WO2023151446A1 - β属冠状病毒融合重组蛋白及其制备方法和应用 - Google Patents

β属冠状病毒融合重组蛋白及其制备方法和应用 Download PDF

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WO2023151446A1
WO2023151446A1 PCT/CN2023/071585 CN2023071585W WO2023151446A1 WO 2023151446 A1 WO2023151446 A1 WO 2023151446A1 CN 2023071585 W CN2023071585 W CN 2023071585W WO 2023151446 A1 WO2023151446 A1 WO 2023151446A1
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protein
covid19
recombinant
fusion
fragment
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蒋永平
王含璐
蒋文宏
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苏州方舟生物科技有限公司
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    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
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    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
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    • C07—ORGANIC CHEMISTRY
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
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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

Definitions

  • the invention belongs to the field of biology, and more specifically, the invention relates to fusion recombinant protein of ⁇ -coronavirus and application thereof.
  • SARS-CoV-2 novel coronavirus
  • COVID-19 infectious pneumonia
  • the new coronavirus vaccines currently under research mainly include inactivated vaccines, adenovirus vector vaccines, nucleic acid vaccines (mRNA vaccines), live attenuated vaccines, etc. These vaccines generally have insufficient specific immunogenicity, and the protective effect varies greatly among populations. There are shortcomings such as antibody-dependent infection enhancement and safety to be considered. In addition, in the face of the rapid mutation of the new coronavirus, such as the widely appearing variants of Delta and Omicron, the specific action time and effect of the vaccine are greatly limited. At present, more than 300 million new crowns have been diagnosed. In view of the high infectivity and mutation of the new crown virus, it is imminent to find a general vaccine to prevent and treat multiple coronaviruses and their variants.
  • S protein plays an important role in the combination and invasion of coronaviruses.
  • the S protein is located on the surface of the coronavirus, forming a unique spike-like structure on the surface of the virus.
  • the S protein is composed of two subunits, S1 and S2, of which S1 forms the globular head of the spike protein and contains the large receptor-binding structure of the S protein.
  • domain N-terminal domain NTD and receptor-binding domain RBD
  • S2 forms the stem of the spike protein and participates in the membrane fusion process.
  • the S2 subunit contains three functional domains, fusion peptide (FP) and peptide repeats (HR1 and HR2), after the RBD at the tip of S1 binds to the receptor, the FP in S2 inserts into the host cell membrane to change conformation, stimulating HR1 and HR2 A six-helix bundle (6HB) is formed, resulting in fusion of the viral membrane with the cellular membrane.
  • FP fusion peptide
  • HR1 and HR2 peptide repeats
  • the S protein has the activity of binding to human upper respiratory tract cell receptors and membrane fusion, and is a key protein that mediates the recognition and infection of human cells by this type of virus.
  • CN113943375A discloses a class of recombinant fusion protein derived from HR region of novel coronavirus S2 protein and its application.
  • This type of novel coronavirus recombinant fusion protein is a recombinant fusion protein obtained by connecting two membrane fusion-related conservative amino acid sequences HR1 and HR2 of the new coronavirus membrane protein S2 protein through a linker peptide.
  • the recombinant fusion protein can be induced and expressed in Escherichia coli, has a high expression level and is easy to purify.
  • the novel coronavirus recombinant fusion protein provided by the present invention can form and maintain a stable trimer structure, simulate the conformation of the novel coronavirus membrane fusion intermediate state, and can be used as a detection material for detecting the novel coronavirus membrane fusion process; it has a good
  • the anti-new coronavirus activity and good immunogenicity have broad application prospects in the development of protein drugs for the prevention or treatment of new coronaviruses, as well as the development of new coronavirus vaccines and anti-new coronavirus antibodies.
  • CN112409469B discloses a fusion protein, a recombinant vector, a recombinant dendritic cell and its application for transmembrane expression of novel coronavirus antigen S2, belonging to the technical field of whole-cell vaccines.
  • the fusion protein includes sequentially linked CD4 signal peptide, Novel coronavirus antigen S2 protein, Flag tag sequence and CD4 transmembrane domain; the invention expresses S2 alone in transmembrane cells, avoiding the risk of ADE that may be caused by other S protein epitopes.
