WO2022121322A1 - 一种新型冠状病毒的重组亚单位疫苗及其应用 - Google Patents

一种新型冠状病毒的重组亚单位疫苗及其应用 Download PDF

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WO2022121322A1
WO2022121322A1 PCT/CN2021/107881 CN2021107881W WO2022121322A1 WO 2022121322 A1 WO2022121322 A1 WO 2022121322A1 CN 2021107881 W CN2021107881 W CN 2021107881W WO 2022121322 A1 WO2022121322 A1 WO 2022121322A1
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fragment
fusion protein
vaccine
seq
rbd
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PCT/CN2021/107881
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French (fr)
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刘永江
张海江
贠炳岭
张爱晶
王艳
陈晓
张尧
银飞
伍树明
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北京康乐卫士生物技术股份有限公司
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Publication of WO2022121322A1 publication Critical patent/WO2022121322A1/zh
Priority to ZA2022/09123A priority Critical patent/ZA202209123B/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

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  • the invention belongs to the technical field of biology and medicine, in particular to a recombinant subunit vaccine of a novel coronavirus and its application, and more particularly to expressing an immunologically active recombinant subunit in a eukaryotic cell from a virus gene artificially synthesized by means of genetic engineering protein, and use the expressed recombinant protein to develop a vaccine method.
  • the new coronavirus is a new type of coronavirus belonging to the genus ⁇ . It has an envelope, and the particles are round or oval, often pleomorphic, with a diameter of 60-140 nm.
  • the structural proteins of the new coronavirus SARS-CoV-2 include S protein, N protein, E protein, and M protein. For SARS-CoV-2, only neutralizing antibodies directed against the S protein can neutralize the virulence of the virus and prevent the virus from infecting the body.
  • the Spike protein (S protein) on the outer envelope of the coronavirus It is a key protein that recognizes host cell receptors during virus infection.
  • the S protein consists of two subunits, S1 and S2, of which the S1 subunit contains a receptor binding domain (Receptor Binding Domain, RBD) to mediate adsorption.
  • the S2 subunit mainly reflects fusion activity.
  • the new coronavirus COVID-19 seriously endangers people's lives and health, and the focus is on prevention. Therefore, vaccines can be very effective in preventing virus infection.
  • the new coronavirus COVID-19 there have been some studies to obtain related vaccines.
  • the dominant antigenic epitope fragments that can be used for the preparation of new coronavirus COVID-19 vaccines have been screened and optimized, and the protein fragments It is fused with human Fc fragment to prepare a new type of virus vaccine.
  • "The Lancet" published the results of my country's first phase I clinical trial of the COVID-19 vaccine due to the Institute of Bioengineering, Academy of Military Medicine, Academy of Military Sciences. The current test results of the vaccine have been reported well.
  • a single dose of the new type 5 adenovirus vector COVID-19 (Ad5-nCoV) vaccine produces specific antibodies and T cells within 14 days, and the related vaccine has applied for a patent.
  • the present invention develops a new type of vaccine by merging and expressing the RBDSD1 sequence in the S1 subunit and the human Fc sequence through gene recombinant expression technology.
  • the SD1 sequence has the effect of stabilizing the RBD structure.
  • further fusion of SD1 can increase a pair of disulfide bonds (at the amino acid positions 591 to 538). time), which can make the whole structure more stable, thus forming the basis of the present invention. Further experiments show that the effect is very good, and the present invention is finally completed.
  • the present invention first provides a fusion protein containing an antigenic epitope of a novel coronavirus COVID-19 vaccine, characterized in that the fusion protein is composed of an antigenic epitope fragment of the novel coronavirus COVID-19 vaccine and an immunoglobulin Fc fragment , wherein the antigenic epitope fragments of the new coronavirus COVID-19 vaccine are the RBD fragment and the SD1 fragment in the S1 subunit.
