CN113549651A - 一种抗新型冠状病毒药物筛选与评价模型的建立方法及应用 - Google Patents

一种抗新型冠状病毒药物筛选与评价模型的建立方法及应用 Download PDF

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CN113549651A
CN113549651A CN202110812726.5A CN202110812726A CN113549651A CN 113549651 A CN113549651 A CN 113549651A CN 202110812726 A CN202110812726 A CN 202110812726A CN 113549651 A CN113549651 A CN 113549651A
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novel coronavirus
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伍义行
周彦铸
廉润通
叶子弘
俞晓平
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China Jiliang University
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Abstract

本发明公开了一种抗新型冠状病毒药物筛选与评价模型的建立方法及应用，基于荧光蛋白Venus的两个非荧光互补片段VN155和VC155重新组合形成荧光蛋白发出荧光信号，并利用2A多肽自我切割特性实现多基因同时表达，来检测新型冠状病毒Spike蛋白与ACE2受体的结合效应，可用于能切断新型冠状病毒吸附并感染人体细胞的抗SARS‑CoV‑2药物的筛选与临床前药效评价。本发明具有简单、快速、微量、直观的优点，结果重现性好，特异性强、灵敏度高，干扰因素少，不易出现假阳性和假阴性，适用于抗SARS‑CoV‑2新药研发。

Description

一种抗新型冠状病毒药物筛选与评价模型的建立方法及应用
技术领域
本发明涉及生物医药技术领域，尤其涉及一种抗新型冠状病毒药物筛选与评价模型的建立方法及应用。
背景技术
新冠肺炎是由新型冠状病毒(SARS-CoV-2)感染引起的传染性疾病，自2019年以来全球蔓延愈演愈烈，至今尚无经临床验证的特效治疗药物批准上市。研发安全有效的抗SARS-CoV-2创新药物需求十分迫切，但缺乏可靠的活性筛选急需的针对病毒感染特异机制的细胞模型。
目前，常用的抗SARS-CoV-2药物的体外筛选及评价方法，主要是通过建立病毒体外感染细胞体系来评价药物的抗病毒作用，即通过分离SARS-CoV-2活病毒，在体外感染培养的Vero-E6细胞来实现。首先，培养Vero-E6细胞，分离SARS-CoV-2，采用病毒稀释液吸附细胞，观察细胞病变，计算病毒半数感染剂量(TCID50)。然后采用相应剂量(TCID50)的病毒感染细胞，再加入最大无毒浓度的试验药物进行干预。由于SARS-CoV-2感染Vero-E6细胞后可形成空斑病变，最后通过计算药物对病毒的半数抑制浓度(IC50)和治疗指数(TI)，来评价药物的抗SARS-CoV-2作用。而鉴于SARS-CoV-2极强的传染能力，出于对研究人员的保护，国家规定使用SARS-CoV-2活病毒，必须在高级别生物安全实验室(P3实验室)进行，但P3实验室建设和运行成本极高，而绝大多数医疗和研究单位都没有P3实验室，无法开展涉及SARS-CoV-2活病毒分离、培养和保存等相关研究工作，给直接采用活病毒感染细胞开展药物活性筛选带来极大障碍和风险。因此，建立适于普通实验室的抗SARS-CoV-2药物筛选细胞模型意义重大。
虽然有关SARS-CoV-2假病毒开展抗SARS-CoV-2的研究已有报道，但假病毒的感染和复制过程不能完全体现活病毒的特性，故利用假病毒系统进行药物筛选更易出现假阳性，经假病毒筛选后的活性化合物仍需使用活病毒确证。鉴于假病毒技术仍存在诸多不足，针对SARS-CoV-2感染的关键分子机制建立新的体外筛选模型仍十分必要。
在SARS-CoV-2跨膜进入宿主细胞，以及在宿主细胞内转录翻译、新病毒体组装、释放过程中的关键蛋白和环节，理论上均可作为抗SARS-CoV-2药物作用的靶点，但阻止病毒进入宿主细胞的环节是抗病毒最优质的作用靶点。阻断SARS-CoV-2通过内吞作用进入宿主细胞的过程所涉及的药物靶点主要有来自宿主的ACE2受体以及来自SARS CoV-2的Spike蛋白。针对SARS-CoV-2进入宿主细胞所必须的Spike蛋白与ACE2受体蛋白的相互作用，建立病毒与宿主细胞相互作用的评价模型用于抗SARS-CoV-2药物筛选与验证，对加快抗SARS-CoV-2新药研发的进程具有重大意义。
发明内容
鉴于上述现有技术中存在的缺陷，本发明基于新型冠状病毒(SARS-CoV-2)Spike蛋白与宿主细胞ACE2受体相互作用是病毒感染细胞的关键环节和抗SARS-CoV-2药物的关键靶点，利用荧光蛋白重组特性，建立了适于普通实验室开展的抗SARS-CoV-2药物筛选与评价的新模型，因此，本发明的目的是提出一种抗新型冠状病毒药物筛选与评价模型的建立方法及应用，解决直接采用活病毒感染细胞开展药物活性筛选带来障碍和风险的问题。
