CN108611395A - 一种抗人乙肝病毒药物的筛选方法 - Google Patents

一种抗人乙肝病毒药物的筛选方法 Download PDF

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CN108611395A
CN108611395A CN201611138490.7A CN201611138490A CN108611395A CN 108611395 A CN108611395 A CN 108611395A CN 201611138490 A CN201611138490 A CN 201611138490A CN 108611395 A CN108611395 A CN 108611395A
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张辉
何欣
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Sun Yat Sen University
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Abstract

本发明公开了一种抗人乙肝病毒药物的筛选方法。具体为将SEQ ID NO：1所示的HBc分别与荧光蛋白的肽段a和肽段b相连，构建得融合蛋白A和B，所述肽段a和肽段b连接共同组成荧光蛋白的全序列；将融合蛋白A和B通过克隆技术共转化同一受体细胞，用待检测药物处理上述转化的受体细胞，同时以未用药物处理的上述转化的受体细胞作为阴性对照；观察细胞中荧光蛋白的信号，如果无荧光蛋白信号或荧光信号低于阴性对照，说明待检测药物可以抗人乙肝病毒，如果荧光信号与阴性对照信号强度相当，说明待检测药物不可以抗人乙肝病毒。本研究通过设计并构建HBc与荧光蛋白的融合蛋白，利用荧光蛋白信号监测HBc组装的过程，简化药物筛选过程中的读出操作，提供了一种用于HBV抗病毒药物的高通量筛选方法。

