CN111607003B - 一种SARS-CoV-2 N/S1(RBD)重组蛋白及制备方法和应用 - Google Patents
一种SARS-CoV-2 N/S1(RBD)重组蛋白及制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种SARS‑CoV‑2 N/S1(RBD)重组蛋白及制备方法和应用，所述SARS‑CoV‑2 N/S1(RBD)重组蛋白是将在原核表达系统中易于表达的SARS‑CoV‑2病毒核衣壳(N)蛋白和SARS‑CoV‑2病毒表面刺突蛋白S1的表达基因片段按照原核E.coli偏好的密码子进行优化，并进行如下改造：将表达病毒核衣壳(N)蛋白和SARS‑CoV‑2病毒表面刺突蛋白S1进行串联表达。本发明所提供的SARS‑CoV‑2 N/S1(RBD)重组蛋白可制备预防SARS‑CoV‑2病毒感染的基因工程疫苗，SARS‑CoV‑2病毒检测试剂盒,及筛选可抑制N或S蛋白结合的抗SARS‑CoV‑2病毒感染的药物。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种SARS-CoV-2 N/S1(RBD)重组蛋白及制备方法和应用。
背景技术
新型冠状病毒肺炎(COVID-19)与2003年爆发的严重急性呼吸综合征 (SARS)均具有广泛的流行性、较强的传染性、较高的致病性,这2种疾病分别是由人类高致病性冠状病毒SARS-Co V-2与SARS-CoV感染所致。对SARS-CoV-2 的来源和进化、形态特征和基因结构、感染和致病分子机制开展深入研究,取得了重大进展,为科学防控COVID-19提供了重要依据。根据上述研究的基础, 文中对COVID-19病毒疫苗、抗体和抑制剂研发提出了设想,在研究防控 COVID-19核心技术上具有一定的参考价值。对病毒性疾病预防用疫苗和治疗用药物是重要组成部分,认识这类药物的研发难点,才有利于抗病毒药物研发。
发明内容
为此，本发明提供了一种SARS-CoV-2 N/S1(RBD)重组蛋白及制备方法和应用，所述SARS-CoV-2 N/S1(RBD)重组蛋白是将在原核表达系统中易于表达的SARS-CoV-2病毒核衣壳(N)蛋白和SARS-CoV-2病毒表面刺突蛋白S1的表达基因片段按照原核E.coli偏好的密码子进行优化，并进行如下改造：将表达病毒核衣壳(N)蛋白和SARS-CoV-2病毒表面刺突蛋白S1进行串联表达。
本发明的第一方面提供了一种SARS-CoV-2 N/S1(RBD)重组蛋白，该重组蛋白的核苷酸序列如SEQ ID NO.1所示。
所述重组蛋白由SARS-CoV-2病毒核衣壳(N)蛋白表达基因片段和 SARS-CoV-2病毒表面刺突蛋白S1蛋白表达基因片段通过Linker融合连接； SARS-CoV-2病毒核衣壳(N)蛋白表达基因片段的核苷酸序列如SEQ ID NO.2 所示，SARS-CoV-2病毒表面刺突蛋白S1蛋白表达基因片段的核苷酸序列如SEQ ID NO.3所示。
所述Linker由0-20个氨基酸组成，优选GS。
本发明第二方面提供了含有SARS-CoV-2 N/S1(RBD)重组蛋白的重组载体，其特征在于，在含有抗卡那霉素的抗性选择基因的表达载体中包含有SEQ ID NO.1核苷酸序列，表达载体优选pET30a载体。
本发明第三方面提供了表达如上述重组载体的宿主菌。
所述宿主菌为具有卡那霉素抗性的表达菌株，优选大肠杆菌E.coli BL21(DE3)。
本发明第四方面提供了含有SARS-CoV-2 N/S1(RBD)重组蛋白的重组载体的制备方法，其特征在于，包括如下步骤：
(1)构建SARS-CoV-2 N/S1(RBD)重组蛋白基因片段，并对pET30a载体进行处理：用限制性内切酶Nde I和限制性内切酶Hind III对pET30a载体双酶切；
(2)以SARS-CoV-2 N/S1(RBD)重组蛋白基因片段为模板进行PCR扩增，回收PCR产物,克隆至pET30a载体中，用限制性内切酶Nde I和限制性内切酶Hind III双酶切后，将基因片段从pUC57载体中切下来，再与处理后的 pET30a载体连接，制得重组载体。
本发明第五方面提供了SARS-CoV-2 N/S1(RBD)重组蛋白的表达方法，其特征在于，包括如下步骤：
(1)按照权利要求8所述的重组载体的制备方法制备含有SARS-CoV-2 N/S1(RBD)重组蛋白的重组载体；
(2)将含有SARS-CoV-2 N/S1(RBD)重组蛋白的重组载体转化至大肠杆菌 E.coliBL21(DE3)，诱导表达SARS-CoV-2 N/S1(RBD)重组蛋白，表达时的诱导温度在15℃，诱导的IPTG的浓度为0.5mM。
