WO2023109979A2 - 一种展示新冠s蛋白的融合蛋白和重组病毒粒子及其应用 - Google Patents

一种展示新冠s蛋白的融合蛋白和重组病毒粒子及其应用 Download PDF

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WO2023109979A2
WO2023109979A2 PCT/CN2023/076443 CN2023076443W WO2023109979A2 WO 2023109979 A2 WO2023109979 A2 WO 2023109979A2 CN 2023076443 W CN2023076443 W CN 2023076443W WO 2023109979 A2 WO2023109979 A2 WO 2023109979A2
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protein
apmv
fusion
recombinant
gene
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陈瑞婷
孙慧敏
毛水花
周孟云
宋家升
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浙江迪福润丝生物科技有限公司
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Definitions

  • the invention belongs to the technical field of biological products, and in particular relates to a fusion protein displaying a new coronavirus S protein, recombinant virus particles and applications thereof.
  • the immunogenicity of the new coronavirus is relatively weak.
  • the S protein of the new coronavirus is a key protein for the virus to bind to the host cell membrane receptor ACE2 and then infect the host cell.
  • S protein is a type I transmembrane protein, and its expression on the cell membrane is related to the amino acid arrangement in the transmembrane region.
  • NDV Newcastle Disease virus
  • SARS-COV-2 new coronavirus
  • transmembrane domain and intracellular domain of the S protein with the transmembrane domain and intracellular domain of the NDV F protein is likely to make the upper membrane of the S protein the same as the upper membrane of the F protein of the NDV virus itself. Form a competitive relationship, interfere with the expression of antigenic proteins on the cell membrane or virus assembly, and affect its immunogenicity.
  • the purpose of the present invention is to provide a fusion protein displaying the S protein of the new coronavirus and the corresponding recombinant virus constructed and its application.
  • the invention provides a fusion protein displaying the S protein of the new coronavirus, comprising the extracellular domain sequence of the S protein of the new coronavirus and the sequence of the transmembrane domain and the intracellular domain of the F protein of the avian Paramyxoviridae virus.
  • the avian Paramyxoviridae virus includes one or more of the following serotypes: APMV-2, APMV-3, APMV-4, APMV-5, APMV-6, APMV-7, APMV-8, APMV -9, APMV-10, APMV-11 and APMV-12.
  • the avian Paramyxoviridae virus includes one or more of the following serotypes: APMV-3, APMV-5, APMV-7 and APMV-8 serotypes.
  • the extracellular domain of the novel coronavirus S protein is derived from the original strain or a mutant strain of the novel coronavirus.
  • the extracellular domain sequence of the novel coronavirus S protein also includes one or more of the following protein coding sequences: S2P, S6P and S2P/GSAS;
  • the S2P is a protein coding sequence formed by performing K986P and V987P mutations on the ectodomain of the new coronavirus S protein;
  • the S2P/GSAS is a protein coding sequence formed by mutating the 682nd to 685th amino acid RRAR into GSAS on the basis of S2P;
  • the S6P is a protein coding sequence formed by carrying out F817P, A892P, A899P and A942P mutations on the basis of S2P.
  • the invention provides a fusion gene encoding the fusion protein.
  • the invention provides a recombinant virus vector comprising the fusion gene.
  • the recombinant virus vector uses Newcastle disease virus vector as the backbone vector.
  • the fusion gene is inserted into the P gene and the M base of the Newcastle disease virus vector in the form of an expression cassette between.
  • the invention provides a recombinant virus particle or vaccine strain prepared from the recombinant virus vector.
  • the present invention provides the application of the fusion protein, the fusion gene, the recombinant virus vector or the recombinant virus particle or vaccine strain in the preparation of a vaccine for the prevention and control of new coronary pneumonia.
  • the invention provides a fusion protein displaying the S protein of the new coronavirus, including the ectodomain sequence of the S protein of the new coronavirus and the transmembrane domain and the intracellular domain coding sequence of the F protein of the avian paramyxoviridae family.
  • the fusion protein formed by replacing the original transmembrane domain and intracellular domain of the new coronavirus S protein with the transmembrane domain and intracellular domain sequence of the Avian Paramyxoviridae F protein in the present invention can solve the following technical problems: 1) Avoid the upper membrane of the fusion protein and the upper membrane of the Newcastle disease virus F protein to form a competitive relationship; 2) Improve the ability of the fusion protein or recombinant Newcastle disease virus to produce neutralizing antibodies after immunizing mice, and weaken the fusion protein or recombinant Newcastle disease virus The ability to generate junk antibodies greatly enhances immunogenicity.
  • the fusion gene or protein produced by the fusion of the coding sequence of the extracellular domain of the S protein and the coding sequence of the transmembrane domain and the intracellular domain of the APMV virus (except NDV) F protein has the potential to become an excellent new crown vaccine candidate antigen huge potential.
  • the present invention specifically limits the types of APMVviridae viruses, wherein the fusion proteins S2P-AP3 (aa3), S2P-AP5, S2P-AP7, S2P-AP8 or their coding genes all have excellent immunogenicity and can be used in Preparation of vaccines for the prevention and control of new coronary pneumonia.
