CN114773487B - 一种流感病毒和新型冠状病毒融合重组蛋白疫苗免疫原及其制备方法 - Google Patents

一种流感病毒和新型冠状病毒融合重组蛋白疫苗免疫原及其制备方法 Download PDF

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CN114773487B
CN114773487B CN202210606093.7A CN202210606093A CN114773487B CN 114773487 B CN114773487 B CN 114773487B CN 202210606093 A CN202210606093 A CN 202210606093A CN 114773487 B CN114773487 B CN 114773487B
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邓磊
刘德建
衣大龙
刘翠翠
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Abstract

本发明属于生物技术领域，具体涉及一种流感病毒和新型冠状病毒融合重组蛋白疫苗免疫原及其制备方法。所述疫苗为通过在流感病毒血凝素蛋白引入血凝素单体间二硫键半胱氨酸突变，并与新型冠状病毒蛋白RBD结构域进行融合表达获得的重组蛋白，该HA‑RBD融合蛋白分别与HA和RBD特异性广谱中和抗体具有较好的特异性结合能力，具备成为可以同时预防流感病毒和新型冠病毒的潜在免疫原。

Description

一种流感病毒和新型冠状病毒融合重组蛋白疫苗免疫原及其 制备方法
技术领域
本发明属于生物技术领域，具体涉及一种流感病毒和新型冠状病毒融合重组蛋白疫苗免疫原及其制备方法。
背景技术
接种疫苗仍是预防流感和新冠病毒感染的最有效手段。甲型和乙型流感病毒血凝素HA以三聚体的形式展示在病毒粒子表面，是流感病毒丰度最高的表面糖蛋白，通过与宿主呼吸道上皮细胞糖蛋白或糖脂上的受体末端的唾液酸结合介导病毒进入细胞，并介导内体中病毒与宿主细胞的膜融合，是适应性免疫反应的主要靶点和流感疫苗设计的热点。
SARS-CoV-2通过其表面刺突蛋白S的受体结合域(RBD)与细胞受体血管紧张素转换酶2(ACE2)结合进入靶细胞引发感染。受生物学功能限制，RBD负责结合宿主受体，是产生中和性抗体的主要靶点，同时也是设计冠状病毒疫苗的重要靶标。
流感病毒和新型冠状病毒都在不断进行突变，在未来新型冠状病毒的预防可能会像流感一样，需要定期接种疫苗提供免疫保护作用，如何实现两种病毒的同时接种，并能产生中和性抗体为当下研究的热点。
发明内容
针对现有技术存在的问题，本发明基于分析流感病毒HA和SARS-CoV-2S蛋白RBD的晶体结构，合理设计构建可溶性HA-RBD融合重组蛋白抗原。通过R327Q突变稳定HA的融合前构象；基于已解析的HA晶体结构，通过结构生物学分析设计引入HA单体间的二硫键增加其三聚体稳定性，并分别与SARS-CoV-2S蛋白RBD结构域融合表达，分别通过变性还原和变性非还原条件的SDS-PAGE考马斯亮蓝染色和WesternBlot分析HA-RBD融合蛋白的表达和三聚体比例，通过ELISA分析HA-RBD融合蛋白分别与HA和RBD特异性广谱中和抗体的结合能力评价其抗原性。
为实现上述发明目的，本发明提供了一种流感病毒和新型冠状病毒融合重组蛋白疫苗免疫原，所述疫苗免疫原为通过在流感病毒血凝素蛋白引入血凝素单体间二硫键半胱氨酸突变，并与新型冠状病毒蛋白RBD结构域进行融合表达获得的重组蛋白。
进一步的，所述流感病毒包括但不限于毒株A/Washington/5/2011(H1N1)、毒株A/New Caledonia/20/1999(H1N1)和与所述毒株A/Washington/5/2011(H1N1)、毒株A/NewCaledonia/20/1999(H1N1)的氨基酸序列具有89％以上同源性的毒株。
进一步的，所述毒株A/Washington/5/2011(H1N1)的突变位点L20C-K47C。
所述毒株A/New Caledonia/20/1999(H1N1)的突变位点L24C-G47C。
进一步的，所述流感病毒和新型冠状病毒融合重组亚单位疫苗的氨基酸序列如SEQ ID NO:2和SEQ ID NO:4所示。
基于同一发明构思的，本发明实施例还提供了一种流感病毒和新型冠状病毒融合重组蛋白疫苗，所述疫苗包括上述流感病毒和新型冠状病毒融合重组蛋白疫苗免疫原；
所述疫苗还包括疫苗载体，所述疫苗载体包括：DNA载体、mRNA载体、水凝胶载体、脱溶剂纳米颗粒载体、蛋白质自组装纳米颗粒载体、腺病毒载体、慢病毒载体、植物病毒载体、减毒人感染病毒载体、人感染复制缺陷型病毒载体，以及破伤风类毒素、白喉类毒素、白喉毒素的无毒变异体、B群脑膜炎球菌外膜蛋白复合体、白介素、病原相关分子模式、损伤相关分子模式融合蛋白载体。