本发明第七方面提供了上述的重组蛋白在制备用于检测和/或预防和/或筛选治疗SARS-CoV-2病毒感染的药物中的应用。
本发明的有益效果是：本发明所提供的SARS-CoV-2 N/S1(RBD)重组蛋白可制备预防SARS-CoV-2病毒感染的基因工程疫苗，SARS-CoV-2病毒检测试剂盒,及筛选可抑制N或S蛋白结合的抗SARS-CoV-2病毒感染的药物。
附图说明
图1显示为SARS-CoV-2 N/S1(RBD)重组蛋白诱导鉴定结果，图中：泳道 M为蛋白(Protein)分子量标准(Marker)，从上到下分别为，200kDa，150 kDa，120kDa，100kDa，85kDa，70kDa，60kDa，50kDa，40kDa，30kDa， 25kDa，20kDa，15kDa，10kDa；泳道1为空白对照；泳道2为15℃ 0.5mM IPTG 16h；泳道3为37℃ 0.5mM IPTG 3h。
图2显示为诱导表达后的SARS-CoV-2 N/S1(RBD)重组蛋白Ni柱纯化鉴定结果，图中：泳道M为蛋白(Protein)分子量标准(Marker)，从上到下分别为，200kDa，150kDa，120kDa，100kDa，85kDa，70kDa，60kDa， 50kDa，40kDa，30kDa，25kDa，20kDa，15kDa，10kDa；泳道1为过滤后的上清液；泳道2为流出液；泳道3为洗脱缓冲液250mM咪唑洗脱液。
图3为纯化后的SARS-CoV-2 N/S1(RBD)重组蛋白电泳结果：泳道M为蛋白(Protein)分子量标准(Marker)，从上到下分别为，200kDa，150kDa， 120kDa，100kDa，85kDa，70kDa，60kDa，50kDa，40kDa，30kDa， 25kDa，20kDa，15kDa，10kDa；泳道1为纯化的SARS-CoV-2 N/S1(RBD) 重组蛋白(实施例中透析后的蛋白)。
图4为含有SARS-CoV-2 N/S1(RBD)重组蛋白的重组载体的分解图。
图5为SARS-CoV-2 N/S1(RBD)重组蛋白产品制备工艺流程图。
图6显示为SARS-CoV-2-N-S1蛋白和人体新冠病毒受体蛋白结合实验结果；
图7显示为SARS-CoV-2-N-S1蛋白和抗2019新型冠状病毒spike蛋白抗体结合实验结果。
具体实施方式
下面结合实施例对本发明作进一步地详细说明，但本发明的实施方式不限于此，在不脱离本发明上述技术思想情况下，根据本领域普通技术知识和惯用手段，做出各种替换和变更，均应包括在本发明的范围内。
实施例1
基因的合成及重组载体的制备：
将在原核表达系统中易于表达的SARS-CoV-2病毒核衣壳(N)蛋白和 SARS-CoV-2病毒表面刺突蛋白S1的表达基因片段按照原核E.coli偏好的密码子进行优化，构建SARS-CoV-2 N/S1(RBD)重组蛋白基因片段：将SARS-CoV-2 病毒核衣壳(N)蛋白表达基因片段和SARS-CoV-2病毒表面刺突蛋白S1蛋白表达基因片段通过Linker融合连接，进行人工合成，Linker为GS，连接方法从N端到C端，依次为：SARS-CoV-2病毒核衣壳(N)-GS-SARS-CoV-2病毒表面刺突蛋白S1，制得改造后表达基因片段；
SARS-CoV-2病毒核衣壳(N)-GS-SARS-CoV-2病毒表面刺突蛋白S1蛋白表达基因片段的核苷酸序列如SEQ ID NO.1所示：
SARS-CoV-2病毒核衣壳(N)蛋白改造后表达基因片段核苷酸序列如SEQ ID NO.2所示：
SARS-CoV-2病毒表面刺突蛋白S1蛋白表达基因片段的核苷酸序列如SEQ ID NO.3所示：
将表达基因片段为模板进行PCR扩增，扩增引物(由生工生物工程(上海) 股份有限公司合成)如下：
F引物taactttaagaaggagatatacaTATGTCAGACAATGGCCC
R引物gtggtgctcgagtgcggccgcaagctTTCATTAGTGATGGT
PCR条件:94℃5min,94℃1min,57℃1min,72℃50s,20个循环后,72℃ 10min。