  • Figure 1 is a schematic diagram of the protein expressed by the fusion gene provided by the present invention, wherein ECD: extracellular domain; TM: transmembrane domain; CT: intracellular domain;
  • Fig. 2 is the relative content of S1 IgG in immune serum detected by ELISA in the embodiment of the present invention
  • Fig. 3 is the pseudovirus neutralization test in the embodiment of the present invention, wherein the abscissa is the serum dilution ratio, and the ordinate is the fluorescence percentage after the pseudovirus infected cells.
  • the invention provides a fusion protein displaying the S protein of the new coronavirus, comprising the extracellular domain sequence of the S protein of the new coronavirus and the transmembrane domain and the intracellular domain sequence of the F protein of the avian paramyxoviridae family.
  • a schematic diagram of the structure of the fusion protein is shown in FIG. 1 .
  • the ectodomain of the new coronavirus S protein is fused with the sequence containing the transmembrane domain and the intracellular domain of the F protein of the avian paramyxoviridae family.
  • the Avian Paramyxoviridae preferably includes one or more of the following serotypes: APMV-2, APMV-3, APMV-4, APMV-5, APMV-6, APMV-7, APMV-8 , APMV-9, APMV-10, APMV-11 and APMV-12, more preferably include one or more of the following serotypes: serotypes APMV-3, APMV-5, APMV-7 and APMV-8.
  • the sources and sequence information of the F proteins of each Avian Paramyxoviridae are shown in Table 1.
  • AP3(aa3) and AP3(aa33) are truncated before the transmembrane domain of F protein, and AP3(aa3) is 3 amino acids truncated before the transmembrane region of F protein (extracellular region of F protein) , while the AP3 (aa33) F protein transmembrane region intercepts 33 amino acid sequences (the extracellular region of the F protein).
  • the SARS-CoV-2 S protein preferably includes the original or mutant SARS-CoV-2 S protein or the S protein with S2P, S6P or S2P/GSAS mutations on the basis of the S protein.
  • the S protein of the new coronavirus is derived from the Delta new coronavirus strain. The source and sequence information of the novel coronavirus S protein are shown in Table 2.
  • the ectodomain of the new coronavirus S protein and the transmembrane domain and intracellular domain sequence of the F protein of the avian Paramyxoviridae are preferably connected by a linker connecting peptide.
  • the present invention has no special limitation on the sequence of the protein linker, and the protein linker well known in the art can be used.
  • the amino acid sequence of the linker connecting peptide is preferably shown in SEQ ID NO: 14 (GGSGS).
  • the invention provides a fusion gene encoding the fusion protein.
  • the sequence of the fusion gene is shown in Table 3.
  • gene synthesis is performed after adding a kozak sequence (GCCACC) at the 5' end.
  • GCCACC kozak sequence
  • the invention provides a recombinant virus vector containing the fusion gene.
  • the backbone vector of the recombinant viral vector is preferably an NDV Lasota strain vector.
  • the fusion gene is inserted between the P gene and the M gene of the NDV viral vector in the form of an expression cassette.
  • the construction method of the recombinant NDV virus vector is to add a transcription termination signal (SEQ ID NO: 24, TTAGAAAAAAA) and a transcription initiation signal (SEQ ID NO: 25, ACGGGTAGAA) to the fusion gene, preferably by Cloned into the backbone vector by homologous recombination to form a recombinant viral vector.
  • a transcription termination signal SEQ ID NO: 24, TTAGAAAAAAA
  • a transcription initiation signal SEQ ID NO: 25, ACGGGTAGAA
  • the invention provides a recombinant virus particle or vaccine strain prepared from the recombinant virus vector.
  • the plasmid expressing the fusion protein (the backbone carrier is the pcDNA3.1(+) carrier) is injected intramuscularly, or the recombinant virion or vaccine strain is immunized into animals by nasal instillation, and the serum binding antibody content and Neutralizing ability against pseudoviruses.
  • the transmembrane domain and intracellular domain of the F protein from different serotypes may indirectly affect the immunogenicity of the fusion protein.
  • the transmembrane domain of the F protein of And the replacement of intracellular domain sequence can improve the neutralizing antibody activity of recombinant Newcastle disease virus;
  • the levels of antibodies produced by plasmid immunization are not consistent, for example, the transmembrane domain and intracellular domain sequences of the F protein from two different strains of APMV-2, the plasmid Immunization possesses different binding antibody production capabilities;
  • the fusion protein formed by the present invention or the antibody produced by recombinant Newcastle disease virus immunized mice with a higher proportion of virus-neutralizing antibodies, laying the foundation for the formation of vaccines in the future and reducing the occurrence of antibody-dependent enhancement effects during multiple immunizations.
  • the present invention provides the application of the recombinant virus vector or the recombinant virus particle or vaccine strain in the preparation of a vaccine for the prevention and control of new coronary pneumonia.
  • the present invention has no special limitation on the preparation method of the vaccine, and the vaccine preparation method well known in the art can be used.
  • the vaccine has strong neutralizing antibody ability, therefore, it has high application value in the prevention and control of new coronary pneumonia.
  • Said vaccine preferably comprises said recombinant virus particle or vaccine strain and an adjuvant.
  • a fusion protein displaying the new coronavirus S protein, a recombinant virus and its application provided by the present invention will be described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.
  • F protein C-terminus comprises transmembrane domain and intracellular domain part of the coding sequence.
