CN113862231B - 一种3a型丙肝病毒亚基因组复制子及应用 - Google Patents
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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3a型丙肝病毒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;所述适应性突变为在突变位点上发生取代、插入或删除一个或多个氨基酸残基。采用本发明的复制子RNA复制非常高效,其中的突变位点可以用于提高3a型全长感染性克隆的复制和病毒传播速度与滴度。本发明的3a型复制子为HCV耐药位点分析、适应性突变功能、HCV致病机制及疫苗研发等领域的研究提供重要研究工具。
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3a型丙肝病毒亚基因组复制子及应用。
背景技术
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。
2012年,Saeed等人用含Neo基因和EMCV IRES的片段替代HCV 3a型S52毒株的20~1032位氨基酸获得新霉素抗性的亚基因组复制子S52/SG-Neo,并用编码萤火虫荧光素酶(Fluc)与Neo的融合基因取代Neo基因,获得亚基因组复制子S52/SG-Feo。为提高基因复制效率,引入位于NS5A蛋白的S2210I氨基酸点突变,使RNA达到5.5×107拷贝/μg RNA。2013年,Saeed等又从一例肝移植后复发的丙型肝炎患者得到3a型HCV毒株S310,用相同的策略建立了S310毒株在Huh7细胞中稳定复制的亚基因组复制子体系。
目前已经有S52、S310和PR87A7三个3a型HCV毒株复制子模型,但是还未有对应高效全长感染性克隆的3a型毒株的复制子模型。
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3a型丙肝病毒亚基因组复制子及应用,采用本发明的复制子RNA复制非常高效,其中的突变位点可以用于提高3a型全长感染性克隆的复制和病毒传播速度与滴度。本发明的3a型复制子为HCV耐药位点分析、适应性突变功能、HCV致病机制及疫苗研发等领域的研究提供重要研究工具。
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3a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;NS3蛋白中适应性突变的位点包括第1470位和/或第1618位,NS4A蛋白中适应性突变的位点包括第1678位,NS4B蛋白中适应性突变的位点包括第1775位、第1825位、第1840位和第1848位中的至少一位,NS5A蛋白中适应性突变的位点包括第2218位、第2256位、第2271位、第2350位和第2426位中的至少一位,NS5B蛋白中适应性突变的位点包括第2880位、第3004位和第3005位中的至少一位,所述适应性突变为在突变位点上发生取代、插入或删除一个或多个氨基酸残基。
本发明在3a型丙肝病毒亚基因组复制子上引入适应性突变,构建3a型丙肝病毒毒株全长基因组对应的带有氨基酸突变的亚基因组复制子SGR-CH3a_14m克隆。转染后G418筛选3周,经过NS5A抗体免疫荧光鉴定和亚基因组扩增测序鉴定,本发明确定得到了稳定表达SGR-CH3a_14m复制子的Huh7.5.1-VISI-mCherry细胞克隆。
作为本发明所述3a型丙肝病毒亚基因组复制子的优选实施方式,所述适应性突变为取代突变,至少包含以下一个或多个氨基酸残基的取代:
1)在NS3蛋白上的第1470位的苯丙氨酸突变为亮氨酸;
2)在NS3蛋白上的第1618位的缬氨酸突变为谷氨酸;
3)在NS4A蛋白上的第1678位的丙氨酸突变为丝氨酸;
4)在NS4B蛋白上的第1775位的缬氨酸突变为丙氨酸;
5)在NS4B蛋白上的第1825位的组氨酸突变为精氨酸;
6)在NS4B蛋白上的第1840位的亮氨酸突变为蛋氨酸;
7)在NS4B蛋白上的第1848位的异亮氨酸突变为缬氨酸;
8)在NS5A蛋白上的第2218位的赖氨酸突变为谷氨酸;
9)在NS5A蛋白上的第2256位的缬氨酸突变为甘氨酸;
10)在NS5A蛋白上的第2271位的天冬氨酸突变为天冬酰胺;
11)在NS5A蛋白上的第2350位的亮氨酸突变为脯氨酸;
12)在NS5A蛋白上的第2426位的丝氨酸突变为精氨酸;
13)在NS5B蛋白上的第2880位的丙氨酸突变为天冬氨酸;
14)在NS5B蛋白上的第3004位的亮氨酸突变为脯氨酸或蛋氨酸;
15)在NS5B蛋白上的第3005位的半胱氨酸突变为酪氨酸。
作为本发明所述3a型丙肝病毒亚基因组复制子的优选实施方式,所述适应性突变选自A~C组合中的任意一种:
A组合:适应性突变为1)~13)及15)的组成;
B组合:适应性突变为1)~13)、14)中亮氨酸突变为脯氨酸及15)的组成;
C组合:适应性突变为适应性突变为1)~13)、14)中亮氨酸突变为蛋氨酸及15)的组成。
作为本发明所述3a型丙肝病毒亚基因组复制子的优选实施方式,所述A组合或B组合还包括3’非翻译区的Δ11nt等突变。
作为本发明所述3a型丙肝病毒亚基因组复制子的优选实施方式,所述复制子的序列如SEQ ID NO:1所示。
优选地,所述3a型丙肝病毒亚基因组复制子还包括用于选择的标记基因和报告基因。更优选地,标记基因是新霉素磷酸转移酶基因,报告基因是荧光素酶。