基于同一发明构思的，本发明实施例还提供了一种流感病毒和新型冠状病毒融合重组亚单位疫苗的制备方法，所述制备方法具体包括以下步骤：
将甲型流感病毒HA的信号肽序列进行置换成昆虫细胞蜂毒素信号肽序列，并在HA胞外基因序列的羧基端添加切割位点和亲和纯化标签序列，依据昆虫细胞宿主优化密码子，合成在pFastBac1载体中，获得带有甲型流感病毒HA序列的重组质粒；
在所述带有甲型流感病毒HA序列的重组质粒的HA胞外域基因序列引入单体间半胱氨酸突变获得HA突变体的pFastBac1重组质粒；
将SARS-CoV-2W-1病毒株的S蛋白昆虫细胞表达质粒与所述HA突变体的pFastBac1重组质粒通过Overlap PCR的方法制备融合的昆虫细胞重组表达质粒；
利用bac-to-bac昆虫杆状病毒表达系统表达所述融合的昆虫细胞重组表达质粒并纯化获得HA-RBD重组蛋白。
进一步的，所述切割位点包括：Factor Xa切割位点、凝血酶裂解位点、HRV 3C蛋白酶切位点、TEV蛋白酶切位点、肠激酶切位点、SUMO蛋白酶切位点；
所述标签序列包括：6×His标签、HA标签、FLAG标签、谷胱甘肽巯基转移酶标签、麦芽糖结合蛋白标签、NusA标签、SUMO标签、Strep-tag标签。
进一步的，所述利用bac-to-bac昆虫杆状病毒表达系统表达所述融合的昆虫细胞重组表达质粒并纯化获得HA-RBD重组蛋白过程具体包括：
将融合的昆虫细胞重组表达质粒转染sf9昆虫细胞，进行培养获得生长状态良好的细胞后进行扩大培养，离心收集上清液；
将所述上清液进行过滤后，并采用亲和树脂层析法进行纯化并进一步洗脱置换，获得HA-RBD重组蛋白。
有益效果：
本发明依据蛋白质结构信息和辅助软件分析结果，通过结构生物学分析在HA单体间引入二硫键，增加其三聚体稳定性，再与SARS-CoV-2S蛋白RBD结构域融合表达，获得可溶性HA-RBD融合重组蛋白抗原，且该HA-RBD融合蛋白分别与HA和RBD特异性广谱中和抗体具有较好的特异性结合能力，具备成为可以同时预防流感病毒和新型冠病毒的潜在免疫原。
附图说明
图1为本发明实施例提供的HA-RBD重组蛋白构建的基因克隆示意图；
图2为本发明实施例提供的SDS-PAGE考马斯亮蓝染色和Western blot检测HA-RBD重组蛋白纯化的分析图；
图3为本发明实施例提供的ELISA检测HA-RBD重组蛋白分别与CR9114和FISW84等识别HA柄部表位广谱中和单克隆抗体的结合能力图；
图4为本发明实施例提供的ELISA检测HA-RBD重组蛋白分别与S309、CR3022、P2B-2F6和B38等识别SARS-CoV-2S蛋白RBD表位广谱中和单克隆抗体的结合能力图。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合具体实施例进行详细描述，但本发明的保护范围并不限于以下具体实施例。
除非另有定义，下文中所使用的所有专业术语与本领域技术人员通常理解含义相同。本文中所使用的专业术语只是为了描述具体实施例的目的，并不是旨在限制本发明的保护范围。
除非另有特别说明，本发明中用到的各种原材料、试剂、仪器和设备等均可通过市场购买得到或者可通过现有方法制备得到。
在本发明实施例中，SARS-CoV-2W-1病毒株的S蛋白昆虫细胞表达质粒由湖南大学葛行义教授馈赠，昆虫细胞sf9购买于ATCC。特异性结合SARS-CoV-2S蛋白RBD的广谱中和抗体S309、CR3022、P2B-2F6和B38等由中山大学陈耀庆教授馈赠。
实施例1
构建HA-RBD昆虫细胞重组表达质粒
以A/Washington/2011(H1N1)和A/New Caledonia/1999(H1N1)病毒为例，将A/Washington/2011(H1N1)和A/New Caledonia/1999(H1N1)病毒HA的信号肽序列置换为昆虫细胞蜂毒素信号肽序列，羧基端添加FactorXa切割位点和6×His标签序列，按照昆虫细胞密码子优化后合成在pFastBac 1载体中，获得pFastBac1-WA11和pFastBac1-NC99胞外域质粒。以合成的pFastBac1-WA11胞外域质粒突变R327Q，接着引入1对单体间半胱氨酸突变组合，包括点突变L20C-K47C获得WA11 Mut；以合成的pFastBac1-NC99胞外域质粒突变R327Q，接着引入1对单体间半胱氨酸突变组合，包括L24C-G47C获得NC99 Mut。上述突变均按照Site-Directed Mutagenesis Kit(NEB,E0554S)说明书操作。将pFastBac1-WA11、pFastBac1-NC99、WA11 Mut、NC99 Mut分别与SARS-CoV-2W-1病毒株的S蛋白昆虫细胞表达质粒通过Overlap PCR的方法【Hilgarth,R.S.,and Lanigan,T.M.(2020).Optimizationofoverlap extension PCR for efficient transgene construction.MethodsX 7,100759】构建多种HA基因片段与RBD基因片段融合的昆虫细胞重组表达质粒pFastBac 1-HA-RBD质粒。