  • amino acid sequence of the RBD fragment is shown in SEQ ID NO: 2; the amino acid sequence of the SD1 fragment is shown in SEQ ID NO: 3.
  • the immunoglobulin Fc fragment is selected from the immunoglobulin Fc fragment of human, mouse, rabbit, bovine, goat, pig, mouse, rabbit, hamster, rat, or guinea pig; further , the immunoglobulin Fc fragment is selected from the Fc fragment of IgG, IgA, IgD, IgE or IgM; wherein preferably, the immunoglobulin Fc fragment is selected from the IgG1 Fc fragment, the IgG2 Fc fragment, the IgG3 Fc fragment, or the IgG4 Fc fragment; especially Preferably, the immunoglobulin Fc fragment is a human IgG Fc fragment, most preferably it is the amino acid sequence shown in SEQ ID NO.5.
  • the fusion protein comprises the amino acid sequence shown in SEQ ID NO.7.
  • the present invention accordingly provides the nucleic acid encoding the fusion protein according to the described fusion protein, preferably, wherein the encoding nucleotide sequence of the RBD fragment is as shown in SEQ ID NO: 1 or its degenerate sequence; the encoding nucleotide sequence of the SD1 fragment As shown in SEQ ID NO: 4 or its degenerate sequence; the nucleotide sequence encoding the immunoglobulin Fc fragment is shown in SEQ ID NO: 6 or its degenerate sequence. Most preferably, the nucleotide sequence of the nucleic acid encoding the fusion protein is shown in SEQ ID NO: 8 or a degenerate sequence thereof.
  • the present invention further provides recombinant vectors and recombinant cells containing the encoding nucleic acid as described.
  • the present invention also provides a novel coronavirus COVID-19 vaccine, characterized in that the vaccine comprises the fusion protein. Further included are adjuvants such as aluminum hydroxide. Wherein, the fusion protein is obtained by chemical synthesis or gene recombination.
  • the fusion of SD1 on the basis of RBD can increase its flexibility and variability, easily form a head-to-tail structure, and the structure is more stable. Advantages; at the same time, due to the fusion with the Fc fragment can form a dimer, it is further speculated that increased immunogenicity and more improved stability.
  • the fusion protein of the antigenic epitope obtained by the present invention has better immune properties and stability, and its effect is significantly better than the effect when only RBD is fused.
  • Figure 1 Plasmid construction nucleic acid gel and double digestion identification (HindIII and XhoI) electropherogram.
  • Sample loading sequence A1: pcDNA3.1-RBD plasmid, A2: pcDNA3.1-RBD double digestion identification (HindIII and XhoI), A3: DNA Marker.
  • C1 pcDNA3.1-RBDSD1-Fc plasmid, C2: pcDNA3.1-RBDSD1-Fc double digestion identification (HindIII and XhoI), C3: DNA Marker.
  • Figure 2 SDS-PAGE electropherogram. Loading sequence: Protein Marker, RBD, RBDSD1-Fc, RBD-Fc.
  • the experimental techniques and experimental methods used in this embodiment are conventional technical methods unless otherwise specified.
  • the materials, reagents, etc. used in this example can be obtained through regular commercial channels unless otherwise specified.
  • Embodiment 1 Vector construction
  • the target gene sequence was synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. in,
  • the RBD nucleotide sequence is as follows (SEQ ID NO: 1):
  • RBD amino acid sequence is as follows (SEQ ID NO: 2):
  • the SD1 nucleotide sequence is as follows (SEQ ID NO: 4):
  • the hFc nucleotide sequence is as follows (SEQ ID NO: 6):
  • hFc amino acid sequence is as follows (SEQ ID NO: 5):
  • RBDSD1-hFc nucleotide sequence is as follows (SEQ ID NO: 8):
  • RBDSD1-hFc amino acid sequence (SEQ ID NO: 7)
  • the above target genes ie RBD, RBD-Fc and RBDSD1-Fc
  • RBD, RBD-Fc and RBDSD1-Fc were constructed into the pcDNA3.1 expression vector by direct synthesis by Gene Company, and the DNA plasmids pcDNA3.1-RBD, pcDNA3.1-RBD-Fc and pcDNA3 were obtained respectively. .1-RBDSD1-Fc. and verified by gel electrophoresis.