为了实现上述目的，本发明采用了如下技术方案：
一种抗新型冠状病毒药物筛选与评价模型的建立方法，以新型冠状病毒Spike蛋白与ACE2受体相互作用为靶点构建的体外药物筛选与评价模型。
进一步的，所用的载体构建方法为双分子荧光互补技术。
进一步的，将新型冠状病毒的Spike蛋白基因，人ACE2基因，以及荧光蛋白Venus的两个非荧光互补片段构建在单一载体或不同载体上转染细胞。
进一步的，1)采用pcDNA4-TO质粒，利用2A多肽的自我切割特性，并以mRFP作为表达对照，将人ACE2基因、新型冠状病毒的Spike蛋白基因，以及荧光蛋白Venus的两个非荧光互补片段VN155和VC155，构建在单一载体上，构建载体pcDNA-ACE2-VN155-P2A-mRFP-T2A-Spike-VC155，基因序列如SEQ ID NO.1所示；
2)培养的Vero-E6细胞贴壁后，将构建的野生型和突变型载体分别转染细胞，根据待测药物的性质及作用机制采用转染前预防给药或转染后药物干预的给药方式处理细胞，同时设立空载体对照、溶剂对照和未处理细胞对照；
3)处理后不同时间，分别用荧光显微镜分析各组细胞的荧光信号强度及蛋白相互作用的亚细胞定位；
4)完成荧光信号强度及蛋白相互作用的亚细胞定位分析后，收集转染细胞检测ACE2和Spike的基因和蛋白表达水平，分析药物干预对转染细胞ACE2和Spike表达的影响。
进一步的，用RT-PCR法检测ACE2和Spike mRNA表达水平；用Western Blotting法检测ACE2和Spike蛋白表达水平。
上述任意一种抗新型冠状病毒药物筛选与评价模型的建立方法，在抗新型冠状病毒药物筛选与评价中的应用。
本发明的突出效果为：
本发明是基于荧光蛋白Venus的两个非荧光互补片段VN155和VC155重新组合形成荧光蛋白发出荧光信号，并利用2A多肽自我切割特性实现多基因同时表达，来检测新型冠状病毒Spike蛋白与ACE2受体的结合效应，可用于能切断新型冠状病毒吸附并感染人体细胞的抗SARS-CoV-2药物的筛选与临床前药效评价。本发明具有简单、快速、微量、直观的优点，结果重现性好，特异性强、灵敏度高，干扰因素少，不易出现假阳性和假阴性，适用于抗SARS-CoV-2新药研发。
附图说明
图1为本发明的新型冠状病毒(SARS-CoV-2)Spike蛋白与宿主细胞ACE2受体相互作用及荧光蛋白重组原理图；
图2为本发明实施例的方法流程图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。
实施例：
主要材料：
1、Vero-E6细胞、感受态细胞DH5a。
2、DMEM培养基(Gibco)、pcDNA4-TO质粒、DNA提取试剂盒(Omega)、PCR纯化试剂盒(Vazyme)、Plasmid Maxi Kit(OMEGA)、dNTPs、高保真pfu聚合酶、
Figure BDA0003169028220000051
2000转染试剂(Sigma)、T7连接酶(Thermo Fisher)、RNase(Invitrogen)、核酸参考Marker(Thermo Fisher)、蛋白Marker(Amresco)；HindIII、BamHⅠ、XbaI、NehⅠ内切酶(InvitrogenTM)；过硫酸铵(APS)(Sigma)、四甲基乙二胺(TEMED)(Sigma)、β-巯基乙醇(Gibco)、ACE2和Spike表达检测试剂等。
3、仪器：
1)倒置荧光显微镜。
2)其它常规分子生物学研究设备。
本实施例的操作流程如图2所示，包括：
一、验证SARS-CoV-2的Spike蛋白与人ACE2受体的结合作用
1、Vero-E6细胞培养：
采用含10％的胎牛血清的DMEM培养基，在含5％二氧化碳的37℃下的培养箱中培养，待细胞长成单层后即可应用。
2、pcDNA-ACE2-VN155-P2A-mRFP-T2A-Spike-VC155载体构建：
采用pcDNA4-TO质粒，利用2A多肽的自我切割特性，并以mRFP作为表达对照，将人ACE2基因、Spike基因，以及荧光蛋白Venus的两个非荧光互补片段VN155和VC155，构建在同时表达多个基因的单一载体上，即pcDNA-ACE2-VN155-P2A-mRFP-T2A-Spike-VC155(基因序列如SEQ ID NO.1所示)。同时，在该载体基础上，构建表达Spike蛋白突变体基因的载体转染Vero-E6细胞作为阴性对照。
3、Spike蛋白与ACE2受体互作的验证：
培养在96孔板里的Vero-E6细胞贴壁后，将构建的野生型和突变型载体分别转染细胞，同时设立空载体对照和未处理细胞对照。转染24和48小时后，分别用荧光显微镜及其软件分析各组细胞的荧光强度和位置。通过观察荧光的强度及位置，可以了解并验证人ACE2受体与新冠病毒Spike蛋白的互作情况及其亚细胞定位。
4、结果判定：
如图1所示，若ACE2受体与Spike蛋白发生相互作用，则引起荧光蛋白Venus的两个非荧光互补片段VN155和VC155重新组合形成荧光蛋白，并发出通过荧光显微镜可以检测到的荧光。通过显微镜观察到荧光，可以证明ACE2受体与Spike蛋白间发生了相互作用；通过观察细胞里荧光出现的位置(如细胞核、细胞质、细胞膜)，可以判断细胞中ACE2受体表达的区域，从而实现亚细胞定位。
二、抗SARS-CoV-2药物活性筛选及评价方法
1、Vero-E6细胞培养：
