CN1249083C - 抑制sars冠状病毒侵染宿主细胞的多肽类药物 - Google Patents
抑制sars冠状病毒侵染宿主细胞的多肽类药物 Download PDFInfo
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- CN1249083C CN1249083C CN 03143037 CN03143037A CN1249083C CN 1249083 C CN1249083 C CN 1249083C CN 03143037 CN03143037 CN 03143037 CN 03143037 A CN03143037 A CN 03143037A CN 1249083 C CN1249083 C CN 1249083C
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Abstract
本发明涉及抑制SARS冠状病毒侵染宿主细胞的多肽类抗病毒药物，所述药物的通式为：X-SEQ-Z，其中SEQ表示SEQ ID NO.1～SEQ ID NO.21所示的21个氨基酸序列。经药效学实验测试，本发明的多肽类药物可抑制SARS冠状病毒侵染宿主细胞。
Description
技术领域
本发明涉及一种抗SARS冠状病毒药物，具体涉及抑制SARS冠状病毒侵染宿主细胞的多肽类抗病毒药物。
背景技术
SARS冠状病毒是引起2003年在我国和世界范围内爆发的“非典型肺炎”的病原体，目前还没有特效的药物能够治疗。
SARS病毒是一种带囊膜的冠状病毒，其侵染细胞的第一步就是病毒囊膜与宿主细胞的融合。多肽类融合阻断分子因其能够阻断病毒囊膜与宿主细胞的融合，在其他病毒药物的开发研制中有着成功的经验，如HIV囊膜蛋白GP41有两段HR结构域，分别形成三个N螺旋和三个C螺旋。在HIV侵入细胞的过程中，这三个N螺旋和三个C螺旋形成Coiled-coil六聚体结构起着至关重要的作用。根据这一原理设计的多肽类药物T-20(商品名Fuzeon)已经上市，它能够有效地阻断由GP41本身的α螺旋形成六聚体，从而阻断HIV膜结构和细胞膜的融合。研究表明，T-20的阻断能力强，对机体副作用低，是一种理想的药物。
SARS囊膜蛋白SPIKE的结构研究表明，在SPIKE蛋白中同样存在能够形成Coiled-coil结构的两段HR结构域。
发明内容：
本发明的目的是开发一类阻断SARS冠状病毒进入宿主细胞的抗病毒药物。
本发明的技术方案是寻找可起到SARS冠状病毒多肽类融合阻断作用的物质，通过该物质与SARS囊膜蛋白Spike的相互作用，阻止SARS囊膜蛋白Spike的构型改变，从而阻断病毒囊膜与宿主细胞的融合，达到预防和治疗SARS病毒感染的目的。
本发明提供的SARS冠状病毒多肽类融合阻断药物的具有如下通式：
X-SEQ-Z
其中，SEQ表示SARS冠状病毒囊膜蛋白SPIKE中的多肽，所述多肽包括如SEQ ID NO.1～SEQ ID NO.21所示的21个氨基酸序列。
在SEQ ID NO.1～SEQ ID NO.21所示的21个氨基酸序列的多肽中：
至少一个氨基酸残基的连接使用非肽键连接方式，例如，以亚氨基、酯、肼、氨基脲或含氮化学键的形式连接；
至少一个氨基酸残基以D构型存在；
至少有一个氨基酸被另一个不同的氨基酸置换，所述置换方式是保守的和/或不保守的；
所述多肽可以是SARS自然突变株相对应氨基酸位置的同源物；
所述多肽可以被包含在一段其它多肽中；
所述多肽可以以单体，二聚体，三聚体和多聚体的形式存在。
X包括氨基、乙酰基，及某些疏水基团和大分子载体基团；
Z包括羧基、氨基及某些疏水基团和大分子载体基团；
所述疏水基团包括9-芴基甲氧羰基(9-fluorenyl methoxy-carbonyl)、苄氧羰基(carbobenzoxyl)1-二甲胺基萘-5-磺酰(dansyl)和叔丁氧羰基(t-butyloxy carbonyl)；
所述大分子载体基团是共轭脂肪酸(Lipid-fatty acid conjuate)、聚乙二醇或碳水化合物类基团。
上述通式中，X和Z也可以不存在。即，如SEQ ID NO.1～SEQ ID NO.21所示的21个氨基酸序列也可用作阻断SARS冠状病毒进入宿主细胞的抗病毒药物。
本发明所述的多肽可以从真核细胞表达系统、原核细胞表达系统或固相化学合成的途径得到。
在采用原核表达系统的实验中，先用PCR的方法扩增SARS囊膜蛋白SPIKE的一段序列，将其克隆到表达载体上。诱导表达后，使用GlutathioneSepharose 4B系统纯化得到多肽样品。
化学合成可选用本技术领域人工合成多肽的常规操作方法。
经药效学实验测试，本发明的多肽类药物对于SARS冠状病毒活病毒感染其易感细胞VeroE6具有阻断能力，表明本发明的多肽类药物可抑制SARS冠状病毒侵染宿主细胞。
具体实施方式
本发明SEQ ID NO.1～SEQ ID NO.21所示的21个氨基酸序列所示的多肽中，SEQ ID NO.1～SEQ ID NO.15采用原核表达系统得到，SEQ ID NO.16～21为人工合成得到。
实施例1大肠杆菌表达多肽
1.SARS病毒样本的总RNA的提取
(1)用1ml trizol(GIBCO)抽提含有SARS病毒样本；
(2)加200μl氯仿，剧烈振荡15秒；
(3)室温静置3-5min，10000g 4℃离心15min；
(4)吸出500μl水相，转移到新管中；
(5)加入500μl异丙醇，充分振荡混匀；室温静置10min
(6)10000g 4℃离心10min，得到SARS病毒RNA沉淀。
2.SARS病毒SPIKE蛋白基因的克隆
(1)将SARS病毒RNA基因组反转录为DNA
反转录引物：SPIRE蛋白antisense primer
反转录酶：MMLV-RT(promega)
(2)通过PCR方法扩增出SPIKE的DNA序列。
扩增引物：