  • the fusion protein constructed by the invention Cellular vaccines can induce higher neutralizing antibody titers in mice.
  • the fragment is COVID19-SF5
  • the sequence is the 880th amino acid to the 1084th amino acid of the S protein of the new coronavirus COVID-19, specifically, the amino acid sequence of the fragment is (SEQ ID NO .SEQ ID NO.13):
  • the present invention fuses and expresses the constant conserved fragment (COVID19-SF5) and the receptor binding domain (RBD) fragment to obtain a ⁇ -coronavirus fusion recombinant protein whose amino acid sequence is as SEQ ID NO.1, thereby providing a more potent , a constant and universal vaccine candidate recombinant fusion protein for this type of coronavirus, providing broader and more favorable protection measures from two dimensions of inhibitory receptor recognition and universal protection.
  • COVID19-SF5 the constant conserved fragment
  • RBD receptor binding domain
  • the RBD region of the S protein of the new coronavirus COVID-19 is a COVID19-SF2 fragment (SEQ ID NO.10), and its amino acid sequence is the 305th amino acid to the 525th amino acid of the S protein of the new coronavirus COVID-19 .
  • the RBD region of the present invention is mainly based on references Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh C-L, Abiona O, et al.Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.Science.2020;
  • the RBD disclosed in 367(6483):1260-3 is the 335th to the 522nd, and the COVID19-SF2 of this application includes the RBD area, and there is a certain overlap with the front and rear areas.
  • amino acid sequence of the fusion recombinant protein is shown in SEQ ID NO.1. Specifically, the amino acid sequence of COVID19-SF2+5 is as follows:
  • the present invention further proposes a gene encoding the above-mentioned fusion recombinant protein.
  • the nucleotide sequence of the gene is SEQ ID NO.2.
  • the present invention also proposes a recombinant vector, which comprises the gene encoding the fusion recombinant protein and the vector.
  • the vectors shown can be pET series vectors, mammalian expression vectors pcDNA3 series, etc.
  • the present application uses the expression vector pQE-3.
  • the present invention also proposes a recombinant bacterium comprising the above-mentioned recombinant vector.
  • the host bacteria can be selected including Escherichia coli BL21, M15, insect cells sf9, mammalian cells CHO, 293 and so on.
  • the present invention also proposes the application of the fusion recombinant protein, the gene encoding the fusion recombinant protein, the recombinant vector, and the recombinant bacteria in the preparation of a universal vaccine and universal antibody for ⁇ -coronavirus.
  • the expression strain construction and protein expression and purification of the COVID19-SF2 protein fragment and COVID19-SF5 protein fragment fusion protein of SARS-CoV-2 are realized by the following methods:
  • PCR of the COVID19-SF2 protein fragment and the COVID19-SF5 protein fragment were carried out, and after the amplification was completed, the PCR products were verified by 2% agarose gel electrophoresis. PCR products were purified using a PCR product purification kit.
  • the target gene is connected to the expression vector pQE-3 through the BamH I and Hind III restriction sites at the 5'-end and 3'-end of the sequence. 1% agarose electrophoresis to verify the digested product. The vector and the target gene were recovered and purified using gel recovery and purification kits to recover and purify the digested products. After purification, the nucleic acid concentration was detected with a One drop spectrophotometer.
  • Transformation the expression vector containing the fusion protein gene is transformed into Escherichia coli M15 strain by a competent method.
  • Selection of positive clones selection of bacterial strains grown on selective plates and performing colony PCR, and induction of protein expression for PCR-positive bacterial strains.
  • Induced expression Take colony PCR-positive clones for expansion culture.
  • the specific method is: pick the positive clones on the plate and culture overnight, take overnight bacteria, add fresh medium to expand culture, culture for about 4 hours, and add a final concentration of 100mM IPTG was induced to express for 4h.
  • the cell pellet was harvested by centrifugation, and the protein expression was verified by SDS-PAGE.
  • Ni-NTA affinity column purification Pack the column according to the steps recommended by the Ni-NTA affinity column manufacturer, and then equilibrate the affinity column with 8M urea (5 column volumes, dissolved in phosphate buffer, pH8.0) , Loading the inclusion body solution dissolved in guanidine hydrochloride at a speed of 5ml/min, after loading, the impurity protein was eluted with sodium phosphate (5 column volumes) of pH 6.0, and then collected with sodium acetate of pH 4.5 target protein.