采用含10％的胎牛血清的DMEM培养基，在含5％二氧化碳的37℃下的培养箱中培养，待细胞长成单层后即可应用。
2、pcDNA-ACE2-VN155-P2A-mRFP-T2A-Spike-VC155载体构建：
采用pcDNA4-TO质粒，利用2A多肽的自我切割特性，并以mRFP作为表达对照，将人ACE2基因、Spike基因，以及荧光蛋白Venus的两个非荧光互补片段VN155和VC155，构建在同时表达多个基因的单一载体上，即pcDNA-ACE2-VN155-P2A-mRFP-T2A-Spike-VC155(基因序列如SEQ ID NO.1所示)。同时，在该载体基础上，构建表达Spike蛋白突变体基因的载体转染Vero-E6细胞作为阴性对照。
3、抗SARS-CoV-2药物活性筛选：
1)根据待测药物的性质及作用机制可采用不同的给药方式(a)或(b)：
(a)转染前预防给药：当DMEM培养基中的Vero-E6细胞贴壁后，先用不同浓度试验药物预处理细胞24小时，然后将构建的野生型和突变型载体分别转染细胞，同时设立空载体对照、溶剂对照和未处理细胞对照。转染24、48和72小时后，分别用荧光显微镜及其相关软件分析各组细胞的荧光信号强度及亚细胞位置。
(b)转染后药物干预：当DMEM培养基中的Vero-E6细胞贴壁后，将构建的野生型和突变型载体分别转染细胞，转染后用不同浓度试验药物处理细胞24小时。药物处理后24、48和72小时，分别用荧光显微镜及其相关软件分析各组细胞的荧光信号强度及亚细胞位置。同时设立空载体对照、溶剂对照和未处理细胞对照。
2)完成荧光信号强度及蛋白互作的亚细胞定位分析后，收集转染细胞用RT-PCR法检测ACE2和Spike mRNA表达水平；用Western Blotting法检测ACE2和Spike蛋白表达水平，分析试验药物干预对转染细胞ACE2和Spike表达的影响。
4、结果判定：
由于试验药物对细胞的处理会影响ACE2与Spike蛋白互作，通过检测荧光的有无、强度及位置，可了解试验药物对ACE2与Spike蛋白结合的干预情况。结合ACE2和Spike的基因和蛋白表达水平，综合评价试验药物的抗SARS-CoV-2活性。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。
序列表
<110>中国计量大学
<120>一种抗新型冠状病毒药物筛选与评价模型的建立方法及应用
<160>1
<210>1
<211>8010
<212>DNA
<213>人工序列
<400>1
atgtcaagct cttcctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60
attgaggaac aggccaagac atttttggac aagtttaacc acgaagccga agacctgttc 120
tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180
aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacacttgcc 240
caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300
cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360
aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420
tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480
aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540
gaagagtatg tggttttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600
gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660
cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720
catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780
tgcctccctg ctcatttgct tggtgatatg tggggtagat tttggacaaa tctgtactct 840
ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900
gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960
cctaatatga ctcaaggatt ctgggaaaat tccatgctaa cggacccagg aaatgttcag 1020
aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080
tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140
tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200
catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260
attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320
aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380
tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440
aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500
gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560
taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620
aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680
ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740
gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800
aattcttttg tgggatggag taccgactgg agtccatatg cagaccaaag catcaaagtg 1860
aggataagcc taaaatcagc tcttggagat aaagcatatg aatggaacga caatgaaatg 1920
tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980
cagatgattc tttttgggga ggaggatgtg cgagtggcta atttgaaacc aagaatctcc 2040
tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100
gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gaatgacaac 2160
agcctagagt ttctggggat acagccaaca cttggacctc ctaaccagcc ccctgtttcc 2220
atatggctga ttgtttttgg agttgtgatg ggagtgatag tggttggcat tgtcatcctg 2280
atcttcactg ggatcagaga tcggaagaag aaaaataaag caagaagtgg agaaaatcct 2340
tatgcctcca tcgatattag caaaggagaa aataatccag gattccaaaa cactgatgat 2400
gttcagacct ccttttaggt gggagccgcg tgtccagcgg gagggaccat ggtgagcaag 2460
ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 2520
ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc 2580
ctgaagctga tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 2640
ctgggctacg gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc 2700
ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac 2760
ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 2820
gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac 2880
aactacaaca gccacaacgt ctatctcacc gccggaggtg gcgggagcgg aggtggcggg 2940
agtcggtcga ccgagatctc ttgatctcag aggaggacct gcttatggcc atggaggccc 3000
gaattcggtc gaccgagatc tctcgaggta ccggtggcgg aggttctggt ggcggaggtt 3060
ctatggcctc ctccgaggac gtcatcaagg agttcatgcg cttcaaggtg cgcatggagg 3120
gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg 3180
gcacccagac cgccaagctg aaggtgacca agggcggccc cctgcccttc gcctgggaca 3240
tcctgtcccc tcagttcacg tacggctcca aggcctacgt gaagcacccc gccgacatcc 3300
ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg 3360
aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct 3420
acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga 3480
ccatgggctg ggaggcctcc accgagcgga tgtaccccga ggacggcgcc ctgaagggcg 3540
agatcaagat gaggctgaag ctgaaggacg gcggccacta cgacgccgag gtcaagacca 3600
cctacatggc caagaagccc gtgcagctgc ccggcgccta caagaccgac atcaagctgg 3660
acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgagcgc gccgagggcc 3720
gccactccac cggcgccgcg gccgcaggtt ctggagaggg cagaggaagt cttctaacat 3780
gcggtgacgt ggaggagaat cccggccctt ctagcatcaa ggccaacttc aagatccgcc 3840
acaacatcga ggacggcggc gtgcagcatg tttgtttttc ttgttttatt gccactagtc 3900
tctagtcagt gtgttaatct tacaaccaga actcaattac cccctgcata cactaattct 3960
ttcacacgtg gtgtttatta ccctgacaaa gttttcagat cctcagtttt acattcaact 4020
caggacttgt tcttaccttt cttttccaat gttacttggt tccatgctat acatgtctct 4080
gggaccaatg gtactaagag gtttgataac cctgtcctac catttaatga tggtgtttat 4140
tttgcttcca ctgagaagtc taacataata agaggctgga tttttggtac tactttagat 4200
tcgaagaccc agtccctact tattgttaat aacgctacta atgttgttat taaagtctgt 4260
gaatttcaat tttgtaatga tccatttttg ggtgtttatt accacaaaaa caacaaaagt 4320
tggatggaaa gtgagttcag agtttattct agtgcgaata attgcacttt tgaatatgtc 4380
tctcagcctt ttcttatgga ccttgaagga aaacagggta atttcaaaaa tcttagggaa 4440
tttgtgttta agaatattga tggttatttt aaaatatatt ctaagcacac gcctattaat 4500
ttagtgcgtg atctccctca gggtttttcg gctttagaac cattggtaga tttgccaata 4560
ggtattaaca tcactaggtt tcaaacttta cttgctttac atagaagtta tttgactcct 4620
ggtgattctt cttcaggttg gacagctggt gctgcagctt attatgtggg ttatcttcaa 4680
cctaggactt ttctattaaa atataatgaa aatggaacca ttacagatgc tgtagactgt 4740
gcacttgacc ctctctcaga aacaaagtgt acgttgaaat ccttcactgt agaaaaagga 4800
atctatcaaa cttctaactt tagagtccaa ccaacagaat ctattgttag atttcctaat 4860
attacaaact tgtgcccttt tggtgaagtt tttaacgcca ccagatttgc atctgtttat 4920
gcttggaaca ggaagagaat cagcaactgt gttgctgatt attctgtcct atataattcc 4980
gcatcatttt ccacttttaa gtgttatgga gtgtctccta ctaaattaaa tgatctctgc 5040
tttactaatg tctatgcaga ttcatttgta attagaggtg atgaagtcag acaaatcgct 5100
ccagggcaaa ctggaaagat tgctgattat aattataaat taccagatga ttttacaggc 5160
tgcgttatag cttggaattc taacaatctt gattctaagg ttggtggtaa ttataattac 5220
ctgtatagat tgtttaggaa gtctaatctc aaaccttttg agagagatat ttcaactgaa 5280
atctatcagg ccggtagcac accttgtaat ggtgttgaag gttttaattg ttactttcct 5340