胶回收试剂盒回收PCR产物,克隆至pET30a载体中(载体质粒pET30a 购自Invitrogen公司，含启动子,N端有一段His Tag/凝血酶/S Tag/肠激酶序列,C端有一可选的His Tag序列,它还含有抗卡那霉素的抗性选择基因)。 SARS-CoV-2-N-S1蛋白表达质粒的构建利用引物上的内切酶位点,用 NdeI/HindIII双酶切,将基因片段从pUC57载体中切下来(在编码重组蛋白引物的5’端引入de I酶切位点,在其3’端引入Hind III酶切位点。PCR分段扩增N蛋白各区段的基因根据SARS-CoV-2-N-S1蛋白基因序列,用DNAstar 软件设计了引物。5’端引物设计了Nde I酶切位点,3’端则设计了Hind III 酶切位点。),再与预先用处理好的pET-30a载体(预先经限制性内切酶Nde I 和限制性内切酶Hind III双酶切)连接,得到重组载体，即最终表达质粒：携带SARS-CoV-2病毒核衣壳(N)蛋白和SARS-CoV-2病毒表面刺突蛋白S1的质粒pET30a-SARS-CoV-2-N-S1，构建过程由百英生物完成，其分解图如图4 所示。限制性内切酶、Taq DNA聚合酶来自百英生物,胶回收试剂盒购自Qigen 公司,
实施例2
重组载体的转化和SARS-CoV-2 N/S1(RBD)重组蛋白的诱导表达
1.将重组载体转化至具有卡那霉素抗性的表达菌株上，本实施例采用的是大肠杆菌E.coli BL21(DE3)，然后将表达SARS-CoV-2-N-S1的大肠杆菌 E.coli BL21(DE3)接种到卡那霉素浓度为50mg/ml的液体培养基中进行摇瓶培养，37℃恒温摇床活化过夜(大概16小时)，活化完成后，将活化菌转移到大瓶中继续培养，在大瓶中继续培养至OD600为0.6-0.8，设置一个实验组，一个对照组，实验组为：加入终浓度为0.5mM IPTG进行15℃过夜(16h) 诱导，诱导完成后，离心收集得到的菌液，待用；对照组1为：加入终浓度为0.5mM IPTG进行37℃诱导3h，诱导完成后，离心收集得到的菌液；对实验组和对照组制备的菌液进行SDS-PAGE凝胶电泳，结果如图1所示，结果表明，实验组条件下制备的菌株中SARS-CoV-2 N/S1(RBD)重组蛋白更稳定存在，更集中在目标分子量约为70kDa。
2.将SARS-CoV-2-N-S1菌体进行超声破碎后。取4L经诱导后的实验组菌液，4℃7000rpm离心15min，弃培养基。离心后的菌体沉淀用柱平衡缓冲液(pH7.4,20mM PB,300nMNaCl)洗涤一遍，4℃、7000rpm离心15min，尽弃上清，加入400mL平衡缓冲液重悬菌体。设置超声破碎仪为功率300w，工作3s，间歇6s，超声时长20min。将菌体重悬液置于冰浴中进行超声破碎，完成一组后，间隔10min进行下一组，总共进行三个循环。破碎后的菌液4℃、11000rpm离心20min，上清用0.45um的滤膜过滤后待用。通过Ni柱(亲和层析介质NiAffinity Resin来自百英生物)纯化SARS-CoV-2-N-S1蛋白,用平衡缓冲液(pH7.4,20mMPB,300nM NaCl)平衡镍柱。将过滤后的上清液过柱，用平衡缓冲液(pH7.4,20mM PB,300nMNaCl)和平衡缓冲液(pH7.4, 20mM PB,300mM NaCl,20mM imidazole)洗杂。
平衡柱子，洗去与柱子结合力弱的杂蛋白。用洗脱缓冲液(pH7.4,20mM PB,300mMNaCl,250mM imidazole)洗脱目的蛋白，收集洗脱液，并透析到最终缓冲液，即得到纯化后的SARS-CoV-2 N/S1(RBD)重组蛋白，进行SDS-PAGE 凝胶电泳，结果如图3所示，其分子量约为70kDa。
实施例3
SARS-CoV-2 N/S1(RBD)重组蛋白产品制备：
制备工艺流程图图5所示，即实施例1+实施例2的制备工艺。
将制备的SARS-CoV-2-N-S1蛋白与Recombinant Human ACE2/hFc
(Biointron，catalog:B232006)(人体新冠病毒受体蛋白)进行结合实验，实验结果如图6所示；
将制备的SARS-CoV-2-N-S1蛋白和Anti-2019-nCov S1 RBD-C01 antibody(Biointron,catalog：LK192)(抗2019新型冠状病毒spike蛋白抗体)进行结合实验，实验结果如图7所示；
由图6和图7可知，SARS-CoV-2-N-S1蛋白浓度在0.0001-0.001ug/ml时，可以与Anti-2019-nCov S1 RBD-C01 antibody(Biointron,catalog：LK192) 有效结合，并与Recombinant Human ACE2/hFc结合，说明SARS-CoV-2-N-S1 蛋白可与Recombinant HumanACE2/hFc和Anti-2019-nCov S1 RBD-C01 antibody结合。其可用于制备用于检测和/或预防和/或筛选治疗SARS-CoV-2 病毒感染的药物。