第二目的,本发明提供了上述3a型丙肝病毒亚基因组复制子的构建方法,包括以下步骤:
在CH3a_Core-NS2上适应性突变K10Q、M375T、M715V、D877G、S933P,将CH3a_Core-NS2驯化5代获得的完全氨基酸突变M375T、D877G、S933P和K10Q、M375T、M715V、D877G、S933P分别引入基因组CH3a_Core-NS2后得到CH3a_C-NS2_3m和CH3a_C-NS2_5m克隆;
用JFH1 NS5B替换掉CH3a NS5B并保留LS突变的克隆CH3a_5-5A,将S52_5-5A病毒的适应性突变D877G、P1118L、F1470L、V1618E、A1678S、H1825R引入了CH3a_5-5A构建了CH3a_5-5A_6m;
将CH3a_5-5A_6m和CH3a_C-NS2_3m拼接获得CH3a_5-5A_9m;在CH3a_5-5A_9m基础上继续引入CH3a_5-5A_6m感染传代获得的的Core-NS2基因区段的适应性突变E72G、F372L、I374V、N533S、N946S和来自CH3a_Core-NS2的适应性突变K10Q得到CH3a_5-5A_15m克隆;
在CH3a_5-5A_15m嵌合病毒实现高效细胞感染培养后,在复制子基因组上引入了CH3a_5-5A_6m独立感染1的3个适应性突变,分别为K2218E、V2256G和L2350P,CH3a_5-5A_9m的1个突变D2271N,和DBN3acc毒株的6个突变V1775A、L1840M、I1848V、S2426R、A2880D、C3005Y,构建了CH3a毒株全长基因组对应的带有14个氨基酸突变F1470L、V1618E、A1678S、V1775A、H1825R、L1840M、I1848V、K2218E、V2256G、D2271N、L2350P、S2426R、A2880D、C3005Y的亚基因组复制子CH3a-SGRep克隆;在亚基因组复制子CH3a-SGRep基础上引入L3004P适应性突变构建成亚基因组复制子CH3a-SGRep+L3004P,在亚基因组复制子CH3a-SGRep基础上引入3’非翻译区的Δ11nt等突变构建成亚基因组复制子CH3a-SGRep+Δ11nt,在亚基因组复制子CH3a-SGRep+L3004P基础上引入3’非翻译区的Δ11nt等突变构建成CH3a-SGRep+L3004P/Δ11nt,在亚基因组复制子CH3a-SGRep基础上引入L3004M适应性突变构建成亚基因组复制子CH3a-SGRep+L3004M。
第三目的,本发明提供了一种病毒颗粒,包括上述的3a型丙肝病毒亚基因组复制子。
第四目的,本发明提供了一种分离细胞,包括上述的3a型丙肝病毒亚基因组复制子。
第五目的,本发明提供了一种3a型丙肝病毒的NS5A蛋白,包括在残基2218位上的谷氨酸、在残基2256位上的甘氨酸、在残基2271位上的天冬酰胺及在残基2350位上的脯氨酸。
第六目的,本发明提供了一种多核苷酸,编码上述的3a型丙肝病毒的NS5A蛋白。
第七目的,本发明提供了上述的3a型丙肝病毒亚基因组复制子在制备抗病毒药物上的应用。
与现有技术相比,本发明具有以下的有益效果:
本发明构建了3a型丙肝病毒亚基因组复制子,筛选得到了稳定表达CH3a非结构蛋白的Huh7.5.1-VISI-mCherry细胞克隆。用该复制子系统检测了CH3a毒株对Daclatasvir、Sofosbuvir的效应浓度,CH3a毒株对Daclatasvir的敏感性比SGR-JFH1(2a)和SGR-CH6a(6a)低~100倍左右,实验说明了本发明的3a型丙肝病毒亚基因组复制子是良好的药物筛选和检测系统。
附图说明
图1为第一段PCR(CH3a-PCR1)氨基酸序列的差异分析图I;
图2为第一段PCR(CH3a-PCR1)氨基酸序列的差异分析图II;
图3为第二段PCR(CH3a-PCR2)氨基酸序列的差异分析图;
图4为CH3a各TA克隆片段序列的拼接方法示意图;
图5为CH3a_C-NS2嵌合克隆基因组结构、CH3a_5-5A基因组结构和氨基酸突变位点示意图以及CH3a_C-NS2嵌合病毒、CH3a_5-5A嵌合病毒的传播速度和滴度的结果图(图5B和图5C的左Y轴表示细胞感染的百分比;右Y轴显示感染率高于80%时,检测上清HCV感染滴度FFU,平均值±SD);
图6为3a型丙肝病毒亚基因组复制子基因组模型和突变位点示意图;
图7为NS5A蛋白抗体检测筛选细胞的亚基因组复制子CH3a-SGRep的表达水平图;
图8为结晶紫染色G418筛选3周的稳定表达3a型丙肝病毒亚基因组复制子的细胞染色图;
图9为3a型丙肝病毒亚基因组复制子细胞内HCV蛋白和RNA的表达水平图;
图10为亚基因组复制子CH3a-SGRep(SGR-CH3a)对DAAs浓度的效应结果图。
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。
本实验选用的HCV GT3a感染者血清用COBAS Ampiprep/COBAS TaqMan滴度3.93×106IU/mL,毒株命名为CH 3a。
实施例1
1.1CH 3a毒株基因组的扩增:Trizol和氯仿法提取病毒RNA,采用常用技术手段扩增CH 3a毒株3’UTR、NS5B段;为了来自一整条病毒RNA的序列,本发明运用巢式引物对CH 3a毒株进行两轮PCR,各段PCR产物加A尾后连接pGEM-T easy载体(TA克隆),转化TOP10感受态细胞,蓝白斑筛选,每个片段选取10个单克隆菌落扩大摇菌,抽提质粒,设计测序引物,Sanger法测序每个反应600~800个碱基,精确测得每个核苷酸序列,比对分析。
