CN111499765B - 一种冠状病毒融合蛋白及其制备方法与应用 - Google Patents
一种冠状病毒融合蛋白及其制备方法与应用 Download PDFInfo
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Abstract
本发明属于抗原制备工艺的技术领域,具体涉及一种冠状病毒融合蛋白及其制备方法与应用。本发明提供的该冠状病毒融合蛋白通过将2019‑nCoV S蛋白RBD结构域核苷酸序列和2019‑nCoV N蛋白核苷酸序列全基因合成后构建至昆虫细胞表达载体中,制备得到含有2019‑nCoV N蛋白和2019‑nCoV S蛋白RBD结构域序列的重组质粒,该重组质粒转化大肠杆菌后获得含有上述融合蛋白基因序列的重组杆粒,重组杆粒转染宿主细胞,进而完成2019‑nCoV N蛋白和2019‑nCoV S蛋白RBD结构域的融合表达。该种融合蛋白的表达过程简单,同时将融合蛋白用于2019‑nCoV抗体检测试剂盒时不仅显著地提高了检测试剂盒的灵敏度,而且也提高了特异性,检出率得到了高效地提升,整体使用性能更为优化。
Description
技术领域
本发明属于抗原制备工艺的技术领域,具体涉及一种冠状病毒融合蛋白及 其制备方法与应用。
背景技术
新型冠状病毒肺炎(CoVID-19)是一种新型冠状病毒,WHO命名为 2019-nCoV。冠状病毒是一个大型病毒家族,可引起感冒以及中东呼吸综合征 (MERS)和严重急性呼吸综合征(SARS)等较严重疾病,而2019-nCoV是以 前从未在人体中发现的冠状病毒新毒株。2019-nCoV属于β属的冠状病毒,有 包膜,颗粒呈圆形或椭圆形,常为多形性,直径60~140nm,其基因特征与 SARSr-CoV和MERSr-CoV有明显区别。研究显示与蝙蝠SARS样冠状病毒 (bat-SL-CoVZC45)同源性达85%以上。
2019-nCoV是一类单股正链RNA冠状病毒,其主要结构蛋白有刺突蛋白 (spikeprotein,S蛋白),核衣壳蛋白(nucleocapsid,N蛋白),膜蛋白(membrane protein,M蛋白),包膜蛋白(envelope protein,E蛋白)。病毒要进入细胞,细 胞上就必须有它对应的受体,S蛋白可与宿主细胞的病毒受体结合,是决定病 毒入侵易感细胞的关键蛋白。该病毒传染性强,目前在全球主要人口集中区均 有发现,已成全球流行趋势。随着最新版新型冠状病毒诊疗方案(试行第七版) 的施行,血清新型冠状病毒特异性抗体IgG和IgM的检测技术也列入确诊手段。 结合磁微粒化学发光免疫检测技术能高效,快速,安全的检测新冠病毒特异性 抗体,为早期疾病的筛查,疾病的确诊,预后的跟踪等提供依据,新冠病毒特 异性抗体IgM/G检测试剂盒的研发将有利于疫情的防控。
目前新冠病毒的检测方法主要是病毒核酸RNA的荧光定量PCR检测技术, 疫情发生以来普通实时定量荧光PCR核酸检测发挥了重要作,但是该检测手段 同时也存在一些问题包括检测阳性率低,约30-50%,假阴性,耗时较长,检测 结果具有很大的不确定性;样本采集相对困难,有气溶胶污染风险,对于医护 人员是严重的威胁。因此,高检测准确度、灵敏度和检出率的新冠病毒特异性 抗体IgM/G磁微粒化学发光检测试剂盒的开发不仅应能为国家和世界的新冠疫 情防控助力,具有重要的社会效益;同时试剂盒的产业化也能促进现代生物技 术行业的进步与显著地发展。
因此,作为新冠病毒特异性抗体IgM/G磁微粒化学发光检测试剂盒的主要 原材料之一的病毒重组抗原,将占据免疫检测试剂盒开发的大量成本。该种融 合蛋白用于2019-nCoV抗体检测试剂盒时不仅显著地提高了检测试剂盒的灵敏 度,而且也提高了特异性,检出率得到了高效地提升,整体使用性能更为优化。
发明内容
为了解决现有技术存在的上述问题,本发明目的在于提供一种冠状病毒融 合蛋白及其制备方法与应用。
本发明所采用的技术方案为:一种冠状病毒融合蛋白,该冠状病毒融合蛋 白包括2019-nCoV S蛋白区段和2019-nCoV N蛋白区段,2019-nCoV S蛋白区 段具有如SEQ IDNO.1所示的核苷酸序列表达,2019-nCoV N蛋白区段具有如 SEQ ID NO.2所示的核苷酸序列表达。
优选地,冠状病毒融合蛋白具有如SEQ ID NO.3所示的氨基酸序列。
一种冠状病毒融合蛋白的制备方法,该制备方法包括:
S1:重组包含2019-nCoV S蛋白区段的核苷酸序列和2019-nCoV N蛋白区 段核苷酸序列的全基因合成序列,将上述全基因合成序列构建至表达载体,获 得重组质粒;
S2:将步骤S1中的重组质粒转化至宿主细胞,获得重组杆粒;
S3:将步骤S2中的重组杆粒转染细胞,进行表达;
S4:将步骤S3中表达得到的细胞培养物进行筛选、纯化,获得融合蛋白。
全基因合成,是一种依照某一蛋白的氨基酸序列,或基因序列,设计全长 引物,利用OVERLAP方法形成模板DNA,再利用PCR扩增的方法得到双链DNA, 然后将PCR产物转化克隆至克隆载体或者表达载体中。该种合成全基因目前是 准确率最高,速度最快的方法,同时可以依据密码子在不同宿主细胞的偏爱性、 不同的实验需求以及对于引物设计时涉及的顺序问题等,来设计基因序列,进 而提高表达水平的一种方法。
优选地,步骤S1中重组包含2019-nCoV S蛋白区段的核苷酸序列和 2019-nCoV N蛋白区段核苷酸序列的全基因合成序列时,采用柔性连接子将 2019-nCoV S蛋白区段的核苷酸序列和2019-nCoV N蛋白区段的核苷酸序列连 接。
优选地,柔性连接子包括Linker连接子。
优选地,Linker连接子具有如SEQ ID NO.4所示的核苷酸序列表达。
优选地,2019-nCoV S蛋白区段的核苷酸序列包括2019-nCoV S蛋白RBD 结构域的核苷酸序列。
结构域,是生物大分子中具有特异结构和独立功能的区域,特指蛋白质中 这样的区域。在球形蛋白中,结构域具有自己特定的三级结构,其功能不依赖 于蛋白质分子中的其余部分,但是同一蛋白质中不同结构域间常可通过不具二 级结构的短序列连接起来。蛋白质分子中不同的结构域常由基因的不同外显子 所编码。2019-nCoV S蛋白RBD结构域是2019-nCoV S蛋白的306-527氨基酸 序列。
优选地,步骤S1中的表达载体包括昆虫细胞表达载体和/或杆状病毒表达 载体。
昆虫细胞表达载体和/或杆状病毒表达载体,用于表达蛋白质的多角体强启 动子,简化克隆的三个读码框,轻松纯化重组融合蛋白的N-端6xHis标签, 在蛋白质纯化后去除组氨酸标签的TEV蛋白酶切割位点。
优选地,步骤S2中的宿主细胞包括大肠杆菌宿主和/或动物性细胞宿主。
一种冠状病毒融合蛋白的应用,利用上述冠状病毒融合蛋白在2019-nCoV 抗体检测试剂盒、疫苗、抗体和诊断抗原应用中的一种或多种。