需要说明的是，在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者终端设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者终端设备所固有的要素。在没有更多限制的情况下，由语句“包括……”或“包含……”限定的要素，并不排除在包括所述要素的过程、方法、物品或者终端设备中还存在另外的要素。此外，在本文中，“大于”、“小于”、“超过”等理解为不包括本数；“以上”、“以下”、“以内”等理解为包括本数。
尽管已经对上述各实施例进行了描述，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例做出另外的变更和修改，所以以上所述仅为本发明的实施例，并非因此限制本发明的专利保护范围，凡是利用本发明说明书及附图内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围之内。
序列表
<110> 泰州市百英生物科技有限公司
<120> 一种SARS-CoV-2 N/S1(RBD)重组蛋白及制备方法和应用
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cgccgccctc agggtctgcc taataatacc gcaagttggt tcactgcgtt gacgcagcac 180
ggcaaggaag atttgaaatt tcctcgtggt caaggagttc ccatcaatac caattcttca 240
ccagacgatc aaattgggta ctatcgccgc gcgacccgtc gcattcgtgg cggtgacggt 300
aagatgaagg atttatcccc tcgttggtac ttctactacc ttggcactgg tccagaggct 360
ggacttcctt acggtgcaaa caaagacggt atcatctggg ttgcaactga gggtgcctta 420
aatacaccta aggatcatat cgggacccgt aacccagcca acaatgctgc aatcgtattg 480
caattacccc aagggacaac tctgccgaag ggattttatg ccgaaggatc acgtggaggg 540
tcgcaggctt ccagccgctc aagctctcgt agccgtaact ctagtcgcaa ctctactccg 600
ggatcgtctc gtggcaccag tccggcacgc atggctggca acggtggaga tgcggccctt 660
gcattgttat tattagaccg tttaaatcag ttggagagca agatgtccgg taaagggcaa 720
caacagcagg gacagaccgt cacgaaaaag tccgctgccg aggcaagcaa gaaaccgcgc 780
caaaaacgca ctgcaacaaa ggcatacaat gtgacccagg catttggtcg ccgtggccct 840
gaacagaccc aaggcaattt tggtgaccaa gaactgattc gtcagggtac agactataaa 900
cactggcctc aaatcgcaca attcgcacct tccgctagcg ctttttttgg catgagtcgt 960
atcggtatgg aggttacccc gtctggcaca tggttgacat atactggcgc tattaaattg 1020
gatgataagg acccgaactt taaagaccag gttattctgc tgaataaaca tattgatgca 1080
tacaagactt tcccaccgac cgaaccgaag aaagacaaaa aaaagaaagc agatgagacg 1140
caagctctgc cgcaacgcca aaagaaacaa caaactgtaa ctctgctgcc cgcggctgac 1200
cttgatgatt tctccaaaca acttcagcag agtatgagtt ccgctgactc cactcaggcc 1260