将克隆测序的核苷酸序列翻译成氨基酸序列,以S52毒株为参照比对差异。本发明将测序克隆的每个位点上出现频次最高的核苷酸定义为共有序列。
第一段PCR(CH3a-PCR1)氨基酸序列的差异分析(只列出差异氨基酸),参考图1和图2。
第一段选取了灰度标记最少的克隆18号克隆(P1-18),进行下一步实验;第二段PCR克隆选取了7627-22(P2-22)和7990-3-4,二者拼接去掉了7990-3-4少见1460位点的N氨基酸;NS5B段因为实验室其他同学课题的需要根据PCR测序结果合成了共有序列,本发明目前阶段采用了该序列;3’UTR扩增选取了序列polyU/UC数目适中,且跟NS5B序列一致性好VR-2克隆序列,比对分析数据此处不做展示;PCR未及部分5’UTR的200bp使用了S52毒株的序列。
第二段PCR(CH3a-PCR2)氨基酸序列的差异分析(只列出差异氨基酸),参考图3。
1.2CH 3a毒株基因组的拼接:选取P1-18、P2-22/7990-3-4、NS5B(FSJ)和VR-2克隆,用Not I-Afl II-BsiW I和BsrGⅠ-Xba I 5个酶,分三大段拼接全场基因组。
1)融合CH3a_5’UTR-AflⅡ的基因片段,NotⅠ和AflⅡ酶切该片段后插入到pS52_5’UTR-NS5A/JFH1;
2)设计点突变去掉7990-3-4克隆内的XbaⅠ突变;
3)限制性内切酶AflⅡ和BsiWⅠ酶切7627-22,拼接到7990-3-4,去掉位点N1460;
4)限制性内切酶BsiW I和BbvCⅠ酶切pJ6_5’UTR-NS2/JFH1_NS3-NS5A/CH3a_NS5B/JFH1-3’UTR作为载体,BsiW I和BsrG I切去除Xba I和N1460位点的7990-3-4,(BsrG I和BbvCⅠ酶切后产生相同的DNA黏性末端)连接两个片段;
5)AflⅡ和Xba I酶切上一部克隆插入第1步的质粒,获得除3’UTR为JFH1的CH3a克隆;
6)选取CH3a-3’UTR的克隆VR-2,用Hpa I和Xba I换JFH1-3’UTR为CH3a序列,获得全基因组为3a的质粒克隆CH3a_FL。CH 3a各TA克隆片段序列的拼接方法示意图如图4所示。
2、CH 3a毒株感染性克隆的构建
2.1CH3a_Core-NS2病毒的培养
参考图5,本发明构建了CH3a_Core-NS3、CH3a_Core-NS2与J6/JFH1_NS5AΔ40-EGFP的嵌合克隆(参考图5A)。前者转染Huh7.5.1后检测到了低水平复制但没有获得强感染性病毒;后者转染获得了传播较快的病毒,病毒转染在第23天达到高峰,滴度为103.8FFU/mL(参考图5B)。
将CH3a_Core-NS2(C-NS2)转染后高峰期病毒感染新Huh7.5.1细胞,然后细胞传代驯化病毒。病毒感染第5代时传播明显变快,取上清RT-PCR扩增病毒的Core-NS2区域,测序鉴定获得了5个适应性的氨基酸突变K10Q(在核心蛋白上的第10位的赖氨酸突变为谷氨酰胺)、M375T(在E1蛋白上的第375位的蛋氨酸突变为苏氨酸)、M715V(在E2蛋白上的第715位的蛋氨酸突变为缬氨酸)、D877G(在NS2蛋白上第877位的天冬氨酸突变为甘氨酸)、S933P(在NS2蛋白上第933位的丝氨酸突变为脯氨酸),将CH3a_C-NS2驯化5代获得的完全氨基酸突变M375T、D877G、S933P和K10Q、M375T、M715V、D877G、S933P分别引入基因组CH3a_C-NS2后得到CH3a_C-NS2_3m和CH3a_C-NS2_5m克隆(参考图5A),转染Huh7.5.1细胞后传代培养。引入适应性突变的基因组病毒传播变快,均在第9天达到感染高峰,上清滴度也分别达到104.3FFU/mL和104.4FFU/mL(参考图5B)。表明培养获得的适应性突变对提高病毒传播速度和滴度有重要作用。
2.2CH3a_5’UTR-NS5A病毒的培养
本发明选用了S52毒株(S52_5-5A病毒)的适应性突变D877G(在NS2蛋白上第877位的天冬氨酸突变为甘氨酸)、P1118L(在NS3蛋白上第1118位的脯氨酸突变为亮氨酸)、F1470L(在NS3蛋白上第1470位的苯丙氨酸突变为亮氨酸)、V1618E(在NS3蛋白上第1618位的缬氨酸突变为谷氨酸)、A1678S(在NS4A蛋白上第1678位的丙氨酸突变为丝氨酸)、H1825R(在NS4B蛋白上第1825位的组氨酸突变为精氨酸)引入了CH3a_5-5A构建了CH3a_5-5A_6m,转染后检测到CH3a_5-5A_6m有持续性的复制感染(参考图5A),并在细胞传代培养第39天时达到感染高峰,滴度为103.3FFU/mL(参考图5C)。本申请人取CH3a_5-5A_6m病毒高峰期的细胞上清感染新的Huh7.5.1和Huh7.5.1-VISI-mCherry细胞,经过细胞长期传代培养驯化病毒,可获得适应性突变增加病毒的复制效率和滴度。
2.3高效CH3a_5-5A病毒的培养