本发明的有益效果为:
本发明提供了一种冠状病毒融合蛋白,其是通过将2019-nCoV S蛋白RBD 结构域核苷酸序列和2019-nCoV N蛋白核苷酸序列全基因合成后构建至昆虫细 胞表达载体中,制备得到含有2019-nCoV N蛋白和2019-nCoV S蛋白RBD结构 域序列的重组质粒,该重组质粒转化大肠杆菌后获得含有上述融合蛋白基因序 列的重组杆粒,重组杆粒转染宿主细胞,进而完成2019-nCoV N蛋白和2019-nCoV S蛋白RBD结构域的融合表达。该种融合蛋白的表达过程简单,同时将融合蛋 白用于2019-nCoV抗体检测试剂盒时不仅显著地提高了检测试剂盒的灵敏度, 而且也提高了特异性,检出率得到了高效地提升,整体使用性能更为优化。
附图说明
图1是本发明实施例2中冠状病毒融合蛋白表达载体的多克隆位点示意图;
图2是本发明实施例4中冠状病毒融合蛋白通过SDS-PAGE电泳的电泳条带 图。
具体实施方式
下面结合具体实施例对本发明做进一步阐释。本领域技术人员将会理解, 下列所描述的实施例是本发明一部分实施例,而不是全部的实施例,仅用于说 明本发明,而不应视为限制本发明的范围。基于本发明中的实施例,本领域普 通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本 发明保护的范围。实施例中未注明具体条件者,按照常规条件进行。所用试剂 均为可以通过市售购买获得的常规产品。
实施例1
2019-nCoV S蛋白和2019-nCoV N蛋白的融合蛋白基因的全序列合成
人工构建融合蛋白基因序列,其包含N端真核KOZAK序列、蜂毒信号肽、 2019-nCoVN蛋白基因(GeneID:43740575)、Linker序列(GGGGS)、2019-nCoV 的RBD结构域基因(S蛋白(GeneID:43740568)的306-527氨基酸序列),人 工融合序列前后分别包含BamH I和EcoR I酶切位点,最后由基因合成公司进 行全基因合成,并构建至常规质粒载体中。
实施例2
实施例1中融合蛋白基因的质粒构建
将获得的蛋白基因S-N的PCR片段与经过限制性内切酶Nco I和EcoR I双 酶切的载体pFastBacTMHT A进行连接。连接产物转化于感受态大肠杆菌DH5α 中,体积不超过感受态细胞的10%,轻轻旋转几次混匀内容物,冰浴30分钟, 将管放入42℃水浴,定时60秒热休克,快速将管转移到冰浴120秒,使细胞 冷却,每管加入400μL LB培养基,37℃缓摇60分钟,使细菌复苏并表达质粒 编码的抗生素抗性标记基因,低速离心2分钟,去上清,留约100μL培养基在 离心管内,重悬菌体,用玻璃铺菌器将菌液在含Amp抗生素的LB琼脂板上铺匀。将平板倒置于37℃恒温培养箱培养12小时后,挑取阳性克隆提取质粒进行双 酶切验证。
实施例3
重组杆粒的构建及融合蛋白的表达
按照Bac-to-Bac说明书方法(Invitrogen),将正确重组质粒转化大肠杆 菌DH10Bac感受态细胞,该转化步骤类似DH5α的化学转化,但复苏时间延长 为2-4h,摇床转速提高至200rpm,用玻璃铺菌器将30μL菌液在含有 Kan+Gent+Tet的LB琼脂板上铺匀,将平板倒置培养于37℃恒温培养箱中。约 30-48小时后可出现蓝白斑菌落,挑取阳性白斑单菌落接入5mL Kan+Gent+Tet 的LB培养液中,37℃培养12-16h,取菌液进行PCR鉴定,结果显示杆粒重组 正确。将重组杆粒经转染试剂转染昆虫细胞sf9,3-5天后离心收取细胞上清液, -80℃保存作为第一代杆状病毒。用第一代杆状病毒感染昆虫细胞sf9约48-96 小时后,收获第二代病毒,用于融合蛋白的表达。使用第二代病毒1:100体积 比感染密度为2x106/mL的昆虫细胞sf9,在SIM SF培养基继续培养72小时后, 8000rpm离心10min收集细胞培养上清。样品-20℃保存待用,取少量上清煮沸 至少3min,用SDS-PAGE检测重组蛋白表达。
实施例4
融合2019-nCoV S蛋白核苷酸序列和2019-nCoV N蛋白核苷酸序列中的 S-N基因序列的纯化
200mL预冷的裂解液(10mM HEPES pH7.5,10mM MgCl2,20mM KCl) 重悬1L细胞培养物离心沉淀,用匀浆器在冰上进行匀浆,匀浆后用超速离心机 离心45分钟,去掉上清。重复洗涤3次,再用高盐溶液重复洗涤三次,提取物 用含甘油的裂解液溶解并液氮速冻,储存在-80℃冰箱。冰上融化解冻提取物, 加入茶碱和碘乙酰胺分别至终浓度为4mM和2mg/mL,冰上放置30分钟后按 照每升提取物加入100mL溶膜缓冲液比例加入溶膜缓冲液,并继续在冰上放置 3小时溶膜,用超速离心机在160000g离心力下离心40分钟。弃沉淀,上清加 至平衡好的Ni-NTA RESIN孵育过夜,次日,弃上清,加入适量平衡缓冲液重 悬填料,将填料转移到自流柱里。冲洗缓冲液1(25mM Tris-Hcl pH8.0;500mM NaCl)洗10个柱体积。冲洗缓冲液2(25mM Tris-Hcl pH8.0;500mM NaCl; 20mM咪唑)洗10个柱体积。冲洗缓冲液3(25mMTris-Hcl pH8.0;500mM NaCl; 250mM咪唑)洗5个柱体积。洗脱液洗脱目的蛋白,纯化得到的目的蛋白保存 在-80℃。SDS-PAGE电泳检测表达蛋白纯度及浓度。
如图1所示,多克隆位点,是载体上含有的一个人工合成的DNA片段,其 上含有多个单一酶切位点,是外源DNA的插入部位,具备条件:是载体中的一 段碱基序列,由数个酶切位点组成,这些位点在载体上都是单一位点。
如图2所示,该示意图是通过利用SDS-PAGE电泳试验,发现细胞上清在 70kDa位置时有明显可溶性蛋白条带,该条带与预测大小融合蛋白的71.8kDa 相一致,说明2019-nCoVS蛋白的RBD结构域和2019-nCoV N蛋白的融合蛋 白在昆虫细胞内成功表达。其中,条带的做左侧为电泳条带的Marker,中部条 带为上清液电泳条带,最右侧为洗脱液电泳条带。
本发明提供了一种冠状病毒融合蛋白,其是通过将2019-nCoV S蛋白RBD 结构域核苷酸序列和2019-nCoV N蛋白核苷酸序列全基因合成后构建至昆虫细 胞表达载体中,制备得到含有2019-nCoV N蛋白和2019-nCoV S蛋白RBD结构 域序列的重组质粒,该重组质粒转化大肠杆菌后获得含有上述融合蛋白基因序 列的重组杆粒,重组杆粒转染宿主细胞,进而完成2019-nCoV N蛋白和2019-nCoV S蛋白RBD结构域的融合表达。该种融合蛋白的表达过程简单,同时将融合蛋 白用于2019-nCoV抗体检测试剂盒时不仅显著地提高了检测试剂盒的灵敏度, 而且也提高了特异性,检出率得到了高效地提升,整体使用性能更为优化。