Description

一种抗人乙肝病毒药物的筛选方法
技术领域
本发明涉及抗病毒药物筛选技术领域，具体地，涉及一种抗人乙肝病毒药物的筛选方法。
背景技术
人乙型肝炎病毒感染是世界范围内的重要公共健康问题。急性乙肝病毒感染后，仍有8％左右发展为慢性乙肝感染，持续性HBV感染将导致肝硬化，甚至肝癌。我国是乙肝大国，乙肝病毒携带者接近1.3亿人，约占总人口的9％。尽管随着乙肝疫苗的大范围普及，新乙肝感染率得到有效控制，但乙肝携带人口基数大，防治乙肝成为我国公共健康问题的重中之重。乙肝传播途径主要通过垂直传播与水平传播。垂直传播是指母婴传播；水平传播主要通过血液传播。
尽管目前市场上有很多抗HBV药物，主要通过使用干扰素或者核苷类似物进行抗病毒治疗。其中核苷类似物通过抑制HBV复制过程中的逆转录酶活性从而抑制HBV产生。逆转录酶抑制剂虽然可以使患者控制乙肝病毒水平，但是随之产生的耐药性、巨额的医药费用、药物严重的副作用等问题也不容小觑。由于HBV逆转录酶体外表达并不产生酶活性，HBV逆转录酶抑制剂的母环结构主要通过对人免疫缺陷病毒(HIV)的逆转录酶化合物改造修饰。针对HBV本身进行从头抗病毒药物研究成为一种迫切需求。想要获得从头抗病毒药物，必须先建立一种合适的药物筛选方法。
发明内容
本发明的目的是为了克服现有技术的上述不足，提供一种抗人乙肝病毒药物的筛选方法。该方法筛选的药物能抑制HBV核心蛋白(HBc)的组装，可以从根本上达到抑制乙肝病毒的作用。
为了实现上述目的，本发明是通过以下技术方案予以实现的：
一种抗人乙肝病毒药物的筛选方法，包括如下步骤：将SEQ ID NO：1所示的HBc分别与荧光蛋白的肽段a和肽段b相连，构建得融合蛋白A和B，所述肽段a和肽段b连接共同组成荧光蛋白的全序列；将融合蛋白A和B通过克隆技术共转化同一受体细胞，用待检测药物处理上述转化的受体细胞，同时以未用药物处理的上述转化的受体细胞作为阴性对照；观察细胞中荧光蛋白的信号，如果无荧光蛋白信号或荧光信号低于阴性对照，说明待检测药物可以抗人乙肝病毒，如果荧光信号与阴性对照信号强度相当，说明待检测药物不可以抗人乙肝病毒。
在HBV病毒生活周期中，病毒包膜蛋白与肝脏细胞膜受体结合，病毒包膜与宿主细胞膜融合，病毒核心颗粒进入胞内并介导病毒基因组进入胞核，通过利用人肝脏细胞内聚合酶将dsDNA基因组补全并转化为cccDNA，cccDNA进一步转录出3.5kb，2.4kb，2.1kb及0.8kb的mRNA，以前基因组RNA(pgRNA)3.5kb为模板逆转录出基因组DNA并作为编码病毒核心蛋白(HBc)和聚合酶蛋白的模板；HBc多聚化包装乙肝DNA基因组形成包装核心颗粒；乙肝表面抗原HBsAg合成后在粗面内质网中多聚化，并转运至高尔基体前腔以包装核心颗粒，装配好的HBV颗粒与亚病毒颗粒转运至高尔基体进行HBsAg的糖基化修饰，最后分泌出宿主细胞而完成生活周期。病毒核心(HBc)颗粒的组装是HBV复制产生过程不可缺少的一步，如果HBc的组装受到抑制，HBV的基因组将不能完成包装从而阻断HBV的复制(见图1)。如何抑制HBc的组装，将是研发抗HBV药物的一个重要靶点。
优选地，所述荧光蛋白为黄色荧光蛋白(yellow fluorescent protein，YFP)，蓝绿色荧光蛋白(cyan fluorescent protein，CFP)或Venus荧光蛋白(Venus fluorescentprotein,VFP)。
更优选地，所述肽段a的氨基酸序列如SEQ IDNO：2所示，肽段b的氨基酸序列如SEQIDNO：3所示；或，肽段a的氨基酸序列如SEQ ID NO：4所示，肽段b的氨基酸序列如SEQ IDNO：5所示；或，肽段a的氨基酸序列如SEQ ID NO：6所示，肽段b的氨基酸序列如SEQ ID NO：7所示；或，肽段a的氨基酸序列如SEQ ID NO：8所示，肽段b的氨基酸序列如SEQ ID NO：9所示；或，肽段a的氨基酸序列如SEQ ID NO：10所示，肽段b的氨基酸序列如SEQ ID NO：11所示；或，肽段a的氨基酸序列如SEQ ID NO：12所示，肽段b的氨基酸序列如SEQ ID NO：13所示。
SEQ ID NO：1所示的HBc分别与荧光蛋白的肽段a和肽段b相连时，使用linker序列连接，所述Linker序列为(GGSG)n；(GGSGG)n；(GSGSG)n；(GSGGG)n；(GGGGS)n；(GGGSG)n；(GSSSG)n；RPACKIPNDLKQKVMNH；RSIAT。
所述受体细胞为HEK293T细胞、HepG2或Huh7。
与现有技术相比，本发明具有如下有益效果：
本发明通过设计并构建HBc与荧光蛋白的融合蛋白，利用荧光蛋白信号监测HBc组装的过程，简化药物筛选过程中的读出操作，提供了一种用于HBV抗病毒药物的高通量筛选方法。
附图说明
图1为HBV病毒生活周期中核心蛋白的组装流程图。
图2为实施例1各处理的荧光信号检测结果。
图3为表1化合物的荧光信号检测结果。
具体实施方式
下面结合说明书附图和具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例1
一种抗人乙肝病毒药物的筛选方法，包括如下步骤：
(1)将SEQ ID NO：1所示的HBc通过Linker序列分别与VFP-N1、VFP-C1相连，得融合蛋白HBc-VFP-N1、HBc-VFP-C1，所述VFP-N1为VFP蛋白1-154位氨基酸组成的肽段，VFP-C1为VFP蛋白155-239位氨基酸组成的肽段。其中，Linker序列包括但不限于(GGSG)n；(GGSGG)n；(GSGSG)n；(GSGGG)n；(GGGGS)n；(GGGSG)n；(GSSSG)n；RPACKIPNDLKQKVMNH；RSIAT等。
(2)在6孔板的HEK293T细胞中，通过克隆技术共转染HBc-VFP-N1 0.5ug和HBc-VFP-C1 0.5ug，或共转染HBc-VFP-N1 0.5ug和VFP-C1 0.5ug，或共转染VFP-N1 0.5ug和HBc-VFP-C1 0.5ug，或共转染VFP-N1 0.5ng和VFP-C1 0.5ng。转染48h后观察VFP的表达(Ex488nm/Em 512nm)结果见图2，该实验说明了，只有当HBc-VFP-1N和HBc-VFP-1C共同存在时，才会产生强荧光信号。HBc的组装使得VFP-N1与VFP-C1互相接近产生完整的VFP蛋白结构，进而产生荧光信号。
(3)基于上述研究，在6孔板的HEK293T细胞中，通过克隆技术共转染HBc-VFP-N10.5ug和HBc-VFP-C1 0.5ug，然后用待检测药物处理上述转化的受体细胞，同时以未用药物处理的上述转化的受体细胞作为阴性对照；观察细胞中荧光蛋白的信号，如果无荧光蛋白信号或荧光信号低于阴性对照，说明待检测药物可以抗人乙肝病毒，如果荧光信号与阴性对照信号强度相当，说明待检测药物不可以抗人乙肝病毒。