ggaggtagtg gaggcggagg ctctggcggg ggtggaagcg ggggaggggg gcctaacatt 1320
acgaatttgt gcccgttcgg ggaggtattt aacgccacac gctttgcaag tgtctatgcg 1380
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tcttttagca ctttcaaatg ttacggtgta tcgcctacaa aacttaacga cttatgcttt 1500
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ggccaaacgg gaaagatcgc cgattataat tataaattac ctgatgactt caccggctgt 1620
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tatcgtttgt ttcgcaagag taacttgaag cctttcgaac gtgacatttc gactgagatc 1740
tatcaggcag gttccacacc atgcaatggt gtagagggct tcaactgtta tttccctctt 1800
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cgccgccctc agggtctgcc taataatacc gcaagttggt tcactgcgtt gacgcagcac 180
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ccagacgatc aaattgggta ctatcgccgc gcgacccgtc gcattcgtgg cggtgacggt 300
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ggacttcctt acggtgcaaa caaagacggt atcatctggg ttgcaactga gggtgcctta 420
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caattacccc aagggacaac tctgccgaag ggattttatg ccgaaggatc acgtggaggg 540
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caacagcagg gacagaccgt cacgaaaaag tccgctgccg aggcaagcaa gaaaccgcgc 780
caaaaacgca ctgcaacaaa ggcatacaat gtgacccagg catttggtcg ccgtggccct 840
gaacagaccc aaggcaattt tggtgaccaa gaactgattc gtcagggtac agactataaa 900
cactggcctc aaatcgcaca attcgcacct tccgctagcg ctttttttgg catgagtcgt 960
atcggtatgg aggttacccc gtctggcaca tggttgacat atactggcgc tattaaattg 1020
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caagctctgc cgcaacgcca aaagaaacaa caaactgtaa ctctgctgcc cgcggctgac 1200
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gttgtcgtcc tgagcttcga acttcttcac gccccagcca cagtttgcgg gcccaaaaag 600
tctcaccacc accaccatca c 621
Claims (8)
1.一种SARS-CoV-2 N/S1(RBD)重组蛋白，其特征在于，该重组蛋白的核苷酸序列如SEQID NO.1所示，所述重组蛋白由SARS-CoV-2病毒核衣壳(N) 蛋白表达基因片段和SARS-CoV-2病毒表面刺突蛋白S1蛋白表达基因片段通过Linker融合连接；SARS-CoV-2病毒核衣壳(N)蛋白表达基因片段的核苷酸序列如SEQ ID NO.2所示，SARS-CoV-2病毒表面刺突蛋白S1蛋白表达基因片段的核苷酸序列如SEQ ID NO.3所示。