尽管已用具体实施例来说明和描述了本发明,然而应意识到,本发明不局 限于上述可选的实施方式,任何人在本发明的启示下都可得出其他各种形式的 产品。上述具体实施方式不应理解成对本发明的保护范围的限制,本领域的普 通技术人员应当理解,在不背离本发明的精神和范围的情况下,可以对前述各 实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等 同替换,与此同时这些修改或者替换,并不会使相应的技术方案的本质脱离本 发明各实施例技术方案的范围;本发明的保护范围应当以权利要求书中界定的 为准,并且说明书可以用于解释权利要求书。
序列表
<110> 四川携光生物技术有限公司
<120> 一种冠状病毒融合蛋白及其制备方法与应用
<140> 202010269998.0
<141> 2020-04-08
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3822
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(3822)
<223> 2019-nCoV S 蛋白核苷酸序列
<400> 1
atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60
agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120
aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc 180
aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240
aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300
ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt 360
aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420
ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480
tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540
ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600
tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660
tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720
ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780
ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840
gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900
tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960
caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020
gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080
tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140
ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200
gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 1260
tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320
cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 1380
ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440
aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500
aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560
ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620
ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680
cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740
acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800
ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 1860
cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920
aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980
gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040
cctcggcggg cacgtagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100
gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160
agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220
tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280
acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa cacccaagaa 2340
gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400
aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460
ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520
cttggtgata ttgctgctag agacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580
ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640
acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700
caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760
aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc actttcttcc 2820
acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880
acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940
ctttcacgtc ttgacaaagt tgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000
cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060
tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120
gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180
gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240
atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300
cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca 3360
tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420
ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480
tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540
aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600
caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt 3660
atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtat gaccagttgc 3720
tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaatttga tgaagacgac 3780
tctgagccag tgctcaaagg agtcaaatta cattacacat aa 3822
<210> 2
<211> 1260
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(1260)
<223> 2019-nCoV N蛋白核苷酸序列
<400> 2
atgtctgata atggacccca aaatcagcga aatgcacccc gcattacgtt tggtggaccc 60
tcagattcaa ctggcagtaa ccagaatgga gaacgcagtg gggcgcgatc aaaacaacgt 120
cggccccaag gtttacccaa taatactgcg tcttggttca ccgctctcac tcaacatggc 180
aaggaagacc ttaaattccc tcgaggacaa ggcgttccaa ttaacaccaa tagcagtcca 240
gatgaccaaa ttggctacta ccgaagagct accagacgaa ttcgtggtgg tgacggtaaa 300
atgaaagatc tcagtccaag atggtatttc tactacctag gaactgggcc agaagctgga 360
cttccctatg gtgctaacaa agacggcatc atatgggttg caactgaggg agccttgaat 420
acaccaaaag atcacattgg cacccgcaat cctgctaaca atgctgcaat cgtgctacaa 480
cttcctcaag gaacaacatt gccaaaaggc ttctacgcag aagggagcag aggcggcagt 540
caagcctctt ctcgttcctc atcacgtagt cgcaacagtt caagaaattc aactccaggc 600
agcagtaggg gaacttctcc tgctagaatg gctggcaatg gcggtgatgc tgctcttgct 660
ttgctgctgc ttgacagatt gaaccagctt gagagcaaaa tgtctggtaa aggccaacaa 720
caacaaggcc aaactgtcac taagaaatct gctgctgagg cttctaagaa gcctcggcaa 780
aaacgtactg ccactaaagc atacaatgta acacaagctt tcggcagacg tggtccagaa 840
caaacccaag gaaattttgg ggaccaggaa ctaatcagac aaggaactga ttacaaacat 900
tggccgcaaa ttgcacaatt tgcccccagc gcttcagcgt tcttcggaat gtcgcgcatt 960
ggcatggaag tcacaccttc gggaacgtgg ttgacctaca caggtgccat caaattggat 1020
gacaaagatc caaatttcaa agatcaagtc attttgctga ataagcatat tgacgcatac 1080
aaaacattcc caccaacaga gcctaaaaag gacaaaaaga agaaggctga tgaaactcaa 1140
gccttaccgc agagacagaa gaaacagcaa actgtgactc ttcttcctgc tgcagatttg 1200