实施例2
一种抗人乙肝病毒药物的筛选方法，基本步骤同实施例1，不同之处在于，将SEQID NO：1所示的HBc通过Linker序列分别与VFP-N2、VFP-C2相连，得融合蛋白HBc-VFP-N2、HBc-VFP-C2，所述VFP-N2为VFP蛋白1-172位氨基酸组成的肽段，VFP-C1为VFP蛋白173-239位氨基酸组成的肽段。
实施例3
一种抗人乙肝病毒药物的筛选方法，基本步骤同实施例1，不同之处在于，将SEQID NO：1所示的HBc通过Linker序列分别与YFP-N1、YFP-C1相连，得融合蛋白HBc-YFP-N1、HBc-YFP-C1，所述YFP-N1为YFP蛋白1-154位氨基酸组成的肽段，YFP-C1为YFP蛋白155-240位氨基酸组成的肽段。
实施例4
一种抗人乙肝病毒药物的筛选方法，基本步骤同实施例1，不同之处在于，将SEQID NO：1所示的HBc通过Linker序列分别与YFP-N2、YFP-C2相连，得融合蛋白HBc-YFP-N2、HBc-YFP-C2，所述YFP-N2为YFP蛋白1-172位氨基酸组成的肽段，YFP-C2为YFP蛋白173-240位氨基酸组成的肽段。
实施例5
一种抗人乙肝病毒药物的筛选方法，基本步骤同实施例1，不同之处在于，将SEQID NO：1所示的HBc通过Linker序列分别与CFP-N1、CFP-C1相连，得融合蛋白HBc-CFP-N1、HBc-CFP-C1，所述CFP-N1为CFP蛋白1-154位氨基酸组成的肽段，CFP-C1为CFP蛋白155-239位氨基酸组成的肽段。
实施例6
一种抗人乙肝病毒药物的筛选方法，基本步骤同实施例1，不同之处在于，将SEQID NO：1所示的HBc通过Linker序列分别与CFP-N2、CFP-C2相连，得融合蛋白HBc-CFP-N2、HBc-CFP-C2，所述CFP-N2为CFP蛋白1-172位氨基酸组成的肽段，CFP-C1为CFP蛋白173-239位氨基酸组成的肽段。
应用例1
使用本发明的方法检测表1中的化合物是否为抗乙肝病毒药物，结果见图3。
表1手性芳杂胺类衍生物的编号及结构
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<210> 7
<211> 85
<212> PRT
<213> CFP-C1 蛋白序列
<400> 7
Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His
1 5 10 15
Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn
20 25 30
Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu
35 40 45
Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His
50 55 60
Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met
65 70 75 80
Asp Glu Leu Tyr Lys
85
<210> 8
<211> 172
<212> PRT
<213> CFP-N2 蛋白序列
<400> 8
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile
165 170
<210> 9
<211> 67
<212> PRT
<213> CFP-C2 蛋白序列
<400> 9
Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro
1 5 10 15
Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr
20 25 30
Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val
35 40 45
Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu
50 55 60
Leu Tyr Lys
65
<210> 10
<211> 154
<212> PRT
<213> VFP-N1 蛋白序列
<400> 10
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Asn Ser His Asn Val Tyr Ile Thr
145 150
<210> 11
<211> 85
<212> PRT
<213> VFP-C1 蛋白序列
<400> 11
Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His
1 5 10 15
Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Asp His Tyr Gln Gln Asn
20 25 30
Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu
35 40 45
Ser Tyr Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His
50 55 60
Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met
65 70 75 80
Asp Glu Leu Tyr Lys
85
<210> 12
<211> 172
<212> PRT
<213> VFP-N2 蛋白序列
<400> 12
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile
165 170
<210> 13
<211> 67
<212> PRT
<213> VFP-C2 蛋白序列
<400> 13
Glu Asp Gly Gly Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro
1 5 10 15
Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr
20 25 30
Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val
35 40 45
Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu
50 55 60
Leu Tyr Lys
65