实施例2
HA-RBD重组蛋白的表达与纯化
利用II转染试剂(Gibco)将多种pFastBac 1-HA-RBD质粒分别转染sf9昆虫细胞，27℃条件下，昆虫细胞在SF-SFM昆虫细胞无血清培养基中静置培养72小时；培养上清液接种生长状态良好的sf9细胞，27℃，110rpm/min条件下，sf9细胞在SF-SFM无血清培养基中震荡培养72h后离心收集培养上清液；继续接种sf9细胞，在27℃，110rpm/min条件下扩大培养，72小时后离心收集培养上清液，经0.45μm滤器过滤后，利用6×His标签亲和层析树脂纯化HA-RBD重组蛋白，利用30kDa超滤管(Millipore)蛋白质洗脱液置换为PBS，离心浓缩后加入20％甘油，-80℃保存。获得多种HA-RBD重组蛋白，具体为：WA11-RBD、WA11Mut-RBD、NC99-RBD、NC99 Mut-RBD，氨基酸序列如SEQ ID NO:1、2、3、4。
另外SARS-CoV-2S蛋白RBD氨基酸序列为：MKFLVNVALVFMVVYISYIYARVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNIEGRHHHHHH。
A/Washington/5/2011(H1N1)野生型HA的氨基酸序列如序列表为：MKFLVNVALVFMVVYISYIYADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKGFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTTADQQSLYQNADTYVFVGTSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTRIYQIEGRHHHHHH。
A/New Caledonia/20/1999(H1N1)野生型H1的氨基酸序列如序列表为：MKFLVNVALVFMVVYISYIYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCLLKGIAPLQLGNCSVAGWILGNPECELLISKESWSYIVETPNPENGTCYPGYFADYEELREQLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKSYVNNKEKEVLVLWGVHHPPNIGNQRALYHTENAYVSVVSSHYSRRFTPEIAKRPKVRDQEGRINYYWTLLEPGDTIIFEANGNLIAPWYAFALSRGFGSGIITSNAPMDECDAKCQTPQGAINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAVGKEFNKLERRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNNECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEGRHHHHHH。
实施例3
SDS-PAGE和WesternBlot分析
在SDS、DTT变性还原和SDS变性非还原条件下对HA-RBD重组蛋白进行10％聚丙烯酰胺SDS-PAGE电泳分析，将蛋白样品转印到PVDF膜上，10％脱脂牛奶(TBS中含0.05％的吐温-20，TBST)室温封闭2小时，His标签鼠源单克隆抗体室温孵育2h，TBST洗涤3遍后，室温孵育羊抗鼠二抗1h，TBST洗涤3遍后，使用ECL化学发光液(Bio-Rad)进行显影曝光。
实施例4
ELISA检测HA-RBD融合蛋白与广谱中和抗体的结合能力
为了检测HA-RBD融合蛋白与特异性识别HA柄部的广谱中和抗体CR9114和FISW84以及特异性识别SARS-CoV-2S蛋白RBD的广谱中和抗体S309、CR3022、P2B-2F6和B38的结合能力，将100ng/孔的可溶性HA-RBD、HA、RBD重组蛋白和BSA包被到ELISA反应板中，4℃过夜；PBST(PBS含0.05％吐温-20)洗涤孔板后，用封闭液(含5％脱脂牛奶PBS)室温封闭1.5h,用PBST洗涤后，加入封闭液倍比稀释的HA和RBD广谱中和抗体(初始浓度5μg/ml,5倍倍比稀释)，室温孵育1h；用PBST洗涤3遍后，羊抗人抗体1：5000稀释后室温孵育1h,用PBST洗3遍后，避光加入TMB单组份显色液，37℃孵育10min后加入等体积的1M H2SO4终止反应。用酶标仪测定OD450 nm处的吸光值。