  • a protein linker was added between the coding sequences of the S2P protein and the C-terminus of the F protein, including the transmembrane domain and the intracellular domain.
  • the transmembrane region prediction software TMHMM-2.0 is used to analyze the APMV family APMV-2, APMV-3, APMV-4, The selected strains of APMV-5, APMV-6, APMV-7 and APMV-8 were predicted for the transmembrane domain of F protein. It should be pointed out that there may be several amino acid deviations in the transmembrane domain and intracellular domain obtained by different protein structure prediction software, and these are within our protection scope.
  • the APMV-2F gene comes from the genome of the APMV-2/Procarduelis nipalensis/China/Suiling/53/2013 virus strain or the APMV-2/Chicken/England/7702/06 virus strain genome, respectively forming S2P-AP2(1) and S2P -AP2(2) fusion gene sequence;
  • APMV-3F gene comes from the APMV3/PKT/Netherland/449/75 virus strain genome, respectively designed to form S2P-AP3(aa3) and S2P-AP3(aa33) fusion gene sequences;
  • APMV- The 4F gene comes from the APMV-4/White-Fronted Goose/Syvaske/Ukraine/6-15-03/2014 virus strain genome, designed to form a S2P-AP4 fusion gene sequence;
  • the APMV-5F gene comes from Avian paramyxovirus 5 strain Budgerigar/Kunitachi /74 virus strain genome, designed to form S2P-AP5
  • a kozak sequence (GCCACC) was added to the 5' end for gene synthesis by Jinweizhi Biotechnology, and cloned into the EcoRI and XhoI restriction enzyme digestion of the pcDNA3.1(+) vector Between the sites, pS2P-AP2(1), pS2P-AP2(2), pS2P-AP3(aa3), pS2P-AP3(aa33), pS2P-AP4, pS2P-AP5, pS2P-AP6, pS2P-AP7 are formed, respectively and pS2P-AP8 expression plasmids.
  • GCCACC kozak sequence
  • a transcription termination signal (TTAGAAAAAA, SEQ ID NO: 24) was added to the 3' end of the fusion gene sequence designed in Example 1, and a transcription initiation signal (ACGGGTAGAA, SEQ ID NO: 25) was added to the 5' end, and the design
  • the primers amplified the fusion gene sequence containing the transcription initiation signal and the transcription termination signal, cloned into the position between the P gene and the M gene of the NDV Lasota strain vector by means of homologous recombination, and constructed a series of recombinant NDV virus vectors.
  • the above-mentioned recombinant NDV virus vector and virus rescue helper plasmids pCI-NP, pCI-P and pCI-L were transfected into BHK21-T7 cells at a ratio of 1:1:1:1, and the plasmid concentration was measured by Nano drop. 72 hours after transfection, the cells were repeatedly frozen and thawed three times, and the mixture was inoculated into 9-11-day-old SPF chicken embryos, 0.2-0.3 mL/embryo, and cultured at 37°C for 24 hours. The dead embryos within 24 hours were discarded, and the allantoic fluid of surviving chicken embryos within 72 hours was harvested and the hemagglutination value was measured.
  • the allantoic fluid of surviving chicken embryos with hemagglutination value (reaching 2log and above) was centrifuged and subpackaged, and stored in a -80°C refrigerator. At the same time, the extracted RNA was analyzed according to the instructions of the HiScript III 1st Strand cDNA Synthesis Kit. Reverse transcription to synthesize first-strand cDNA. Afterwards, the synthesized cDNA was identified by PCR gel electrophoresis, and the PCR reaction system and reaction conditions were set according to the instructions of HiscriptII One Step RT-PCR kit.
  • NDV-S2P-AP3(aa3) and NDV-S2P-AP7 were named NDV-S2P-AP3(aa3) and NDV-S2P-AP7.
  • transmembrane domain and the intracellular domain sequence of the S2P protein are fused with the sequences containing the NDV virus F protein transmembrane domain and the intracellular domain to obtain a fusion protein (SEQ ID NO: 28), which is synthesized according to the method of Example 1 for encoding fusion Protein fusion gene (SEQ ID NO: 29), construct recombinant vector and rescue virus according to the method of embodiment 2, and finally obtained recombinant Newcastle disease virion is named NDV-S2P/F34.
  • the plasmid containing the fusion gene constructed in Example 1 and the recombinant NDV virus constructed in Example 2 were used to immunize 4-5 week-old Babl/c mice, and each plasmid or recombinant virus was used to immunize four mice respectively.
  • the procedure for plasmid immunization was intramuscular injection of 100 ⁇ g/mouse (with the addition of sterile PBS, the final injection volume was not less than 100 ⁇ L/mouse), and blood was collected 10 days after immunization.
  • the immunization procedure of the recombinant NDV virus was intranasal 50 ⁇ L per mouse, and the immunization was carried out on the 0th day and the 7th day respectively, and the blood was collected on the 14th day.
  • the control group was immunized with sterile PBS.
  • the test kit instructions require plate washing, sample incubation, antibody incubation, color development and termination of the reaction, and finally read the OD 450 value under the microplate reader. Divide the OD 450 reading of each sample by the background OD 450 reading to obtain Relative IgG antibody data.
  • VSV-S ⁇ 24-GFP (Xiong HL, Wu YT, Cao JL, et al.