gatgatttct ccaaacaatt gcaacaatcc atgagcagtg ctgactcaac tcaggcctaa 1260
<210> 3
<211> 649
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr
1 5 10 15
Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg
20 25 30
Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn
35 40 45
Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu
50 55 60
Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro
65 70 75 80
Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly
85 90 95
Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr
100 105 110
Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp
115 120 125
Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp
130 135 140
His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln
145 150 155 160
Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser
165 170 175
Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn
180 185 190
Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala
195 200 205
Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu
210 215 220
Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln
225 230 235 240
Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys
245 250 255
Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln
260 265 270
Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp
275 280 285
Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile
290 295 300
Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile
305 310 315 320
Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala
325 330 335
Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu
340 345 350
Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro
355 360 365
Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln
370 375 380
Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu
385 390 395 400
Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser
405 410 415
Thr Gln Ala Gly Gly Gly Gly Ser Phe Thr Val Glu Lys Gly Ile Tyr
420 425 430
Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe
435 440 445
Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr
450 455 460
Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys
465 470 475 480
Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe
485 490 495
Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr
500 505 510
Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln
515 520 525
Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu
530 535 540
Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu
545 550 555 560
Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg
565 570 575
Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr
580 585 590
Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr
595 600 605
Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr
610 615 620
Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro
625 630 635 640
Ala Thr Val Cys Gly Pro Lys Lys Ser
645
<210> 4
<211> 15