  • Enzyme digestion of plasmid Take plasmid DNA, carry out enzyme digestion with Xho I enzyme and Hand III enzyme according to the enzyme digestion reaction system in the table, react at 37°C in a constant temperature incubator for 2 hours, and then conduct gel electrophoresis.
  • the cell viability rate is greater than 95%, and the viable cell density is greater than or equal to 4.0 ⁇
  • 10 6 cells/mL can be used directly; if the cell density is lower than 4.0 ⁇ 10 6 cells/mL, the cells can be collected by centrifugation (800 rpm, 5min), and the cells can be resuspended in Transpro CD01 at a density of 4.0 ⁇ 10 6 cells/mL in the culture medium;
  • VPA valproic acid
  • RBD-Fc the purification of RBDSD1-Fc adopts the following Protein A affinity chromatography method
  • Chromatography column AT Protein A Diamond affinity chromatography medium, column volume CV: 5ml, flow rate: 5ml/min, pressure limit: ⁇ 0.3MPa.
  • Pretreatment Rinse with purified water for at least 5 CVs.
  • Loading take the filtered CHO suspension cell culture and load it onto a well-equilibrated chromatography column, collect the sample flow through, and sample 100 ⁇ l therefrom for electrophoresis detection.
  • Elution Elute with 100% elution buffer, elute to the UV baseline level, collect each elution fraction in separate tubes, and add an appropriate volume of neutralization buffer according to the collection volume in each collection tube and mix well.
  • Chromatography column SP cation exchange chromatography column, column volume CV: 5ml, flow rate: 5ml/min, pressure limit: ⁇ 0.3MPa.
  • Pretreatment Rinse with 1M NaOH for at least 3 CV and then with purified water for at least 10 CV.
  • Loading take the G25 solution to collect samples and load them to a well-equilibrated chromatography column, collect the sample flow through, and sample 100 ⁇ l therefrom for electrophoresis detection.
  • Embodiment four SDS PAGE electrophoresis detection
  • the electrophoresis conditions are: gel concentration 10%, first Run at constant voltage of 100V for 15min, then change to constant voltage of 200V to run to the front just before running out, then stain with Coomassie brilliant blue, decolorize with boiling water, and take pictures to record the electrophoresis results (the electrophoresis diagram is shown in Figure 2).
  • Use Labworks software to analyze the electrophoretic purity of the corresponding lanes of the samples collected by Protein A affinity chromatography, and record the analysis results.
  • Embodiment 5 Determination of protein concentration by BCA method
  • mice were immunized.
  • Thirty 6-8 week old female BALB/c mice were purchased and randomly divided into 3 groups (RBD-Fc, RBDSD1-Fc and negative control), 10 mice in each group.
  • Each mouse was immunized with 10ug protein.
  • Booster immunizations were given every two weeks for a total of two booster immunizations. From the first immunization, mouse blood was collected every two weeks, and the supernatant was collected by centrifugation for neutralizing antibody titer detection.
  • Cell preparation One day before the experiment, the cells to be infected were seeded in a 96-well cell culture plate with an inoculation amount of about 1 ⁇ 10 4 cells/well.
  • Pseudovirus infection Take out the cryopreserved pseudovirus and set it to thaw. After it is completely thawed, absorb the required amount of pseudovirus and add it to the cell culture system to infect the target cells. 6-8h after virus infection, replace the fresh medium to continue culturing.
  • the neutralizing antibody titer was taken as the maximum dilution of serum when the infection was inhibited by 50%.