Sense：5’-cgggatccaacgaacatgtttattttcttattatttc-3’
antisense：5’-cggaattcgtttatgtgtaatgtaatttgacaccc-3’
(3)将PCR产物连接到pcDNA3.1+载体中
连接方式：BamHI，EcoRI双切连接。
3.克隆得到下列SEQ ID NO.1～SEQ ID NO.15的多肽的序列及其PCR引物如下：
SEQ ID NO.1：NGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTA
AATGGCATTGGAGTTACC
TGC AGT TGA TGT TGT TGT AAG
SEQ ID NO.2：PDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQ
CCAGATGTTGATCTTGGC
TTGAAGGTCAATGAGTGATTC
SEQ ID NO.3：GDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQ
GGCGACATTTCAGGCATTAAC
TTGAAGGTCAATGAGTGATTC
SEQ ID NO.4：ISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPW
A TTT CAG GCA TTA ACG CTT C
CCAAGGCCATTTAATATATTGC
SEQ ID NO.5：INASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPW
ATTAACGCTTCTGTCGTCAAC
CCAAGGCCATTTAATATATTGC
SEQ ID NO.6：VVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPW
G TCG TCA ACA TTC AAA AAG
CCAAGGCCATTTAATATATTGC
SEQ ID NO.7：IQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPW
A TTC AAA AAG AAA TTG ACC GC
CCAAGGCCATTTAATATATTGC
SEQ ID NO.8：IDRLNEVAKNLNESLIDLQELGKYEQYIKWPW
A TTG ACC GCC TCA ATG AGG TC
CCAAGGCCATTTAATATATTGC
SEQ ID NO.9：ISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGK
A TTT CAG GCA TTA ACG CTT C
TTTTCCCAATTCTTGAAG
SEQ ID NO.10：INASVVNIQKEIDRLNEVAKNLNESLIDLQELGK
ATTAACGCTTCTGTCGTCAAC
TTTTCCCAATTCTTGAAG
SEQ ID NO.11：VVNIQKEIDRLNEVAKNLNESLIDLQELGK
G TCG TCA ACA TTC AAA AAG
TTTTCCCAATTCTTGAAG
SEQ ID NO.12：IQKEIDRLNEVAKNLNESLIDLQELGK
A TTC AAA AAG AAA TTG ACC GC
TTTTCCCAATTCTTGAAG
SEQ ID NO.13：ISGINASVVNIQKEIDRLNEVAKNLNESLIDL
A TTT CAG GCA TTA ACG CTT C
AAG GTC AAT GAG TGA TTC A
SEQ ID NO.14：INASVVNIQKEIDRLNEVAKNLNESLIDL
ATTAACGCTTCTGTCGTCAAC
AAG GTC AAT GAG TGA TTC A
SEQ ID NO.15：VVNIQKEIDRLNEVAKNLNESLIDL
G TCG TCA ACA TTC AAA AAG
AAG GTC AAT GAG TGA TTC A
4，多肽的表达
(1)将PCR产物回收纯化后连接到pGEX-6p-2载体(Amersham Pharmacia Biotech.27-4598-01)的smaI切点。
(2)将正确构建的重组质粒转入到JM109(DE3)感受态细胞中，用含抗生素的LB培养基过夜培养。
(3)过夜培养物1∶50接400ml新鲜培养基，2小时后加80ml 1MIPTG诱导，继续培养4小时。
(4)4℃，8000rpm 6min离心培养的细菌，加入15ml Lysis Buffer重悬沉淀，加入溶菌酶至终浓度为100mg/ml，置冰上30min，冰上超声12次(300W，10s/10s)
(5)4℃，12000rpm离心15min，取上清以5-10mgGST/ml的比例加入Glutathione Sepharose 4B(Amersham Pharmacia Biotech.17-0756-01)，在冰上结合1h，使样品液过层析柱(Bio-Rad 74306)使用5倍体积的Hepes500洗涤GST柱子，之后使用10倍体积的Hepes100洗涤，再用0.5ml含10mMGSH的Hepes100洗涤柱料4次，收集流出液。
(6)在酶切Buffer终透析过夜，除去GSH，使用分光光度计定量(1OD280＝0.5mg/ml)，按照每100mg加入1ml(2units)preScissionProtease(Amersham Pharmacia Biotech.27-0843-01)，4℃酶切3h。以5-10mg GST/ml的比例加入Glutathione Sepharose 4B(Amersham PharmaciaBiotech.17-0756-01)，在冰上结合1h，使样品液过层析柱(Bio-Rad74306)，收集流出液，使用0.5ml酶切Buffer再洗一次，收集流出液。