Claims (5)

1.一种抗人乙肝病毒药物的筛选方法，其特征在于，包括如下步骤：将SEQ ID NO：1所示的HBc分别与荧光蛋白的肽段a和肽段b相连，构建得融合蛋白A和B，所述肽段a和肽段b连接共同组成荧光蛋白的全序列；将融合蛋白A和B通过克隆技术共转化同一受体细胞，用待检测药物处理上述转化的受体细胞，同时以未用药物处理的上述转化的受体细胞作为阴性对照；观察细胞中荧光蛋白的信号，如果无荧光蛋白信号或荧光信号低于阴性对照，说明待检测药物可以抗人乙肝病毒，如果荧光信号与阴性对照信号强度相当，说明待检测药物不可以抗人乙肝病毒。
2.根据权利要求1所述的筛选方法，其特征在在于，所述荧光蛋白为黄色荧光蛋白、蓝绿色荧光蛋白或Venus荧光蛋白。
3.根据权利要求1所述的筛选方法，其特征在在于，所述肽段a的氨基酸序列如SEQ IDNO：2所示，肽段b的氨基酸序列如SEQ ID NO：3所示；或，肽段a的氨基酸序列如SEQ ID NO：4所示，肽段b的氨基酸序列如SEQ ID NO：5所示；或，肽段a的氨基酸序列如SEQ ID NO：6所示，肽段b的氨基酸序列如SEQ ID NO：7所示；或，肽段a的氨基酸序列如SEQ ID NO：8所示，肽段b的氨基酸序列如SEQ ID NO：9所示；或，肽段a的氨基酸序列如SEQ ID NO：10所示，肽段b的氨基酸序列如SEQ ID NO：11所示；或，肽段a的氨基酸序列如SEQ ID NO：12所示，肽段b的氨基酸序列如SEQ ID NO：13所示。
4.根据权利要求1所述的筛选方法，其特征在在于，SEQ ID NO：1所示的HBc分别与荧光蛋白的肽段a和肽段b相连时，使用linker序列连接，所述Linker序列为(GGSG)n；(GGSGG)n；(GSGSG)n；(GSGGG)n；(GGGGS)n；(GGGSG)n；(GSSSG)n；RPACKIPNDLKQKVMNH；RSIAT。
5.根据权利要求1所述的筛选方法，其特征在在于，所述受体细胞为HEK293T细胞、HepG2或Huh7。
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CN114751990B (zh) * 2022-04-01 2023-10-20 中国科学技术大学 酰胺高丝氨酸内酯受体-增强绿色荧光融合蛋白及其制备方法与应用
CN114875052A (zh) * 2022-06-27 2022-08-09 齐鲁工业大学 一种能够检测透明质酸的融合蛋白及检测方法
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