  • Embodiment 7 Placement stability
  • RBDSD1-Fc can still induce the body to produce high titers of neutralizing antibodies after being placed in an incubator at 37 °C for 15 days, indicating that SD1 has the effect of stabilizing the protein structure of RBDSD1-Fc.

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Abstract

本发明公开一种含有新型冠状病毒COVID-19疫苗的抗原表位的融合蛋白，所述融合蛋白由新型冠状病毒COVID-19疫苗的抗原表位片段和免疫球蛋白Fc片段构成，其中新型冠状病毒COVID-19疫苗的抗原表位片段是S1亚基中的RBD片段和SD1片段。本发明经过研究和探索，发现在RBD的基础上融合SD1，可以增加其柔韧和可变性，容易形成头对尾的结构，结构更加稳定，相对于仅融合RBD更具有优势。进一步通过实验表明本发明获得的抗原表位的融合蛋白具较好的免疫特性和稳定性，其效果显著优于仅融合RBD时的效果。

Description

一种新型冠状病毒的重组亚单位疫苗及其应用 技术领域：
本发明属于生物及医药技术领域，具体涉及新型冠状病毒的重组亚单位疫苗及其应用，更具体涉及在通过基因工程手段人工合成的病毒基因在真核细胞中表达出有免疫活性的重组亚单位蛋白，并且利用表达出的重组蛋白研制成疫苗的方法。
背景技术：
新型冠状病毒属于β属的新型冠状病毒，有包膜，颗粒呈圆形或椭圆形，常为多形性，直径60-140nm。新冠病毒SARS-CoV-2的结构蛋白包括S蛋白、N蛋白、E蛋白、M蛋白。对于SARS-CoV-2而言，只有直接针对S蛋白的中和抗体才能够中和病毒的毒力、阻止病毒对机体的感染，冠状病毒外套膜上的刺突蛋白(Spike protein，S蛋白)是病毒感染过程中识别宿主细胞受体的一种关键蛋白，S蛋白由S1和S2两个亚基组成，其中S1亚基中包含受体结合结构域(Receptor Binding Domain,RBD)介导吸附作用，S2亚基主要体现融合活性。
新型冠状病毒COVID-19严重危害人们的生命健康，重在预防，因此疫苗能够非常有效的预防护病毒感染。针对新型冠状病毒COVID-19，已经有一些研究获得相关的疫苗，例如CN111333704A的专利申请中筛选和优化了可以用于制备新型冠状病毒COVID-19疫苗的优势抗原表位片段，并将该蛋白片段与人Fc片段融合，制备成新型的病毒疫苗。而2020年5月22日《The Lancet》重磅刊发我国首个由于军事科学院军事医学研究院生物工程研究所COVID-19疫苗I期临床试验结果，结果表明国内进入人体试验阶段的新型冠状病毒疫苗目前试验结果反馈良好，单一剂量的新5型腺病毒载体COVID-19(Ad5-nCoV)疫苗在14天内产生的特异抗体和T细胞，相关的疫苗已申请专利。
但是基于新型冠状病毒的特点，仍需开发有效的结果可靠的疫苗。
发明内容：
对此，本发明通过基因重组表达技术，把S1亚基中的RBDSD1序列和人Fc序列融合表达，研制出一种新型的疫苗。本发明经过研究和探索，通过同源建模，推断SD1序列有稳定RBD结构的作用，与仅融合单独的RBD相比，进一步融合SD1可以增加一对二硫键(在氨基酸第 591到538位间)，如此可让整个结构更加稳定，由此构成本发明的基础。进一步通过实验表明其效果非常好，最终完成本发明。