  • VSV SARS-CoV-2 S -Protein-Bearing Stomatis Virus
  • an excellent antigen should be able to produce more neutralizing antibodies and as few non-neutralizing antibodies as possible.
  • the fusion genes S2P-AP3 (aa3), S2P-AP5 and S2P-AP7 are slightly weaker than the S2P gene to produce binding antibodies, But the ability to produce neutralizing antibodies is stronger. Since the present invention selects a fusion gene with a relatively good plasmid immunization effect for testing during the rescue process of the recombinant virus, this cannot rule out that other fusion genes with a general plasmid immunization effect cannot obtain similar excellent immune effects.
  • the fusion gene or protein produced by the fusion of the coding sequence of the extracellular domain of the S protein and the coding sequence of the transmembrane domain and the intracellular domain of the F protein of APMVviridae viruses has the potential to become an excellent novel coronavirus. Great potential for vaccine candidate antigens.

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Abstract

本发明提供了一种展示新冠S蛋白的融合蛋白和重组病毒粒子及其应用，本发明属于生物制品技术领域。本发明提供了一种表达新冠S蛋白的融合基因及融合蛋白，该融合基因或融合蛋白包含新冠S蛋白的胞外结构域序列与禽副黏病毒科F蛋白的跨膜结构域和胞内结构域序列。同时，含有该序列的重组NDV病毒具有较强的中和抗体的能力。可见，S蛋白的胞外域编码序列和APMV病毒科病毒(除NDV外)F蛋白的跨膜结构域和胞内结构域编码序列相融合所产生的融合基因或蛋白，具有成为优秀新冠疫苗候选抗原的巨大潜力。

Description

一种展示新冠S蛋白的融合蛋白和重组病毒粒子及其应用
本申请要求于2021年12月16日提交中国专利局、申请号为202111541851.3、发明名称为“一种展示新冠S蛋白的融合蛋白和重组病毒粒子及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物制品技术领域，具体涉及一种展示新冠S蛋白的融合蛋白以及重组病毒粒子及其应用。
背景技术
新冠病毒的免疫原性相对较弱，在以活病毒为载体构建疫苗株时，理想情况下，希望病毒粒子表面或被病毒感染进而表达细胞膜抗原的细胞表面有更多的抗原蛋白展示。新冠病毒的S蛋白是病毒与宿主细胞膜受体ACE2结合，进而感染宿主细胞的关键蛋白。S蛋白属于I型跨膜蛋白，其在细胞膜上的表达量与跨膜区氨基酸排列有关。以新城疫(Newcastle Disease virus,NDV)病毒新型冠状病毒重组疫苗株为例，前期研究发现，将新型冠状病毒(SARS-COV-2)的S蛋白的跨膜结构域和胞内结构域更换为NDV F蛋白的跨膜结构域和胞内结构域后所拯救得到的重组NDV-COVS-F病毒，其免疫原性比原始S蛋白直接插入NDV病毒载体所形成的重组病毒更好。然而，将S蛋白的跨膜结构域和胞内结构域更换为NDV F蛋白的跨膜结构域和胞内结构域，有极大可能使得S蛋白的上膜与NDV病毒本身F蛋白的上膜形成竞争关系，干扰细胞膜上抗原蛋白的表达或者病毒组装，影响其免疫原性。
发明内容
有鉴于此，本发明的目的在于提供一种展示新冠病毒S蛋白的融合蛋白和构建的相应重组病毒及其应用。
本发明提供了一种展示新冠病毒S蛋白的融合蛋白，包含新冠病毒S蛋白的胞外域序列和含禽副黏病毒科病毒F蛋白的跨膜结构域和胞内结构域的序列。
优选的，所述禽副黏病毒科病毒包括以下一种或几种血清型：APMV-2、APMV-3、APMV-4、APMV-5、APMV-6、APMV-7、APMV-8、APMV-9、APMV-10、APMV-11和APMV-12。
优选的，所述禽副黏病毒科病毒包括以下一种或几种血清型：APMV-3、APMV-5、APMV-7和APMV-8血清型。