在CH3a_5-5A_6m的基础上引入了CH3a_C-NS2病毒传代培养获得的适应性突变M375T(在E1蛋白上的第375位的蛋氨酸突变为苏氨酸)、M715V(在E2蛋白上的第715位的蛋氨酸突变为缬氨酸)、S933P(在NS2蛋白上第933位的丝氨酸突变为脯氨酸)构建了CH3a_5-5A_9m克隆(参考图5A)。两个克隆一起转染Huh7.5.1-VISI-mCherry细胞,CH3a_5-5A_9m传播比CH3a_5-5A_6m增快,第19天可以达到高峰,上清滴度增高至103.6FFU/mL(参考图5C)。为了获得复制效率更高的病毒,申请人在CH3a_5-5A_9m基础上继续引入CH3a_5-5A_6m感染传代获得的的Core-NS2基因区段的适应性突变E72G(在核心蛋白上的第72位的谷氨酸突变为甘氨酸)、F372L(在E1蛋白上的第372位的苯丙氨酸突变为亮氨酸)、I374V(在E1蛋白上的第374位的异亮氨酸突变为缬氨酸)、N533S(在E2蛋白上的第533位的天冬酰胺突变为丝氨酸)、N946S(在NS2蛋白上第946位的天冬酰胺突变为丝氨酸)和来自CH3a_C-NS2的适应性突变K10Q(在核心蛋白上的第10位的赖氨酸突变为谷氨酰胺)构建新克隆CH3a_5-5A_15m(参考图5A)。该克隆转染Huh7.5.1-VISI-mCherry细胞后第5天即可以达到感染高峰,上清滴度达到105.1FFU/mL。
3、CH3a复制子的筛选
在本毒株CH3a_5-5A_15m嵌合病毒实现高效细胞感染培养后,申请人又在复制子基因组上引入了CH3a_5-5A_6m独立感染1的3个适应性突变,分别为K2218E(在NS5A蛋白上的第2218位的赖氨酸突变为谷氨酸)、V2256G(在NS5A蛋白上的第2256位的缬氨酸突变为甘氨酸)和L2350P(在NS5A蛋白上的第2350位的亮氨酸突变为脯氨酸),CH3a_5-5A_9m的1个突变D2271N(在NS5A蛋白上的第2271位的天冬氨酸突变为天冬酰胺),文献报道的DBN3acc的6个突变V1775A(在NS4B蛋白上的第1775位的缬氨酸突变为丙氨酸)、L1840M(在NS4B蛋白上的第1840位的亮氨酸突变为蛋氨酸)、I1848V(在NS4B蛋白上的第1848位的异亮氨酸突变为缬氨酸)、S2426R(在NS5A蛋白上的第2426位的丝氨酸突变为精氨酸)、A2880D(在NS5B蛋白上的第2880位的丙氨酸突变为天冬氨酸)、C3005Y(在NS5B蛋白上的第3005位的半胱氨酸突变为酪氨酸),构建了CH3a毒株全长基因组对应的带有14个氨基酸突变F1470L、V1618E、A1678S、V1775A、H1825R、L1840M、I1848V、K2218E、V2256G、D2271N、L2350P、S2426R、A2880D、C3005Y的亚基因组复制子CH3a-SGRep克隆(序列如SEQ ID NO:1)。在亚基因组复制子CH3a-SGRep基础上引入L3004P(在NS5B蛋白上的第3004位的亮氨酸突变为脯氨酸)适应性突变构建成亚基因组复制子CH3a-SGRep+L3004P,在亚基因组复制子CH3a-SGRep基础上引入3’非翻译区的Δ11nt等突变构建成亚基因组复制子CH3a-SGRep+Δ11nt,在亚基因组复制子CH3a-SGRep+L3004P基础上引入3’非翻译区的Δ11nt等突变构建成CH3a-SGRep+L3004P/Δ11nt,在亚基因组复制子CH3a-SGRep基础上引入L3004M(在NS5B蛋白上的第3004位的亮氨酸突变为蛋氨酸)适应性突变构建成亚基因组复制子CH3a-SGRep+L3004M,上述复制子模型和突变位点的示意图如图6所示。上述复制子分别转染后G418筛选3周,经过NS5A抗体免疫荧光鉴定和亚基因组扩增测序鉴定,确定得到了稳定表达3a型丙肝病毒亚基因组复制子的Huh7.5.1-VISI-mCherry细胞克隆。
实施例2
验证亚基因组复制子CH3a-SGRep(即SGR-CH3a)对DAAs浓度的效应:
实验: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和Sofosbuvir处理,72h后用NS5A蛋白的抗体做免疫荧光检测复制子的表达水平(±SEM)。SGR-JFH1亚基因组复制子为对照组,SGR-CH6a为6a型HCV毒株的亚基因组复制子,6a型HCV毒株为中国专利CN110129341A中的CH6a毒株。
参考图7为NS5A蛋白抗体检测筛选细胞的亚基因组复制子CH3a-SGRep的表达水平图。G418筛选的有Neo抗性细胞用NS5A蛋白抗体做免疫荧光,Hoechst定位细胞核。
参考图8为G418筛选3a型丙肝病毒亚基因组复制子3周后,表达复制子细胞的结晶紫染色图。
参考图9为3a型丙肝病毒亚基因组复制子细胞内HCV蛋白和RNA的表达水平图。
本发明亚基因组复制子CH3a-SGRep对Daclatasvir、Sofosbuvir的EC50(nM,95%CI)分别是2.74(2.26-3.32)和263.1(216.4-319.7)(参考图10,对照组为SGR-JFH1)。亚基因组复制子CH3a-SGRep对Daclatasvir的EC50是其他毒株的~100倍左右,与该型对一些DAAs有特殊的耐受性。实验说明亚基因组复制子CH3a-SGRep是良好的药物筛选和检测系统。
采用本发明的复制子RNA复制非常高效,其中的突变位点可以用于提高3a型全长感染性克隆的复制和病毒传播速度与滴度。因此,本发明的3a型复制子可以为HCV耐药位点分析、适应性突变功能、HCV致病机制及疫苗研发等领域的研究提供重要研究工具。
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3a型丙肝病毒亚基因组复制子及应用
<130> 2021.09.29
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 8020