本发明首先提供一种含有新型冠状病毒COVID-19疫苗的抗原表位的融合蛋白，其特征在于，所述融合蛋白由新型冠状病毒COVID-19疫苗的抗原表位片段和免疫球蛋白Fc片段构成，其中新型冠状病毒COVID-19疫苗的抗原表位片段是S1亚基中的RBD片段和SD1片段。
优选地，所述RBD片段的氨基酸序列如SEQ ID NO：2所示；所述SD1片段的氨基酸序列如SEQ ID NO：3所示。
在具体实施方式中，所述免疫球蛋白Fc片段选自于人、小鼠、兔子、牛、山羊、猪、小鼠、兔、仓鼠、大鼠、或豚鼠的免疫球蛋白Fc片段；进一步地，所述免疫球蛋白Fc片段选自IgG、IgA、IgD、IgE或IgM的Fc片段；其中优选地，所述免疫球蛋白Fc片段选自IgG1Fc片段、IgG2Fc片段、IgG3Fc片段、或IgG4Fc片段；尤其优选地，所述免疫球蛋白Fc片段是人IgG Fc片段，最优选地其是如SEQ ID NO.5所示的氨基酸序列。
最优选地，所述融合蛋白包含SEQ ID NO.7所示的氨基酸序列。
本发明相应提供根据所述的融合蛋白的编码核酸，优选地，其中RBD片段的编码核苷酸序列如SEQ ID NO：1所示或其简并序列；所述SD1片段的编码核苷酸序列如SEQ ID NO：4所示或其简并序列；所免疫球蛋白Fc片段编码核苷酸序列如SEQ ID NO：6所示或其简并序列。最优选地，融合蛋白的编码核酸的核苷酸序列如SEQ ID NO：8所示或其简并序列。
本发明进一步提供含有如所述的编码核酸的重组载体、重组细胞。
本发明还提供一种新型冠状病毒COVID-19疫苗，其特征在于，所述疫苗包含所述的融合蛋白。进一步还包括佐剂，例如氢氧化铝。其中，所述融合蛋白通过化学合成，或基因重组方法获得。
本发明经过研究和探索，通过同源建模和实验发现在RBD的基础上融合SD1，可以增加其柔韧和可变性，容易形成头对尾的结构，结构更加稳定，相对于仅融合RBD更具有优势；同时，由于与Fc片段融合可以形成二聚体，进一步推测增加免疫原性和更加提高的稳定性。本发明步通过实验表明本发明获得的抗原表位的融合蛋白具较好的免疫特性和稳定性，其效果显著优于仅融合RBD时的效果。
附图说明
图1：质粒构建核酸胶以及双酶切鉴定(HindIII和XhoI)电泳图。加样顺序：A1：pcDNA3.1-RBD质粒，A2：pcDNA3.1-RBD双酶切鉴定(HindIII和XhoI)，A3：DNA Marker。B1： pcDNA3.1-RBD-Fc质粒，B2：pcDNA3.1-RBD-Fc双酶切鉴定(HindIII和XhoI)，B3：DNA Marker。C1：pcDNA3.1-RBDSD1-Fc质粒，C2：pcDNA3.1-RBDSD1-Fc双酶切鉴定(HindIII和XhoI)，C3：DNA Marker。
图2：SDS-PAGE电泳图。加样顺序：蛋白Marker，RBD，RBDSD1-Fc，RBD-Fc。
具体实施方式
本发明的以下实施例和附图仅说明实现本发明的具体实施方案，这些方案和附图不可以理解为对本发明的限制，任何在不脱离本发明的原理和实质的情况下所做的任何改变，均落在本发明的保护范围之内。
本实施例中所用到的实验技术与实验方法，如无特殊说明均为常规技术方法。本实施例中所使用的材料、试剂等，如无特殊说明，均可通过正规商业渠道获得。
实施例一、载体构建
通过化学合成的方法，由北京六合华大基因科技有限公司合成目的基因序列。其中，
RBD核苷酸序列如下(SEQ ID NO：1)：
Figure PCTCN2021107881-appb-000001
其编码的RBD氨基酸序列如下(SEQ ID NO：2)：