结果分析
如图1所示，其为HA-RBD重组抗原基因构建示意图，昆虫细胞表达载体pFastBac 1的PH启动子之后依次串联表达蜂毒素信号肽基因、HA胞外域基因片段、SARS-CoV-2S蛋白RBD基因片段、FactorXa切割位点和6×His标签序列。
SDS-PAGE考马斯亮蓝染色和WesternBlot鉴定HA-RBD重组抗原
如图2所示，NC99-RBD、NC99 Mut-RBD、WA11-RBD和WA11Mut-RBD融合重组蛋白均表达成功，SDS变性和DTT还原条件下，各个HA-RBD融合重组蛋白的单体条带纯度均较高；SDS变性非还原条件下，各个HA-RBD融合重组蛋白均检测到一定比例的三聚体条带，相较于NC99-RBD和WA11-RBD融合重组蛋白，NC99Mut-RBD和WA11 Mut-RBD融合重组蛋白中三聚体形式蛋白所占比例均有一定程度的提高。
HA-RBD融合蛋白与HA和RBD广谱中和抗体具有较好的特异性结合能力
WA11-RBD、WA11 Mut-RBD、NC99-RBD和NC99 Mut-RBD融合重组蛋白与HA柄部特异性广谱中和抗体CR9114和FISW84(图3)，以及SARS-CoV-2S蛋白RBD特异性广谱中和抗体S309、CR3022、P2B-2F6和B38等(图4)均能较好的特异性结合,表明上述HA-RBD融合重组蛋白中HA和RBD的抗原性均保持完好，提示上述HA-RBD融合重组蛋白具备成为可以同时预防流感病毒和新型冠病毒的潜在免疫原。
在其他氨基酸突变、添加或减少等操作的情况下，如果未显著提升融合蛋白的抗原性，仍受本专利保护。
以上所述实施例，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的技术范围内，根据本发明的技术方案及其构思加以等同替换或改变，都应涵盖在本发明的保护范围内。
序列表
<110> 湖南大学
<120> 一种流感病毒和新型冠状病毒融合重组蛋白疫苗免疫原及其制备方法
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Val Leu Val Leu Trp Gly Val His His Pro Pro Asn Ile Gly Asn Gln
195 200 205
Arg Ala Leu Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser
210 215 220
His Tyr Ser Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val
225 230 235 240
Arg Asp Gln Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro
245 250 255
Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp
260 265 270
Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser
275 280 285
Asn Ala Pro Met Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly
290 295 300
Ala Ile Asn Ser Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile
305 310 315 320
Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr
325 330 335
Gly Leu Arg Asn Ile Pro Ser Ile Gln Ser Gln Gly Leu Phe Gly Ala
340 345 350
Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp
355 360 365
Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp
370 375 380
Gln Lys Ser Thr Gln Asn Ala Ile Asn Cys Ile Thr Asn Lys Val Asn
385 390 395 400
Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu
405 410 415
Phe Asn Lys Leu Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp
420 425 430
Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