<212> DNA
<213> 人工合成
<400> 1
gcctgcctct tacgaggcga cactccacca tggatcactc ccctgtgagg aacttctgtc 60
ttcacgcgga aagcgcctag ccatggcgtt agtacgagtg tcgtgcagcc tccaggaccc 120
cccctcccgg gagagccata gtggtctgcg gaaccggtga gtacaccgga atcgctgggg 180
tgaccgggtc ctttcttgga gcaacccgct caatacccag aaatttgggc gtgcccccgc 240
gagatcacta gccgagtagt gttgggtcgc gaaaggcctt gtggtactgc ctgatagggt 300
gcttgcgagt gccccgggag gtctcgtaga ccgtgcaaca tgagcacact tcctaaaccc 360
caaagaaaaa ccaaaagaaa caccatcggc gcgccaatga ttgaacaaga tggattgcac 420
gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca 480
atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt 540
gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg 600
tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga 660
agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct 720
cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg 780
gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg 840
gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc 900
gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat 960
ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac 1020
tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt 1080
gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct 1140
cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agtttaaaca 1200
gaccacaacg gtttccctct agcgggatca attccgcccc tctccctccc ccccccctaa 1260
cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc 1320
caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac 1380
gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt 1440
gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg 1500
caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata 1560
agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga 1620
aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt 1680
accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc 1740
gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac 1800
acgataatac catggctccg atcacagcat acacccagca aactaggggc cttcttggga 1860
ctatcgttac cagcttgact ggcagggata agaatgtggt gaccggtgaa gtgcaggtac 1920
tttctacagc cacccagacc ttcctaggta caacagtggg gggggttatg tggactgttt 1980
accatggtgc aggttcgaga acactcgcgg gcgctaaaca tccagcgctc cagatgtaca 2040
caaatgtgga tcaggacctc gtcgggtggc cagcccctcc aggggccaag tctcttgaac 2100
cgtgcgcctg cggatccgca gacttatact tggttacccg cgaagctgat gtcatccctg 2160