优选的，所述新冠病毒S蛋白的胞外结构域来源于新冠病毒原始毒株或变异株。
优选的，所述新冠病毒S蛋白的胞外结构域序列还包括以下一种或几种蛋白编码序列：S2P，S6P和S2P/GSAS；
所述S2P是将所述新冠病毒S蛋白胞外域进行K986P和V987P突变所形成的蛋白编码序列；
所述S2P/GSAS是在S2P基础上将第682～685位氨基酸RRAR突变为GSAS所形成的蛋白编码序列；
所述S6P是在S2P基础上进行F817P、A892P、A899P和A942P突变所形成的蛋白编码序列。
本发明提供了一种编码所述融合蛋白的融合基因。
本发明提供了一种包含所述融合基因的重组病毒载体。
优选的，所述重组病毒载体以新城疫病毒载体为骨架载体。
优选的，所述融合基因以表达盒子形式插入新城疫病毒载体的P基因和M基 因之间。
本发明提供了一种由所述重组病毒载体制备得到的重组病毒粒子或疫苗株。
本发明提供了所述融合蛋白、所述融合基因、所述重组病毒载体或所述重组病毒粒子或疫苗株在制备防控新冠肺炎的疫苗中的应用。
本发明提供的一种展示新冠病毒S蛋白的融合蛋白，包括新冠病毒S蛋白的胞外域序列和禽副黏病毒科F蛋白的跨膜结构域和胞内结构域编码序列。本发明采用禽副黏病毒科F蛋白的跨膜结构域和胞内结构域序列替换新冠病毒S蛋白的原始跨膜结构域和胞内结构域后形成的融合蛋白可以解决以下技术问题：1)避免融合蛋白的上膜与新城疫病毒F蛋白的上膜形成竞争关系；2)提高了融合蛋白或重组新城疫病毒免疫小鼠后产生中和抗体的能力，减弱了融合蛋白或重组新城疫病毒产生垃圾抗体的能力，极大地增强了免疫原性。可见，S蛋白的胞外域编码序列和APMV病毒科病毒(除NDV外)F蛋白的跨膜结构域和胞内结构域编码序列相融合所产生的融合基因或蛋白，具有成为优秀新冠疫苗候选抗原的巨大潜力。
进一步的，本发明具体限定了APMV病毒科病毒的种类，其中融合蛋白S2P-AP3(aa3)、S2P-AP5、S2P-AP7、S2P-AP8或其编码基因都具有优异的免疫原性，可用于防控新冠肺炎的疫苗的制备。
附图说明
图1为本发明提供的融合基因所表达蛋白示意图，其中ECD：胞外结构域；TM：跨膜结构域；CT：胞内结构域；
图2为本发明实施例中ELISA检测免疫血清S1IgG相对含量；
图3为本发明实施例中假病毒中和试验，其中横坐标为血清稀释比，纵坐标为假病毒感染细胞后荧光百分比。
具体实施方式
本发明提供了一种展示新冠病毒S蛋白的融合蛋白，包含新冠病毒S蛋白的胞外域序列和禽副黏病毒科F蛋白的跨膜结构域和胞内结构域序列。所述融合蛋白结构示意图见图1。将新冠病毒S蛋白的胞外域与含禽副黏病毒科F蛋白的跨膜结构域和胞内结构域的序列相融合。
在本发明中，所述禽副黏病毒科优选包括以下一种或几种血清型：APMV-2、APMV-3、APMV-4、APMV-5、APMV-6、APMV-7、APMV-8、APMV-9、APMV-10、APMV-11和APMV-12，更优选包括以下一种或几种血清型：血清型APMV-3、APMV-5、APMV-7和APMV-8。其中各禽副黏病毒科F蛋白的来源及序列信息见表1。
表1 各禽副黏病毒科F蛋白的来源及序列信息
Figure PCTCN2023076443-ftappb-I100001

Figure PCTCN2023076443-ftappb-I100002
其中，AP3(aa3)和AP3(aa33)的区别在于F蛋白跨膜结构域前截取了不同氨基酸数量，AP3(aa3)为F蛋白跨膜区前截取3个氨基酸(F蛋白的胞外区)，而AP3(aa33)F蛋白跨膜区前截取33个氨基酸序列(F蛋白的胞外区)。
在本发明中，所述新冠病毒S蛋白优选包括原始或变异株的新冠病毒S蛋白或在S蛋白的基础上发生S2P、S6P或S2P/GSAS突变的S蛋白。本发明实施例中，所述新冠病毒S蛋白来源于Delta新冠病毒株。所述新冠病毒S蛋白的来源及序列信息见表2。
表2 S蛋白的来源及序列信息
Figure PCTCN2023076443-ftappb-I100003
在本发明中，新冠病毒S蛋白的胞外域和禽副黏病毒科F蛋白的跨膜结构域和胞内结构域序列之间优选用linker连接肽连接。本发明对所述蛋白linker的序列没有特殊限制，采用本领域所熟知的蛋白linker即可。所述linker连接肽的氨基酸序列优选见SEQ ID NO:14(GGSGS)。
本发明提供了一种编码所述融合蛋白的融合基因。所述融合基因的序列见表3。
Figure PCTCN2023076443-ftappb-I100004
在本发明中，所述融合基因经过密码子优化后，在5’端添加kozak序列(GCCACC)后进行基因合成。
本发明提供了一种含所述融合基因的重组病毒载体。所述重组病毒载体的骨架载体优选为NDV Lasota株载体。所述融合基因以表达盒子形式插入NDV病毒载体的P基因和M基因之间。
在本发明中，所述重组NDV病毒载体的构建方法，将所述融合基因添加转录终止信号(SEQ ID NO:24，TTAGAAAAAA)和转录起始信号(SEQ ID NO:25，ACGGGTAGAA)，优选通过同源重组方式克隆至骨架载体中，形成重组病毒载体。
本发明提供了一种由所述重组病毒载体制备的重组病毒粒子或疫苗株。