Figure PCTCN2021107881-appb-000002
SD1核苷酸序列如下(SEQ ID NO：4)：
Figure PCTCN2021107881-appb-000003
其编码的SD1氨基酸序列如下(SEQ ID NO：3)：
Figure PCTCN2021107881-appb-000004
hFc核苷酸酸序列如下(SEQ ID NO：6)：
Figure PCTCN2021107881-appb-000005
其编码的hFc氨基酸序列如下(SEQ ID NO：5)：
Figure PCTCN2021107881-appb-000006
RBDSD1-hFc核苷酸序列如下(SEQ ID NO：8)：
Figure PCTCN2021107881-appb-000007
Figure PCTCN2021107881-appb-000008
RBDSD1-hFc氨基酸序列(SEQ ID NO：7)
Figure PCTCN2021107881-appb-000009
由基因公司直接合成的方式将上述目的基因(即RBD、RBD-Fc和RBDSD1-Fc)构建到pcDNA3.1表达载体，分别获得DNA质粒pcDNA3.1-RBD、pcDNA3.1-RBD-Fc、pcDNA3.1-RBDSD1-Fc。并通过凝胶电泳进行了验证。
质粒的酶切：取质粒DNA，按下表中的酶切反应体系Xho I酶、Hand III酶进行酶切，于37℃，恒温培养箱中反应2h，随后进行凝胶电泳。
  双酶切
质粒DNA 4μl
10×buffer4 1μl
Hind III 0.5μl
Xho I 0.5μl
注射用水 4μl
总体积 10μl
琼脂糖凝胶的制备及电泳步骤如下：
(1)用电子天平称取0.5g琼脂糖，加入1×TAE 50mL，在微波炉中加热溶解，混匀，冷却至55℃左右时，倒入已准备好的制胶槽中，插上样品梳。
(2)待胶凝固后，拨去梳子，放入加有足够1×TAE电泳缓冲液的电泳槽中，缓冲液高出凝胶表面约1mm。
(3)将经酶切后的质粒DNA加入2μl 6×载样缓冲液，混匀，然后用微量移液器加入样品孔中，同时用合适的DNA分子量标准品作对照。
(4)接通电极，在110v/cm凝胶的电压下恒压进行电泳，DNA由负极向正极移动。
(5)当加样缓冲液中的溴酚蓝迁移至足够分离DNA片段的距离时，关闭电源。
(6)准备一个容器，倒入50mL的1×TAE电泳缓冲液，加入15μl GelredTM，混匀，把电泳胶放入容器中，避光染色30min。
(7)在紫外凝胶成像仪下观察，酶切片段的大小。预期pCDNA3.1大小为5.6Kb，RBD的大小为715bp，RBDhFc大小为1399bp，RBDSD1hFc的大小为1549bp。如图1所示，验证正确的情况下进行后续的实验。
实施例二、细胞瞬转
①转染前一天按照2.0×10 6cells/mL密度接种，培养第二天细胞密度可至4.0×
10 6cells/mL左右；
②培养第二天细胞计数后，细胞活率＞95％，活细胞密度≥4.0×
10 6cells/mL，可直接使用；若细胞密度低于4.0×10 6cells/mL，可通过离心(800rpm，5min)收集细胞，将细胞以4.0×10 6cells/mL密度重悬于Transpro CD01培养基中；
③按照优化后的瞬转工艺，制备DNA和PEI混合液；
④将混合液加入到培养液中，进行培养；
⑤培养18h后，建议补加一次终浓度为2mM丙戊酸(VPA)和初始培养体积5％+0.5％DN feed2+DN feed B2，可进一步提高活细胞密度和蛋白表达量，瞬转后培养过程若出现细胞结团现象，可以添加0.05-0.10g/L硫酸葡聚糖。