cacggcgcag gggggactcc acagcgagct tgctcagtcc tagacctctc gcctgcctta 2220
agggttcctc tggaggtcct gttatgtgcc cttcggggca tgtcgcgggg atctttagag 2280
ctgctgtgtg caccagaggt gtagcaaaag ctttacagtt cataccagtg gaaaccctta 2340
gcacacaggc taggtctcca tctttctctg acaattcaac tcctcccgct gttccacaga 2400
gctaccaagt ggggtacctt catgccccga ccggcagcgg taagagcaca aaggtcccgg 2460
ccgcttatgt agcacaagga tataacgttc tcgtgctgaa tccatcagtg gcggccacac 2520
tgggcttcgg ctctttcatg tcacgtgctt atgggatcga ccctaacatc cgcactggga 2580
accgcaccat tacaactggc gccaagctga cctattccac ctatggtaag ttccttgcgg 2640
acgggggttg ctccggagga gcatatgatg tgattatttg tgatgaatgc catgcccaag 2700
acgctactag catattgggt ataggcacgg tcctagatca ggctgagaca gccggggtga 2760
ggctgacggt tctagcgaca gcaactcccc caggcagcat cactgtgcca cattctaaca 2820
tcgaagaagt ggccctgggc tccgaaggtg agatcccttt ctacggcaag gctataccga 2880
tagacctgct caaggggggg aggcacctta tgttttgcca ttccaagaaa aagtgtgatg 2940
agttagcatc caaactcaga ggcatggggc tcaacgctgt agcgtactat aggggtctcg 3000
atgtgtccgt cataccaaca acaggagacg tcgtagtttg cgctactgac gctctcatga 3060
ctggattcac cggagacttc gattctgtca tagactgcaa cgtggctgtt gagcagtacg 3120
ttgacttcag tttggaccct actttctcca tcgagactcg cactgctccc caagacgcag 3180
tctcccgcag ccaacgtcgt ggccgtacgg gccgaggtag actcggtacg taccggtatg 3240
ttacccccgg tgaaagaccg tctggaatgt ttgactcggt tgttctctgt gagtgttatg 3300
acgcgggctg ttcgtggtac gatctgcagc ccgccgagac cacagtcaga ctgagagctt 3360
acttgtccac gccggggtta cctgtctgcc aagaccattt agacttttgg gagagcgtct 3420
tcactggact aactcacata gatgcccact ttttgtcaca gactaagcag cagggactca 3480
atttctcgta cctgactgcc taccaagcca cagtgtgcgc ccgcgcgcag gctccccccc 3540
caagttggga cgagacgtgg aagtgcctcg tgcggcttaa accaacacta catggaccca 3600
cgccccttct atatcggttg gggcctgtcc aaaatgaagt ctgcttgaca caccccgtca 3660
caaagtacat catggcatgc atgtcagctg atttggaagt aaccaccagt acctgggtgt 3720
tgcttggagg ggtcctcgcg gccctagcgg cctactgctt gtcggtcggc tgcgttgtga 3780
ttgtgggtca tatcgagctg gggggcaagc cggcactcgt gccagacaga gaggtgttgt 3840
atcaacaata cgatgagatg gaggagtgct cacgagctgc cccatacgtc gaacaagctc 3900
aggtaatagc ccaccagttc aaagaaaagg tccttggatt gctgcagcga gccacccaac 3960
aacaagctgt cattgagcct atagtagcta ccaactggca aaaacttgag gccttctggc 4020
acaagtacat gtggaatttt gtgagtggga tccagtacct agcaggcctc tccactttgc 4080
ccggcaaccc tgctgtggcg tctcttatgg cgttcactgc ttcagtcacc agtcccctga 4140