在本发明中，将表达融合蛋白的质粒(骨架载体为pcDNA3.1(+)载体)采用肌肉注射，或者所述重组病毒粒子或疫苗株以鼻腔滴注方式免疫动物，检测血清结合抗体含量以及对假病毒的中和能力。结果显示，1)不同血清型病毒来源的F蛋白的跨膜区与胞内区可能间接影响融合蛋白的免疫原性发挥，与S2P蛋白相比，禽副黏病毒科F蛋白的跨膜结构域和胞内结构域序列的替换能够提高重组新城疫病毒中和抗体活性；
2)同一种血清型下的两株病毒，其质粒免疫产生的抗体水平并不一致，例如APMV-2的两种不同的病毒株来源的F蛋白的跨膜结构域和胞内结构域序列，质粒免疫拥有不同的结合抗体产生能力；
3)质粒免疫时pS2P-AP3(aa3)、pS2P-AP7、pS2P-AP5、pS2P-AP8结合抗体产生能力不及pS2P，但血清中和抗体能力更加强劲。重组病毒免疫时，NDV-S2P-AP7与NDV-S2P结合抗体水平相当，但血清中和抗体表现更优异，可见，本发明所形成的融合蛋白或重组新城疫病毒免疫小鼠所产生的抗体中，有更高的比例的病毒中和抗体，为日后形成疫苗和多次免疫时降低抗体依赖增强效应的发生奠定基础。
基于所述融合蛋白具有优异的免疫原性，本发明提供了所述重组病毒载体或所述重组病毒粒子或疫苗株在制备防控新冠肺炎的疫苗中的应用。
本发明对所述疫苗的制备方法没有特殊限制，采用本领域所熟知的疫苗制备方法即可。所述疫苗具有较强的中和抗体能力，因此，在防控新冠肺炎中具有较高的应用价值。所述疫苗优选包括所述重组病毒粒子或疫苗株和佐剂。
下面结合实施例对本发明提供的一种展示新冠S蛋白的融合蛋白和重组病毒及其应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
1.融合基因设计及合成
对新冠病毒(新冠Delta变异株)的S蛋白的胞外域编码序列(氨基酸1-1213，SEQ ID NO:17)进行K986P和V987P位突变后，分别融合APMV-2、APMV-3、APMV-4、APMV-5、APMV-6、APMV-7、APMV-8血清型病毒种病毒F蛋白的C端包含跨膜结构域和胞内结构域部分的编码序列。在S2P蛋白和F蛋白C端包含跨膜结构域和胞内结构域部分的编码序列之间添加蛋白linker。因APMV病毒科除NDV外，其他病毒株的F蛋白都未收录进入Swiss-Prot数据库中，所以使用跨膜区域预测软件TMHMM-2.0对APMV病毒科APMV-2、APMV-3、APMV-4、APMV-5、APMV-6、APMV-7、APMV-8的选中病毒株进行F蛋白的跨膜结构域预测。需要指出的是，不同蛋白结构预测软件得到的跨膜结构域和胞内结构域可能存在几个氨基酸的偏差，这些都属于我们的保护范围。其中，APMV-2F基因来自于APMV-2/Procarduelis nipalensis/China/Suiling/53/2013病毒株或APMV-2/Chicken/England/7702/06病毒株基因组，分别形成S2P-AP2(1)和S2P-AP2(2)融合基因序列；APMV-3F基因来自于APMV3/PKT/Netherland/449/75病毒株基因组，分别设计形成S2P-AP3(aa3)和S2P-AP3(aa33)融合基因序列；APMV-4F基因来自于APMV-4/White-Fronted Goose/Syvaske/Ukraine/6-15-03/2014病毒株基因组，设计形成S2P-AP4融合基因序列；APMV-5F基因来自于Avian paramyxovirus 5 strain Budgerigar/Kunitachi/74病毒株基因组，设计形成S2P-AP5融合基因序列；APMV-6F基因来自于Avian paramyxovirus 6 isolate teal/Novosibirsk region/455/2009病毒株基因组，设计形成S2P-AP6融合基因序列；APMV-7F基因来自于APMV-7/dove/Tennessee/4/75病毒株基因组，设计形成S2P-AP7融合基因序列；APMV-8F基因来自于Avian paramyxovirus 8 strain goose/Delaware/1053/76病毒株基因组，设计形成S2P-AP8融合基因序列。
以上所设计形成的融合基因序列经过密码子优化后，在5’端添加kozak序列(GCCACC)通过金唯智生物进行基因合成，并克隆到pcDNA3.1(+)载体的EcoRI和XhoI限制性酶切位点之间，分别形成pS2P-AP2(1)，pS2P-AP2(2)，pS2P-AP3(aa3)，pS2P-AP3(aa33)，pS2P-AP4，pS2P-AP5，pS2P-AP6，pS2P-AP7和pS2P-AP8表达质粒。
实施例2
重组NDV病毒载体的构建及重组NDV病毒拯救
在实施例1设计得到的融合基因序列的3'端分别添加转录终止信号(TTAGAAAAAA，SEQ ID NO:24)，在5’端添加转录起始信号(ACGGGTAGAA,，SEQ ID NO:25)，设计引物扩增含有转录起始信号和转录终止信号的融合基因序列，通过同源重组的方式克隆进入NDV Lasota株载体的P基因和M基因位置之间，构建得到一系列重组NDV病毒载体。