ttacaatcat atggtttcca acccactaat ggtgttggtt accaaccata cagagtagta 5400
gtactttctt ttgaacttct acatgcacca gcaactgttt gtggacctaa aaagtctact 5460
aatttggtta aaaacaaatg tgtcaatttc aacttcaatg gtttaacagg cacaggtgtt 5520
cttactgagt ctaacaaaaa gtttctgcct ttccaacaat ttggcagaga cattgctgac 5580
actactgatg ctgtccgtga tccacagaca cttgagattc ttgacattac accatgttct 5640
tttggtggtg tcagtgttat aacaccagga acaaatactt ctaaccaggt tgctgttctt 5700
tatcaggatg ttaactgcac agaagtccct gttgctattc atgcagatca acttactcct 5760
acttggcgtg tttattctac aggttctaat gtttttcaaa cacgtgcagg ctgtttaata 5820
ggggctgaac atgtcaacaa ctcatatgag tgtgacatac ccattggtgc aggtatatgc 5880
gctagttatc agactcagac taattctcct cggcgggcac gtagtgtagc tagtcaatcc 5940
atcattgcct acactatgtc acttggtgca gaaaattcag ttgcttactc taataactct 6000
attgccatac ccacaaattt tactattagt gttaccacag aaattctacc agtgtctatg 6060
accaagacat cagtagattg tacaatgtac atttgtggtg attcaactga atgcagcaat 6120
cttttgttgc aatatggcag tttttgtaca caattaaacc gtgctttaac tggaatagct 6180
gttgaacaag acaaaaacac ccaagaagtt tttgcacaag tcaaacaaat ttacaaaaca 6240
ccaccaatta aagattttgg tggttttaat ttttcacaaa tattaccaga tccatcaaaa 6300
ccaagcaaga ggtcatttat tgaagatcta cttttcaaca aagtgacact tgcagatgct 6360
ggcttcatca aacaatatgg tgattgcctt ggtgatattg ctgctagaga cctcatttgt 6420
gcacaaaagt ttaacggcct tactgttttg ccacctttgc tcacagatga aatgattgct 6480
caatacactt ctgcactgtt agcgggtaca atcacttctg gttggacctt tggtgcaggt 6540
gctgcattac aaataccatt tgctatgcaa atggcttata ggtttaatgg tattggagtt 6600
acacagaatg ttctctatga gaaccaaaaa ttgattgcca accaatttaa tagtgctatt 6660
ggcaaaattc aagactcact ttcttccaca gcaagtgcac ttggaaaact tcaagatgtg 6720
gtcaaccaaa atgcacaagc tttaaacacg cttgttaaac aacttagctc caattttggt 6780
gcaatttcaa gtgttttaaa tgatatcctt tcacgtcttg acaaagttga ggctgaagtg 6840
caaattgata ggttgatcac aggcagactt caaagtttgc agacatatgt gactcaacaa 6900
ttaattagag ctgcagaaat cagagcttct gctaatcttg ctgctactaa aatgtcagag 6960
tgtgtacttg gacaatcaaa aagagttgat ttttgtggaa agggctatca tcttatgtcc 7020
ttccctcagt cagcacctca tggtgtagtc ttcttgcatg tgacttatgt ccctgcacaa 7080
gaaaagaact tcacaactgc tcctgccatt tgtcatgatg gaaaagcaca ctttcctcgt 7140
gaaggtgtct ttgtttcaaa tggcacacac tggtttgtaa cacaaaggaa tttttatgaa 7200
ccacaaatca ttactacaga caacacattt gtgtctggta actgtgatgt tgtaatagga 7260
attgtcaaca acacagttta tgatcctttg caacctgaat tagactcatt caaggaggag 7320