cgaccaacca aactattttt tttaacatac tcggggggtg ggttgcaacc catttggcag 4200
ggccccagag ctcttccgca tttgtggtaa gcggcttagc tggcgctgcc atagggggca 4260
taggcctggg caaggtcttg cttgacatcc tggcaggata cggggctggc gtctcaggcg 4320
ccctggtagc ttttaagatc atgggaggag aactccccac tactgaggac atggtcaacc 4380
tgttacccgc catactatct ccaggcgccc tcgtcgttgg tgtaatatgc gctgccatac 4440
tgcgtcgcca cgtaggacct ggggagggag cggttcagtg gatgaacagg cttattgcat 4500
tcgcatcccg gggtaaccac gtctcaccaa cacactatgt ccccgagagc gatgctgcag 4560
cgagggtcac cgcgttgctg agttctctaa ctatcacaag cctgctccgg cggttgcacc 4620
agtggatcaa tgaggactac ccaagtccct gcagcggcga ttggctgcgt gacatctggg 4680
actgggtttg cacagtgttg tccgacttca aaacatggct ctctgctaag attataccag 4740
cgctccctgg actgcccttc atttcatgtc agaggggata caagggcgtg tggcggggag 4800
acggtgtgat gtcgacacgc tgtccttgcg ggtcatcaat aactggccat gtgaagaatg 4860
ggtccatgcg gcttgcaggg ccgcgtacat gtgctaacat gtggtacggt acctttccca 4920
tcaatgagta caccaccgga cccagcacac cttgcccatc acccaactac actcgcgcac 4980
tgtggcgcgt ggctgccaac agctacgttg aggtgcgccg ggtgggagac ttccattaca 5040
ttacaggggc cacagaagat gaactcaagt gtccgtgcca agtgccggct gccgagtttt 5100
tcactgaagt ggatggggtg agactccacc gttacgcccc tccatgtaag cccttgttga 5160
gagatgacat cactttcatg gtggggttga attcctacgt gataggatct caactcccct 5220
gtgagcctga accagatgtt tctgtgctga cctcgatgct aagagaccct tcccatatca 5280
ccgccgagac ggcggcgcgc cgtctcgcgc gcgggtctcc tccatcggag gcaagctcat 5340
ccgccagcca actatcggct ccgtcgttga aagccacctg ccagacgcat aggcctcatc 5400
cagacgctga gctagtagac gccaacttgt tatggcggca agagatgggc agcaacatca 5460
cgcgggtaga gtctgagacg aaggttgtga ttcttgattc attcgaacct ctgagagccg 5520
aacctgatga cggcgagctc tcggtggctg cagagtgttt caagaaacct cccaagtacc 5580
ctccggctct tcctatatgg gctaggccag attacaaccc tccactgtta gaccgctgga 5640
aagcaccgga ttatgaacca ccaactgtcc atgggtgcgc cttaccacca cgaggcgctc 5700
caccggtgcc tcctcctcgg aggaaaagaa caatccagct ggatggctcc aacgtgtccg 5760
cggcgctagc cgcgctagcg gaaaaatcat ttccatcctc gaaaccacag gaggagaata 5820
gctcgtcctc tggggtcgac acacagtcca gcactacttc caaggcgctc ccttctccgg 5880
gaggggagtc tgactcagag tcatgttcgt ccatgcctcc tcttgaggga gagccgggcg 5940
atccagactt gagttgcgac tcttggtcca ccgttagcga cagcgaggag cagagtgtgg 6000
tctgctgctc tatgtcgtac tcttggaccg gcgccctgat aacaccatgt agtgctgagg 6060
aggagaaact gcctatcagc ccactcagca actccttgtt gagacatcat aacatggtct 6120
attcaacgtc gtcaagaagc gcttctcagc gccagaagaa ggttaccttc gataggctgc 6180