实施例2多肽抑制SARS病毒感染靶细胞实验
一、实验材料及方法
1病毒的获取和培养
取含有SARS病毒的上清加到长成单层的VeroE6细胞上，37℃感染1小时，加入DMEM+2％FBS，继续于37℃培养观察CPE，待CPE完全时收获上清。
2病毒滴度的测定
将病毒上清连续稀释，将只有一半细胞样品出现CPE，而下一个稀释度没有CPE的稀释度定为最大稀释度。
3测定多肽对病毒的抑制活性
每份测试细胞加入多肽样品终浓度1μM，加入病毒200TCID50，24小时后观察CPE判断细胞是否被感染。
二、实验结果
所观察到CPE细胞未被感染，证明受试多肽可阻断SARS冠状病毒进入宿主细胞。
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<211>35
<212>PRT
<213>SARS冠状病毒
<400>3
Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu
1 5 10 15
Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile
20 25 30
Asp Leu Gln
35
<210>4
<211>46
<212>PRT
<213>SARS冠状病毒
<400>4
lle Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp
1 5 10 15
Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
20 25 30
Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp
35 40 45
<210>5
<211>43
<212>PRT
<213>SARS冠状病毒
<400>5
Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn
1 5 10 15
Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu
20 25 30
Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp
35 40
<210>6
<211>39
<212>PRT
<213>SARS冠状病毒
<400>6
Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys
1 5 10 15
Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu
20 25 30
Gln Tyr Ile Lys Trp Pro Trp
35
<210>7
<211>36
<212>PRT
<213>SARS冠状病毒
<400>7
Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn
1 5 10 15
Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile
20 25 30
Lys Trp Pro Trp
35
<210>8
<211>32
<212>PRT
<213>SARS冠状病毒
<400>8
Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile
1 5 10 15
Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp
20 25 30
<210>9
<211>37
<212>PRT
<213>SARS冠状病毒
<400>9
Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp
1 5 10 15
Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser LeuIle Asp Leu
20 25 30
Gln Glu Leu Gly Lys
35
<210>10
<211>34
<212>PRT
<213>SARS冠状病毒
<400>10
Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn
1 5 10 15
Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu
20 25 30
Gly Lys
<210>11
<211>30
<212>PRT
<213>SARS冠状病毒
<400>11
Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys
1 5 10 15
Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys
20 25 30
<210>12
<211>27
<212>PRT
<213>SARS冠状病毒
<400>12
Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn
1 5 10 15
Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys
20 25
<210>13
<211>32
<212>PRT
<213>SARS冠状病毒
<400>13
Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp
1 5 10 15
Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
20 25 30
<210>14