ttagataaat attttaagaa tcatacatca ccagatgttg atttaggtga catctctggc 7380
attaatgctt cagttgtaaa cattcaaaaa gaaattgacc gcctcaatga ggttgccaag 7440
aatttaaatg aatctctcat cgatctccaa gaacttggaa agtatgagca gtatataaaa 7500
tggccatggt acatttggct aggttttata gctggcttga ttgccatagt aatggtgaca 7560
attatgcttt gctgtatgac cagttgctgt agttgtctca agggctgttg ttcttgtgga 7620
tcctgctgca aatttgatga agacgactct gagccagtgc tcaaaggagt caaattacat 7680
tacacataag tgggagccgc gtgtccagcg ggagggaccg acaagcagaa gaacggcatc 7740
aaggccaact tcaagatccg ccacaacatc gaggacggcg gcgtgcagct cgccgaccac 7800
taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg 7860
agctaccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg 7920
gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggaggtggc 7980
gggagcggag gtggcgggag tagaattcgg 8010

Claims (6)

1.一种抗新型冠状病毒药物筛选与评价模型的建立方法，其特征在于：以新型冠状病毒Spike蛋白与ACE2受体相互作用为靶点构建的体外的药物筛选与评价模型。
2.根据权利要求1所述的一种抗新型冠状病毒药物筛选与评价模型的建立方法，其特征在于：所用的载体构建方法为双分子荧光互补技术。
3.根据权利要求2所述的一种抗新型冠状病毒药物筛选与评价模型的建立方法，其特征在于：将新型冠状病毒的Spike蛋白基因，人ACE2基因，以及荧光蛋白Venus的两个非荧光互补片段构建在单一载体或不同载体上转染细胞。
4.根据权利要求3所述的一种抗新型冠状病毒药物筛选与评价模型的建立方法，其特征在于：1)采用pcDNA4-TO质粒，利用2A多肽的自我切割特性，并以mRFP作为表达对照，将人ACE2基因、新型冠状病毒的Spike蛋白基因，以及荧光蛋白Venus的两个非荧光互补片段VN155和VC155，构建在单一载体上，构建载体pcDNA-ACE2-VN155-P2A-mRFP-T2A-Spike-VC155，基因序列如SEQ ID NO.1所示；
2)培养的Vero-E6细胞贴壁后，将构建的野生型和突变型载体分别转染细胞，根据待测药物的性质及作用机制采用转染前预防给药或转染后药物干预的给药方式处理细胞，同时设立空载体对照、溶剂对照和未处理细胞对照；
3)处理后不同时间，分别用荧光显微镜分析各组细胞的荧光信号强度及蛋白相互作用的亚细胞定位；
4)完成荧光信号强度及蛋白相互作用的亚细胞定位分析后，收集转染细胞检测ACE2和Spike的基因和蛋白表达水平，分析药物干预对转染细胞ACE2和Spike表达的影响。
5.根据权利要求4所述的一种抗新型冠状病毒药物筛选与评价模型的建立方法，其特征在于：用RT-PCR法检测ACE2和Spike mRNA表达水平；用Western Blotting法检测ACE2和Spike蛋白表达水平。
6.权利要求1-5所述的任意一种抗新型冠状病毒药物筛选与评价模型的建立方法，在抗新型冠状病毒药物筛选与评价中的应用。
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CN108265078A (zh) * 2016-12-29 2018-07-10 中国计量大学 一种基于宿主细胞靶点的抗乙肝模型建立及应用
CN111662884A (zh) * 2020-06-18 2020-09-15 中吉当康(北京)基因技术有限公司 一种假病毒及其包装方法和药物评估系统

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CN111662884A (zh) * 2020-06-18 2020-09-15 中吉当康(北京)基因技术有限公司 一种假病毒及其包装方法和药物评估系统

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* Cited by examiner, † Cited by third party
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CN115414364A (zh) * 2022-09-23 2022-12-02 四川大学 一种以rna g-四链体为靶点的抗新冠病毒药物及其筛选方法

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