aagtgctcga cgaccattac agggttgtat taaaggaggt aaaggagcga gcgtccaagg 6240
tgaaggctcg catgcttacc atcgaggaag cgtgcgcgct cgtccctcct cactctgccc 6300
gatcgaagtt cgggtatagt gcgaaggacg ttcgctcctt gtccagcaag gccattaacc 6360
agatccgctc cgtctgggag gacttgctgg aagacaccac aactccaatt ccaactacca 6420
taatggcgaa gaacgaggta ttttgtgtgg accctgtcaa agggggccgc aaacccgctc 6480
gcctcattgt gtaccctgac ctgggggtgc gtgtctgtga gaaacgcgcc ctatatgacg 6540
tgatacagaa gttgtcaatt gagacgatgg gttccgccta tggattccaa tattcgcctc 6600
aacagcgggt cgaacgtcta ctgaagatgt gggcctcaaa gaaaacccct ctggggttct 6660
cgtatgacac ccgctgcttt gactcaactg tcactgaaca ggacatcagg gtggaagagg 6720
agatatacca atgctgtgat cttgaaccgg aggccaggaa agtgatctcc tccctcacgg 6780
agcggcttta ctgcgggggt cctatgttca acagcaaggg gacccagtgt ggttatcgcc 6840
gttgccgtgc cagtggagtt ctgcccacca gcttcggcaa tacgatcact tgttacatca 6900
aggccacagc ggctgcaaag gccgcaggcc tccaaaaccc ggactttctt gtttgcgggg 6960
acgacctggt cgtggtggct gagagtgttg gcgtcgaaga ggatagagca gccctgagag 7020
ctttcacgga ggctatgacc aggtattctg ctccacctgg ggatgctccg cagcccacct 7080
acgaccttga gctcattaca tcttgctcct ccaatgtctc cgtggcacgg gacgaaaagg 7140
ggaagaggta ttattacctc acccgtgatg ccaccactcc cctaagccgt gcggcttggg 7200
agacagctcg tcacactcca gttaactcct ggctgggtaa tatcatcatg tacgcgccta 7260
ccatctgggt gcgcatggta atgatgacac acttcttctc catactccaa tcccaggaga 7320
tacttgatcg gcccctcgac tttgaaatgt acggggccac ttactctgtc actccgctgg 7380
atttaccagc aatcattgaa agactccatg gtctgagcgc gttcacgctc cacagttact 7440
ctccagtaga actcaatagg gtcgcgggga cactcaggaa acttgggtgc ccccccctac 7500
gagcgtggag acatcgggca cgagcagtgc gcgccaagct tatcgcccag ggggggaagg 7560
ccaaaatatg tggtctttac ctctttaatt gggcggtacg caccaagacc aaactcactc 7620
cactaccggc cgctggccag ctggacttat ccagctggtt tacggttggt gtcggcggga 7680
acgacattta tcacagcgtg tcacgtgccc gaacccgcca tttgctgctt tgcctactcc 7740
tactagcggt aggggtaggc atctttctcc tgccagcacg gtagagcggc acacactagg 7800
tacactccat agctaactgt tccttttttt tttttttttt tttttttttt tttttttttt 7860
ttttttcttt tttttttttt tccctctttc ttcccttctc atcttattct actttctttc 7920
ttggtggctc catcttagcc ctagtcacgg ctagctgtga aaggtccgtg agccgcatga 7980
ctgcagagag tgccgtaact ggtctctctg cagatcatgt 8020
Claims (3)
1.一种3a型丙肝病毒亚基因组复制子,其特征在于,所述复制子的序列如SEQ ID NO:1所示。
2.一种分离细胞,其特征在于,包括如权利要求1所述的3a型丙肝病毒亚基因组复制子。
3.如权利要求1所述的3a型丙肝病毒亚基因组复制子在制备抗病毒药物中的应用。
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