<211>29
<212>PRT
<213>SARS冠状病毒
<400>14
Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn
1 5 10 15
Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
20 25
<210>15
<211>25
<212>PRT
<213>SARS冠状病毒
<400>15
Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys
1 5 10 15
Asn Leu Asn Glu Ser Leu Ile Asp Leu
20 25
<210>16
<211>35
<212>PRT
<213>SARS冠状病毒
<400>16
Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys
1 5 10 15
Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu
20 25 30
Ile Asp Leu
35
<210>17
<211>35
<212>PRT
<213>SARS冠状病毒
<400>17
Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu
1 5 10 15
Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys
20 25 30
Trp Pro Trp
35
<210>18
<211>36
<212>PRT
<213>SARS冠状病毒
<400>18
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
1 5 10 15
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu
20 25 30
Ala Ala Thr Lys
35
<210>19
<211>36
<212>PRT
<213>SARS冠状病毒
<400>19
Gln Asn Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn
1 5 10 15
Lys Ala Ile Ser Gln Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala
20 25 30
Leu Gly Lys Leu
35
<210>20
<211>35
<212>PRT
<213>SARS冠状病毒
<400>20
Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys
1 5 10 15
Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu
20 25 30
Gln Tyr Ile
35
<210>21
<211>35
<212>PRT
<213>SARS冠状病毒
<400>21
Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp
1 5 10 15
Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu
20 25 30
Gln Glu Leu
35
Claims (6)
1.抑制SARS冠状病毒侵染宿主细胞的多肽类药物，具有如下通式：
X-SEQ-Z
式中，SEQ表示SARS冠状病毒囊膜蛋白SPIKE中的多肽，所述多肽是SEQ ID NO.1～SEQ ID NO.21所示的21个氨基酸序列；
X是氨基、乙酰基及疏水基团和大分子载体基团；
Z是羧基、氨基及疏水基团和大分子载体基团；
所述疏水基团是9-芴基甲氧羰基、苄氧羰基、1-二甲胺基萘-5-磺酰和叔丁氧羰基；
所述大分子载体基团是共轭脂肪酸、聚乙二醇或碳水化合物类基团。
2.权利要求1所述的多肽类药物，其中所述多肽的至少一个氨基酸残基以D构型存在。
3.权利要求1所述的多肽类药物，其中所述多肽的形式包括单体、二聚体、三聚体和多聚体。
4.权利要求1所述的多肽类药物在制备预防和治疗SARS冠状病毒药物中的用途。
5.SEQ ID NO.1～SEQ ID NO.21所示序列的多肽。
6.权利要求5所述的多肽在制备预防和治疗SARS冠状病毒药物中的用涂。
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03143037 CN1249083C (zh) | 2003-06-12 | 2003-06-12 | 抑制sars冠状病毒侵染宿主细胞的多肽类药物 |
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| CN 03143037 CN1249083C (zh) | 2003-06-12 | 2003-06-12 | 抑制sars冠状病毒侵染宿主细胞的多肽类药物 |
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| CN1566142A CN1566142A (zh) | 2005-01-19 |
| CN1249083C true CN1249083C (zh) | 2006-04-05 |
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| CN114437184B (zh) * | 2021-08-16 | 2022-11-18 | 中国科学院微生物研究所 | 一种抗新型冠状病毒的多肽及其应用 |
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