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(15)
<223> linker连接子核苷酸序列
<400> 4
ggaggcggcg gatct 15
<210> 5
<211> 675
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(675)
<223> 2019-nCoV S蛋白RBD结构域的核苷酸序列
<400> 5
ttcactgtag aaaaaggaat ctatcaaact tctaacttta gagtccaacc aacagaatct 60
attgttagat ttcctaatat tacaaacttg tgcccttttg gtgaagtttt taacgccacc 120
agatttgcat ctgtttatgc ttggaacagg aagagaatca gcaactgtgt tgctgattat 180
tctgtcctat ataattccgc atcattttcc acttttaagt gttatggagt gtctcctact 240
aaattaaatg atctctgctt tactaatgtc tatgcagatt catttgtaat tagaggtgat 300
gaagtcagac aaatcgctcc agggcaaact ggaaagattg ctgattataa ttataaatta 360
ccagatgatt ttacaggctg cgttatagct tggaattcta acaatcttga ttctaaggtt 420
ggtggtaatt ataattacct gtatagattg tttaggaagt ctaatctcaa accttttgag 480
agagatattt caactgaaat ctatcaggcc ggtagcacac cttgtaatgg tgttgaaggt 540
tttaattgtt actttccttt acaatcatat ggtttccaac ccactaatgg tgttggttac 600
caaccataca gagtagtagt actttctttt gaacttctac atgcaccagc aactgtttgt 660
ggacctaaaa agtct 675
<210> 6
<211> 225
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln
1 5 10 15
Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro
20 25 30
Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp
35 40 45
Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr
50 55 60
Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr
65 70 75 80
Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val
85 90 95
Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys
100 105 110
Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val
115 120 125
Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr
130 135 140
Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu
145 150 155 160
Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn
165 170 175
Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe
180 185 190
Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu
195 200 205
Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys
210 215 220
Ser
225
Claims (8)
1.一种冠状病毒融合蛋白,其特征在于,所述冠状病毒融合蛋白包括2019-nCoV S蛋白区段和2019-nCoV N 蛋白区段,所述2019-nCoV S蛋白区段如SEQ ID NO.1所示的核苷酸序列表达,2019-nCoV N 蛋白区段如SEQ ID NO.2所示的核苷酸序列表达;
所述冠状病毒融合蛋白如SEQ ID NO.3所示的氨基酸序列。
2.根据权利要求1所述的一种冠状病毒融合蛋白的制备方法,其特征在于,所述制备方法包括:
S1:重组包含2019-nCoV S蛋白区段的核苷酸序列和2019-nCoV N 蛋白区段核苷酸序列的全基因合成序列,将上述全基因合成序列构建至表达载体,获得重组质粒;
S2:将步骤S1中的重组质粒转化至宿主细胞,获得重组杆粒;
S3:将步骤S2中的重组杆粒转染细胞,进行表达;
S4:将步骤S3中表达得到的细胞培养物进行筛选、纯化,获得融合蛋白。
3.根据权利要求2所述的一种冠状病毒融合蛋白的制备方法,其特征在于,所述步骤S1中重组包含2019-nCoV S蛋白区段的核苷酸序列和2019-nCoV N 蛋白区段核苷酸序列的全基因合成序列时,采用柔性连接子将2019-nCoV S蛋白区段的核苷酸序列和2019-nCoV N蛋白区段的核苷酸序列连接。
4.根据权利要求3所述的一种冠状病毒融合蛋白的制备方法,其特征在于,所述柔性连接子如SEQ ID NO.4所示的核苷酸序列表达。
5.根据权利要求2所述的一种冠状病毒融合蛋白的制备方法,其特征在于,所述2019-nCoV S蛋白区段的核苷酸序列包括2019-nCoV S蛋白RBD结构域的核苷酸序列。
6.根据权利要求2所述的一种冠状病毒融合蛋白的制备方法,其特征在于,所述步骤S1中的表达载体包括昆虫细胞表达载体和/或杆状病毒表达载体。
7.根据权利要求2所述的一种冠状病毒融合蛋白的制备方法,其特征在于,所述步骤S2中的宿主细胞包括大肠杆菌宿主和/或动物性细胞宿主。
8.根据权利要求1所述的一种冠状病毒融合蛋白在制备2019-nCoV 抗体检测试剂盒、疫苗、抗体或诊断抗原中的应用。
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