⑥培养至7天，或者活力低于60％，结束培养，收集细胞上清，用于蛋白纯化。
实施例三、蛋白纯化
一、对于RBD-Fc，RBDSD1-Fc的纯化采取下述Protein A亲和层析方法
层析柱：AT Protein A Diamond亲和层析介质，柱体积CV：5ml，流速：5ml/min，限压：≤0.3MPa。
预处理：用纯化水冲洗至少5个CV。
平衡：先用洗脱缓冲液淋洗1个CV，后用结合缓冲液充分平衡至少10个CV备上样。
上样：取过滤后CHO悬浮细胞培养物上样至平衡好的层析柱，收集上样流穿，并从中取样100μl用于电泳检测。
淋洗：上样完成后流速提高至5ml/min，用结合缓冲液淋洗至少6个CV至UV基线平。
洗脱：用100％洗脱缓冲液进行洗脱，洗脱至UV基线平，分管收集各洗脱组分，同时在各收集管内根据收集体积加入适量收集体积的中和缓冲液并混匀。
二、对于RBD的纯化采取下述SP阳离子交换层析
层析柱：SP阳离子交换层析柱，柱体积CV：5ml，流速：5ml/min，限压：≤0.3MPa。
预处理：先用1M NaOH冲洗至少3个CV，后用纯化水冲洗至少10个CV。
平衡：先用SP-B Buffer淋洗2个CV，后用SP-A Buffer充分平衡至少10个CV备上样。
上样：取G25换液收集样品上样至平衡好的层析柱，收集上样流穿，并从中分别取样100μl用于电泳检测。
淋洗：上样完成后流速提高至5ml/min，用SP-A Buffer淋洗至少6个CV至UV基线平。
洗脱：先用0-20％SP-B Buffer，20个CV进行线性洗脱，接着用20-100％SP-B Buffer，10个CV进行线性洗脱，分管收集各洗脱组分。
实施例四、SDS PAGE电泳检测
取纯化过程中收集的各样品按100μl样品+25μl Loading dye(还原型)的比例配制各待电泳样品(沸水浴5min)，然后取适量上样进行电泳，电泳条件为：胶浓度10％，先恒压100V跑15min，后改恒压200V跑至前沿刚好跑出，接着考马斯亮蓝染色，沸水脱色，拍照记录电泳结果(电泳图如图2所示)。用Labworks软件对Protein A亲和层析收集样品对应泳道进行电泳纯度分析，记录分析结果。
实施例五、BCA法蛋白浓度测定
将供试样品用注射用水稀释不同倍数，使稀释后样品浓度在编准品浓度范围内，并记录稀释倍数。将稀释好的对照品溶液、供试品溶液以20ul/孔量加入微孔板，每个样品平行上样3复孔。将甲液(2，2'-联喹啉-4，4'二羧酸钠溶液)：乙液(硫酸铜)按50:1的比例配置适量碱性铜试液，混匀后200ul/孔加入微孔板，37℃温育30min。确保孔中无气泡、孔底洁净后，用酶标仪在570nm处测定吸光度值。各样品3复孔平均值减去空白孔3孔平均值计算最终的光吸收值，用对照品值。对其浓度作标准曲线，A＝a*c+b，计算质控样品和供试品的浓度，结果如下表所示。
  RBD RBD-Fc RBDSD1-Fc
转染体系 40ml 40ml 40ml
上清蛋白浓度 0.03mg/ml 0.23mg/ml 0.44mg/ml
纯化后蛋白浓度 0.22mg/ml 0.72mg/ml 1.61mg/ml
总量 0.3mg 3.5mg 10.2mg
实施例六、小鼠免疫和中和抗体检测
将各重组蛋白分别与氢氧化铝佐剂混合，并免疫小鼠。采购30只6-8周雌性BALB/c小鼠，随机分成3组(RBD-Fc，RBDSD1-Fc和阴性对照)，每组10只。每只小鼠肌肉免疫10ug蛋白。每隔两周加强免疫一次，共加强免疫两次。从第一次免疫开始，每隔两周采集小鼠血液，离心取上清，进行中和抗体滴度检测。
