagc ttccaataaa tcctatgagc aactccctta taagacacca taacatggtg 6120
tattccacca catcacgcag cgccagcctc cgccagaaga aggtcacatt tgacagactg 6180
caagtgtttg accaacacta ccaggatgta ctaaaggaga ttaagcttcg agcgtccacg 6240
gtgcaggcaa aactcctatc catagaggaa gcctgtgacc ttacgccatc gcactcagca 6300
cggtccaagt atggatatgg tgcgcaggac gttagaagcc atgctagcaa ggccgttgac 6360
cacatccgct ccgtgtggga ggacttgcta gaagactctg ataccccaat tcccacgacc 6420
atcatggcta agaatgaagt cttctgcgta gatccgtcga agggtggacg caagccggca 6480
cgcctaatag ttttcccaga cttgagcgtg cgggtctgcg agaagatggc cctatacgac 6540
gtgacgcgga agttaccaca ggccgtgatg ggttcagcat acggattcca gtactcccct 6600
aaccagaggg ttgagtactt gctcaaaatg tggcggtcaa agaaggtacc catgggcttt 6660
tcttacgaca ccaggtgttt tgattcaacc gtcaccgagc gggatatccg gactgagaac 6720
gaaatctatc agtcttgcca gctggacccc gtggcaagga gggcagtatc atccctaaca 6780
gagcggctct acgtaggcgg ccccatggta aactccaagg gacagccatg tggctaccgt 6840
agatgccgcg ccagtggggt gctgcccacg agcatgggaa acaccctcac atgctatctg 6900
aaggcacagg ccgcctgcag ggcggccaac atcaaaaact atgacatgtt ggtgtgcgga 6960
gatgacttag tggtcatttg tgagagtgct ggcgtccagg aggacactgc gtcactgcga 7020
gcattcacgg atgctatgac caggtattca gctccccctg gagacgcccc gcaacctaca 7080
tatgaccttg agctcataac atcatgctca tccaacgtct ccgtcgccca cgacggcaac 7140
gggaagagat attactacct cacacgtgac tgcactactc cacttgcgcg ggctgcatgg 7200
gagacagccc gccacactcc agtcaactcg tggttgggca acatcatcat gtttgccccc 7260
acgatatggg tgcgtatggt tctgatgacc cattttttct ccatactcca gtcgcaagaa 7320
caattggaga aagcactcga ctttgacatc tacggagtga cctattctgt ctctccactt 7380
gatctcccag caatcattca acgactccat ggcatggcag cattttcact ccacggatac 7440
tctccagttg agctcaatag ggtaggggct tgcctcagga aacttggggt acctcccttg 7500
agagcctgga gacatcgagc cagagctgtc agagccaaac tcattgccca gggggggaaa 7560
gcggctatat gcggtaagta cctctttaac tgggcggtga agaccaaatt aaaactcact 7620
ccactggtct ccgcgagcaa gcttgacttg tcaggctggt tcgtggcagg ctacgacggg 7680
ggggacattt atcacagcgt gtcccaggct cgaccccgtc tcttacttct tggcctactc 7740
ctactctcag taggggtagg catctttttg ctccccgctc ggtagagcgg cacacactag 7800
gtacactcca tagctaactg ttcctttttt tttttttttt tttttttttt tttttttttt 7860
tttttttctt tttttttttt ttccctcttt cttcccttct catcttattc tactttcttt 7920
cttggtggct ccatcttagc cctagtcacg gctagctgtg aaaggtccgt gagccgcatg 7980
actgcagaga gtgccgtaac tggtctctct gcagatcatg t 8021
<210> 2
<211> 20
<212> DNA
<213> 人工合成
<400> 2
aatggctctc ctcaagcgta 20
<210> 3
<211> 22
<212> DNA
<213> 人工合成
<400> 3
gctttacatg tgtttagtcg ag 22
<210> 4
<211> 20
<212> DNA
<213> 人工合成
<400> 4
agatgcctac ccctactgag 20
<210> 5
<211> 22
<212> DNA
<213> 人工合成
<400> 5
gccacgaacc agcctgataa gt 22
<210> 6
<211> 17
<212> DNA
<213> 人工合成
<400> 6
tcccgtttgg cacgaga 17
<210> 7
<211> 20
<212> DNA
<213> 人工合成
<400> 7
gaaaagtttg gcttgcaacc 20
<210> 8
<211> 24
<212> DNA
<213> 人工合成
<400> 8
tccgcaggtt acatcagtgg gtca 24
<210> 9
<211> 22
<212> DNA
<213> 人工合成
<400> 9
ccccttgcgc tacctcatgg tt 22
Claims (7)
1.一种6a型丙肝病毒亚基因组复制子，其特征在于，所述复制子的核苷酸全长序列如SEQ ID NO:1所示。
2.一种多核苷酸，其特征在于，包括如权利要求1所述的6a型丙肝病毒亚基因组复制子。
3.一种表达载体，其特征在于，所述表达载体包括如权利要求2所述的多核苷酸。
4.一种基因工程化的细胞，其特征在于，所述细胞包括如权利要求2所述的多核苷酸或如权利要求3所述的表达载体。
5.如权利要求4所述的细胞，其特征在于，所述细胞为Huh7.5。
6.如权利要求1所述的6a型丙肝病毒亚基因组复制子在制备抗病毒药物中的应用。
7.如权利要求1所述的6a型丙肝病毒亚基因组复制子在体外筛选抗HCV感染药物中的应用。
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| WO2019154472A1 (en) * | 2018-02-09 | 2019-08-15 | Hvidovre Hospital | Efficient cell culture system for hepatitis c virus genotype 6a |
| CN110129341A (zh) * | 2019-01-24 | 2019-08-16 | 中山大学 | 丙肝病毒基因6a型中国毒株全长感染性克隆及其应用 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019154472A1 (en) * | 2018-02-09 | 2019-08-15 | Hvidovre Hospital | Efficient cell culture system for hepatitis c virus genotype 6a |
| CN110129341A (zh) * | 2019-01-24 | 2019-08-16 | 中山大学 | 丙肝病毒基因6a型中国毒株全长感染性克隆及其应用 |
Non-Patent Citations (3)
| Title |
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| Mingxiao Chen et al..Development of an Infectious Cell Culture System for Hepatitis C Virus Genotype 6a Clinical Isolate Using a Novel Strategy and Its Sensitivity to Direct-Acting Antivirals.Frontiers in Microbiology.2018,第9卷1-18. * |
| Santseharay Ramirez et al..Current status and future development of infectious cell-culture models for the major genotypes of hepatitis C virus: Essential tools in testing of antivirals and emerging vaccine strategies.Antiviral Research.2018,第158卷264-287. * |
| 李义平 ; .HCV细胞感染模型研究进展及其在药物研究中的应用.临床肝胆病杂志.2015,第31卷(第11期),1825-1830. * |
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