435 440 445
Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn
450 455 460
Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile
465 470 475 480
Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met
485 490 495
Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
500 505 510
Ser Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met
515 520 525
Gly Val Tyr Gln Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro
530 535 540
Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg
545 550 555 560
Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val
565 570 575
Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys
580 585 590
Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
595 600 605
Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile
610 615 620
Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
625 630 635 640
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp
645 650 655
Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys
660 665 670
Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln
675 680 685
Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe
690 695 700
Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln
705 710 715 720
Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala
725 730 735
Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys
740 745 750
Val Asn Phe Asn Ile Glu Gly Arg His His His His His His
755 760 765

Claims (3)

1.一种流感病毒和新型冠状病毒融合重组蛋白疫苗免疫原，其特征在于，所述疫苗免疫原为通过在流感病毒血凝素蛋白引入血凝素单体间二硫键半胱氨酸突变，并与新型冠状病毒蛋白RBD结构域进行融合表达获得的重组蛋白；其中，所述流感病毒包括毒株A/Washington/5/2011（H1N1）、毒株A/New Caledonia/20/1999（H1N1）；所述毒株A/Washington/5/2011（H1N1）的突变位点L20C-K47C；所述毒株A/New Caledonia/20/1999（H1N1）的突变位点L24C-G47C；所述流感病毒和新型冠状病毒融合表达的重组蛋白的氨基酸序列如SEQ ID NO:2和SEQ ID NO:4所示。
2.一种流感病毒和新型冠状病毒融合重组蛋白疫苗，其特征在于，所述疫苗包括权利要求1所述的流感病毒和新型冠状病毒融合重组蛋白疫苗免疫原；
所述疫苗还包括疫苗载体，所述疫苗载体包括：DNA载体、mRNA载体、水凝胶载体、脱溶剂纳米颗粒载体、蛋白质自组装纳米颗粒载体、腺病毒载体、慢病毒载体、植物病毒载体、减毒人感染病毒载体、人感染复制缺陷型病毒载体，以及破伤风类毒素、白喉毒素的无毒变异体、B群脑膜炎球菌外膜蛋白复合体、白介素、病原相关分子模式、损伤相关分子模式融合蛋白载体。
3.根据权利要求2所述的一种流感病毒和新型冠状病毒融合重组蛋白疫苗，其特征在于，所述疫苗载体包括白喉类毒素。
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