将上述构建的重组NDV病毒载体和病毒拯救辅助质粒pCI-NP、pCI-P和pCI-L以1：1：1：1比例，经Nano drop测定质粒浓度后，转染BHK21-T7细胞。转染后72h，反复冻融细胞3次，并将混合物接种9～11日龄SPF鸡胚，0.2～0.3mL/胚，37℃培养24h。弃去24h内的死胚，收获72h内存活的鸡胚尿囊液并测量血凝价。将有血凝价(达到2log及以上)且存活的鸡胚尿囊液离心后分装，冻存于-80℃冰箱，同时根据HiScript III 1st Strand cDNA Synthesis Kit试剂盒说明书要求对提取的RNA进行反转录，合成首链cDNA。之后对合成的cDNA进行PCR凝胶电泳鉴定，PCR反应体系和反应条件按照喏唯赞HiscriptII One Step RT-PCR kit说明书要求设置。鉴定上游引物NDV-F3153：AAGGTCCAACTCTCCAAGCGG(SEQ ID NO:26)，下游引物NDV-R3454：GTCCTCCTTACTATCAGTCCACA(SEQ ID NO:27)。最终形成的重组NDV病毒疫苗株命名为NDV-S2P-AP3(aa3)和NDV-S2P-AP7。
对比例1
将S2P蛋白的跨膜域和胞内域序列与含NDV病毒F蛋白跨膜域和胞内域的序列相融合，得到融合蛋白(SEQ ID NO:28)，按照实施例1的方法合成编码融合蛋白的融合基因(SEQ ID NO:29)，按照实施例2的方法构建重组载体以及拯救病毒，最终得到的重组新城疫病毒粒子命名为NDV-S2P/F34。
实施例3
小鼠免疫试验
分别使用实施例1构建的含融合基因的质粒和实施例2构建的重组NDV病毒对4-5周龄Babl/c小鼠进行免疫，每种质粒或重组病毒分别免疫四只小鼠。质粒免疫程序为肌肉注射100μg/只(可补加无菌PBS，最终注射体积不低于100μL/只)，免疫后10天采血。重组NDV病毒免疫程序为滴鼻50μL/只，分别在第0天和第7天进行免疫，第14天采血，免疫时重组病毒血凝价为6～7log2。对照组采用无菌PBS进行免疫。
实施例4
ELISA检测免疫血清新冠S蛋白结合抗体
提前将96孔酶标板放置室温平衡，使用义翘神州(SARS-CoV-2(2019-nCoV)Spike S1抗体滴度检测试剂盒中稀释液对样本血清及阳性对照分别做20倍稀释。按照检测试剂盒说明书要求进行洗板、样本孵育、抗体孵育、显色和终止反应，最后在酶标仪下读取OD450数值。分别以每个样品的OD450读数除以背景OD450读数即得到相对IgG抗体数据。
结果如图2所示。可见，免疫小鼠后，含有融合基因S2P-AP3(aa3)、S2P-AP5、S2P-AP7、S2P-AP8的质粒和重组NDV病毒NDV-S2P-AP3(aa3),NDV-S2P-AP7结合抗体相对较高。特别是NDV-S2P-AP3(aa3)，其抗体水平达到NDV-S2P的大约7 倍之多。然而，并非质粒的结合抗体高，所形成的重组NDV病毒其结合抗体就高，这可能与病毒出芽及包装机制有关。同时，从实验结果可以发现，对于APMV-2，即同一种血清型下的两株病毒，其产生的抗体水平并不一样，可见，S蛋白胞外域融合某一毒株F蛋白的跨膜结构域和胞内结构域所形成的融合蛋白，免疫结果并不能反应所有该血清型下的其他病毒株的免疫效果，鉴于APMV病毒科囊括的病毒株太多，这里仍然有广阔的深入发掘研究空间。另外，pS2P-AP3(aa3)和pS2P-AP3(aa33)的免疫效果差异较大，这两种融合基因所表达蛋白的跨膜结构域和胞内结构域氨基酸序列一致，不同的是，在跨膜结构域之前所选择的延伸区段长度不同。我们推测，出现这一结果的原因很可能是由于跨膜结构域之前所选择的延伸区段影响了整个融合蛋白的构象。
实施例5
假病毒中和试验
取15μL待测血清加入96孔板，加入135μL DMEM培养基进行稀释。随后进行4个梯度的2倍比稀释。在稀释后的血清中加入50μL假病毒液37℃孵育1h，假病毒使用VSV-S△24-GFP(Xiong HL,Wu YT,Cao JL,et al.Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus(VSV)pseudovirus and ACE2-overexpressing BHK21 cells.Emerg Microbes Infect.2020；9(1):2105-2113.doi:10.1080/22221751.2020.1815589)是由VSV病毒载体G囊膜蛋白删除后插入新冠S△24基因以及GFP基因经病毒拯救而来，S△24基因删除了新冠S蛋白C端的24个氨基酸，使得S蛋白可以更多地呈现在VSV病毒囊膜上。孵育30min时，消化Vero-E6细胞，孵育结束后，每孔加入100μL细胞，37℃，5％CO2细胞培养箱中培养24h。同时设置无血清细胞对照(150μL MEDM培养基+100μL细胞)和无血清病毒对照(100μL MEDM培养基+50μL假病毒+100μL细胞)。最后在荧光显微镜下拍照，记录每孔细胞荧光百分比数。
结果见图3和表4。
表4 含融合基因的质粒和重组NDV病毒中和试验结果
Figure PCTCN2023076443-ftappb-I100005
由图3可见，质粒pS2P-AP3(aa3)、pS2P-AP8、pS2P-AP5、pS2P-AP7以及NDV-S2P-AP7免疫小鼠后，血清中和抗体水平均高于pS2P或NDV-S2P免疫后小鼠。