2.根据权利要求1所述的重组蛋白，其特征在于，所述Linker为GS。
3.一种含有SARS-CoV-2 N/S1(RBD)重组蛋白的重组载体，其特征在于，在含有抗卡那霉素的抗性选择基因的表达载体中包含有SEQ ID NO.1核苷酸序列，表达载体为pET30a载体。
4.一种表达如权利要求3所述重组载体的宿主菌。
5.根据权利要求4所述的宿主菌，其特征在于，所述宿主菌为具有卡那霉素抗性的大肠杆菌E.coli BL21(DE3)。
6.根据权利要求3中所述的含有SARS-CoV-2 N/S1(RBD)重组蛋白的重组载体的制备方法，其特征在于，包括如下步骤：
（1）构建SARS-CoV-2 N/S1(RBD)重组蛋白基因片段，并对pET30a载体进行处理：用限制性内切酶Nde I和限制性内切酶Hind III对pET30a载体双酶切；
（2）以SARS-CoV-2 N/S1(RBD)重组蛋白基因片段为模板进行PCR扩增，回收PCR产物,克隆至pET30a载体中，用限制性内切酶Nde I和限制性内切酶Hind III双酶切后，将基因片段从pUC57载体中切下来，再与处理后的pET30a载体连接，制得重组载体。
7.SARS-CoV-2 N/S1(RBD)重组蛋白的表达方法，其特征在于，包括如下步骤：
（1）按照权利要求6所述的重组载体的制备方法制备含有SARS-CoV-2 N/S1(RBD)重组蛋白的重组载体；
（2）将含有SARS-CoV-2 N/S1(RBD)重组蛋白的重组载体转化至大肠杆菌E.coli BL21(DE3)，诱导表达SARS-CoV-2 N/S1(RBD)重组蛋白，表达时的诱导温度在15℃，诱导的IPTG的浓度为0.5mM。
8.权利要求1-2任一项所述的重组蛋白在制备用于检测和/或预防和/或筛选治疗SARS-CoV-2病毒感染的药物中的应用。
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| KR20230019166A (ko) | 2020-06-03 | 2023-02-07 | 리제너론 파아마슈티컬스, 인크. | 항-SARS-CoV-2 스파이크 당단백질 항체를 이용한 SARS-CoV-2 감염 및 COVID-19 치료 또는 예방 방법 |
| US20220056111A1 (en) * | 2020-08-11 | 2022-02-24 | Yurogen Biosystems Llc | MONOCLONAL ANTIBODY AGAINST SPIKE S1 PROTEIN OF SARS-CoV-2 AND USE THEREOFOF |
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| WO2004110483A1 (en) * | 2003-05-31 | 2004-12-23 | Yiyou Chen | Method and composition of a novel vaccine design for the prevention and treatment of sars |
| CA2532196A1 (en) * | 2003-06-18 | 2005-01-20 | Chinese National Human Genome Center At Shanghai | Characterization of the earliest stages of the severe acute respiratory syndrome (sars) virus and uses thereof |
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| GB0911794D0 (en) * | 2009-07-07 | 2009-08-19 | Animal Health Inst | Chimaeric protein |
| CN111024954A (zh) * | 2020-03-09 | 2020-04-17 | 深圳市易瑞生物技术股份有限公司 | 联合检测covid-19抗原和抗体的胶体金免疫层析装置及其使用法 |
| CN111089962B (zh) * | 2020-03-25 | 2020-07-17 | 中山生物工程有限公司 | 联合检测新型冠状病毒IgM/IgG抗体的胶体金试剂盒及制备方法 |
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