中和抗体滴度检测实验方法如下：
1.细胞准备：实验前一天，将约为1x10 4个细胞/孔的接种量把待感染细胞接种于96孔细胞培养板中。
2.假病毒感染：取出冻存的假病毒置融化，待完全融化后，吸取所需量假病毒加入细胞培养体系中感染目的细胞，病毒感染后6-8H后更换新鲜培养基继续培养。
3.感染检测：细胞感染假病毒48-72H后,通过观察检测荧光素酶的活性判定感染效率。
4.以抑制50％的感染时血清的最大稀释倍数作为中和抗体滴度。
实验结果如下表所示，可以发现RBDSD1-Fc中和抗体滴度远高于RBD-Fc，而且高滴度中和抗体维持时间长。
Figure PCTCN2021107881-appb-000010
实施例七、放置稳定性
将纯化的蛋白RBD-Fc和RBDSD1-Fc放入37℃温箱，放置15天(相当于4℃放置两年)，参照实施例六免疫小白鼠，并检测中和抗体，发现37℃温箱放置15天的RBDSD1-Fc蛋白仍然能诱导机体产生高滴度的中和抗体，而37℃温箱放置15天的RBD-Fc蛋白诱导机体的中和抗体滴度相对较低(结果见下表)。
Figure PCTCN2021107881-appb-000011
Figure PCTCN2021107881-appb-000012
由此可见，RBDSD1-Fc在37℃温箱放置15天，仍然能诱导机体产生高滴度的中和抗体，说明SD1有稳定RBDSD1-Fc蛋白结构的作用。

Claims (10)

  1. 一种含有新型冠状病毒COVID-19疫苗的抗原表位的融合蛋白，其特征在于，所述融合蛋白由新型冠状病毒COVID-19疫苗的抗原表位片段和免疫球蛋白Fc片段构成，其中新型冠状病毒COVID-19疫苗的抗原表位片段是S1亚基中的RBD片段和SD1片段。
  2. 如权利要求1所述的融合蛋白，其特征在于，所述RBD片段的氨基酸序列如SEQ ID NO：2所示；所述SD1片段的氨基酸序列如SEQ ID NO：3所示。
  3. 如权利要求1所述的融合蛋白，其特征在于，所述免疫球蛋白Fc片段选自于人、小鼠、兔子、牛、山羊、猪、小鼠、兔、仓鼠、大鼠、或豚鼠的免疫球蛋白Fc片段；进一步地，所述免疫球蛋白Fc片段选自IgG、IgA、IgD、IgE或IgM的Fc片段；其中优选地，所述免疫球蛋白Fc片段选自IgG1 Fc片段、IgG2 Fc片段、IgG3 Fc片段、或IgG4 Fc片段；尤其优选地，所述免疫球蛋白Fc片段是人IgG Fc片段，最优选地其是如SEQ ID NO.5所示的氨基酸序列。
  4. 根据权利要求3所述的融合蛋白，其特征在于，所述融合蛋白包含SEQ ID NO.7所示的氨基酸序列。
  5. 根据权利要求1至4任一项所述的融合蛋白的编码核酸，优选地，其中RBD片段的编码核苷酸序列如SEQ ID NO：1所示或其简并序列；所述SD1片段的编码核苷酸序列如SEQ ID NO：4所示或其简并序列；所免疫球蛋白Fc片段编码核苷酸序列如SEQ ID NO：6所示或其简并序列。
  6. 如权利要求5所述的编码核酸，其特征在于，其核苷酸序列如SEQ ID NO：8所示或其简并序列。
  7. 含有如权利要求5所述的编码核酸的重组载体、重组细胞。
  8. 一种新型冠状病毒COVID-19疫苗，其特征在于，所述疫苗包含权利要求1-4任一项所述的融合蛋白。
  9. 如权利要求8所述的疫苗，其特征在于，其还包括佐剂，例如氢氧化铝。
  10. 如权利要求8所述的疫苗，其特征在于，所述融合蛋白通过化学合成，或基因重组方法获得。
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