优秀的抗原应该能产生出更多的中和抗体，以及尽量少的非中和活性的抗体。 综合本发明的结合抗体以及假病毒中和抗体试验结果可以发现，在质粒免疫时，融合基因S2P-AP3(aa3)、S2P-AP5以及S2P-AP7相对于S2P基因产生结合抗体的程度稍弱，但产生中和抗体的能力更强。由于本发明在进行重组病毒的拯救过程中，选择了质粒免疫结果相对较好的融合基因进行测试，但这不能排除其他质粒免疫效果一般的融合基因不能得到类似优良的免疫效果。由此得出，S蛋白的胞外域编码序列和APMV病毒科病毒(除NDV外)F蛋白的跨膜结构域和胞内结构域编码序列相融合所产生的融合基因或蛋白，具有成为优秀新冠疫苗候选抗原的巨大潜力。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。对这些实施例的多种修改对本领域的专业技术人员来说是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (16)

  1. 一种展示新冠病毒S蛋白的融合蛋白，其特征在于，包含新冠病毒S蛋白的胞外域序列和含禽副黏病毒科病毒F蛋白的跨膜结构域和胞内结构域的序列。
  2. 根据权利要求1所述融合蛋白，其特征在于，所述禽副黏病毒科病毒包括以下一种或几种血清型：APMV-2、APMV-3、APMV-4、APMV-5、APMV-6、APMV-7、APMV-8、APMV-9、APMV-10、APMV-11和APMV-12。
  3. 根据权利要求1所述融合蛋白，其特征在于，所述禽副黏病毒科病毒包括以下一种或几种血清型：APMV-3、APMV-5、APMV-7和APMV-8血清型。
  4. 根据权利要求1所述的融合蛋白，其特征在于，所述新冠S蛋白的胞外结构域来源于新冠原始毒株或变异株。
  5. 根据权利要求4所述的融合蛋白，其特征在于，所述新冠S蛋白的胞外结构域序列还包括以下一种或几种蛋白编码序列：S2P、S6P和S2P/GSAS；
    所述S2P是将所述新冠S蛋白胞外域进行K986P和V987P突变所形成的蛋白编码序列；
    所述S2P/GSAS是在S2P基础上将第682～685位氨基酸RRAR突变为GSAS所形成的蛋白编码序列；
    所述S6P是在S2P基础上进行F817P、A892P、A899P和A942P突变所形成的蛋白编码序列。
  6. 一种编码权利要求1～5中任意一项所述融合蛋白的融合基因。
  7. 一种包含权利要求6所述融合基因的重组病毒载体。
  8. 根据权利要求7所述重组病毒载体，其特征在于，所述重组病毒载体以新城疫病毒载体为骨架载体。
  9. 根据权利要求8所述重组病毒载体，其特征在于，所述融合基因以表达盒子形式插入所述新城疫病毒载体的P基因和M基因之间。
  10. 根据权利要求7所述重组病毒载体，其特征在于，包括S2P-AP3(aa3)、S2P-AP8、S2P-AP5或S2P-AP7融合基因的5'端添加转录起始信号，3'端添加转录终止信号后，通过同源重组方式分别克隆进入NDV Lasota株载体的P基因和M基因位置之间形成的重组病毒载体；
    S2P-AP3(aa3)融合基因的核苷酸序列如SEQ ID NO:17所示；
    S2P-AP8融合基因的核苷酸序列如SEQ ID NO:23所示；
    S2P-AP5融合基因的核苷酸序列如SEQ ID NO:20所示；
    S2P-AP7融合基因的核苷酸序列如SEQ ID NO:22所示。
  11. 一种由权利要求7～10中任意一项所述重组病毒载体制备得到的重组病毒粒子或疫苗株。
  12. 根据权利要求11所述重组病毒粒子或疫苗株，其特征在于，包括NDV-S2P-AP3(aa3)和/或NDV-S2P-AP7；
    所述NDV-S2P-AP3(aa3)为表达S2P-AP3(aa3)融合基因的重组新城疫病毒载体经病毒拯救得到；所述S2P-AP3(aa3)融合基因的核苷酸序列如SEQ ID NO:17所示；
    所述NDV-S2P-AP7为表达S2P-AP7融合基因的重组新城疫病毒载体经病毒拯救得到；所述S2P-AP7融合基因的核苷酸序列如SEQ ID NO:22所示。
  13. 权利要求1～5中任意一项所述融合蛋白、权利要求6所述融合基因、权利要求7～10中任意一项所述重组病毒载体或权利要求11或12所述重组病毒粒子或疫苗株在制备防控新冠肺炎的疫苗中的应用。
  14. 一种防控新冠肺炎的疫苗，其特征在于，活性成分包括权利要求1～5中任意一项所述融合蛋白、权利要求7～10中任意一项所述重组病毒载体或权利要求11或12所述重组病毒粒子或疫苗株。
  15. 权利要求1～5中任意一项所述融合蛋白、权利要求6所述融合基因、权利要求7～10中任意一项所述重组病毒载体或权利要求11或12所述重组病毒粒子或疫苗株在防控新冠肺炎中的应用。
  16. 一种防控新冠肺炎的方法，其特征在于，将权利要求7～10中任意一项所述重组病毒载体采用肌肉注射方式免疫或权利要求11或12所述重组病毒粒子或疫苗株采用鼻喷方式免疫。
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