  • 8M urea 5 column volumes, dissolved in phosphate buffer, pH8.0
  • Loading the inclusion body solution dissolved in guanidine hydrochloride at a speed of 5ml/min after loading, the impurity protein was eluted with sodium phosphate (5 column volumes) of pH 6.0, and then collected with sodium acetate of pH 4.5 target protein.
  • Refolding step using urea gradient solution dialysis Dilute the above purified protein solution to 0.3mg/ml with 3M urea (in sodium acetate buffer, 15PH4.5), dialyze with different concentrations of urea at 4°C Each solution was dialyzed once for 24 hours each time.
  • the ratio of the inner and outer fluids of the dialysis bag was 1:5, the inner fluid was 3.5M urea-sodium acetate buffer, and the outer fluids were 3M, 2.5M, 1.5M, 1M, and 0.5M in sequence. , 0M and 0M urea in dialysis buffer.
  • a high-efficiency and general-purpose coronavirus fusion protein was obtained, named "COVID19-SF2+5", and its antibody has certain cross-reactivity with each S protein fragment, especially with COVID19-SF2 and COVID19-SF5 High binding ability. It is suggested that the fusion protein not only retains the RBD region, but can induce the production of IgG antibodies that specifically block the binding of the virus to the receptor, and at the same time includes a constant conservative fragment COVID19-SF5, which can induce a wider variety of S protein fragments. Cross-reactive broad-spectrum IgG antibodies.
  • the antiserum of the "COVID19-SF2+5" fusion protein was obtained by immunization of mice, the serum comprehensive antibody IgG was obtained by purification, and the serum comprehensive antibody IgG of the new coronavirus SARS-CoV-2S protein COVID19-SF2+COVID19-SF5 fusion protein was prepared.
  • the present invention further proposes a method for industrialized fermentation to prepare recombinant fusion proteins, comprising the following steps:
  • the recombinant bacteria are used as the seed bacteria, and the seed bacteria are amplified as the seed liquid through night shaking bacteria;
  • the seed liquid is fermented and cultivated in 2 ⁇ YT medium. After fermentation, the bacteria are collected by an industrial automatic continuous centrifuge. The collected bacteria are first made into a suspension with the extract A, and then cracked with an aqueous lyase. Then treat with extract B, collect the precipitated inclusion body after centrifugation, dilute with buffer solution, and centrifuge, the precipitate is insoluble inclusion body;
  • the collected samples were dialyzed in a chromatographic cabinet at 4°C.
  • the dialyzed samples were centrifuged to obtain the supernatant, which was concentrated by an ultrafiltration concentrator, and then purified by AKTA protein purification system Sephadex G-75 chromatography; according to The protein peak collection sample of the AKTA protein purification system is the purified fusion protein.
  • Fermentation tanks and pipelines are sterilized. Empty tanks need to be sterilized at 121°C for 30 minutes before each fermentation, and the prepared medium is put into the tank and sterilized again at 121°C for 30 minutes. After cooling to the required temperature of 37°C, inoculate the seed solution ( The dominant expression strains that were frozen before, the seed bacteria were amplified by night shaking); 40L fermenter was added with 500mL of seed liquid and 35000mL of medium (overnight bacteria were used as seed liquid, and the medium was 2 ⁇ YT medium), of which, 2 ⁇ YT Culture medium: 1L of culture medium contains 16g of tryptone, 10g of yeast extract, and 5g of sodium chloride. Stir evenly and then sterilize under high temperature and high pressure.
  • Fermentation conditions such as parameters such as temperature, pH, oxygen flow rate and fermentation time, are all controlled by the supporting computer operating system of the fermenter. Set the temperature at 37°C, pH 7.0, and ferment for about 7 hours. (Dissolved oxygen value or dissolved oxygen concentration: DO value 60%, temperature 37 ° C, pH 7.0. Add inducer IPTG when the bacterial concentration reaches the peak value, and the total incubation time is 7 hours.)
  • the bacterial cells were collected by an industrialized automatic continuous centrifuge at a centrifugation speed of 10,000 g and a temperature of 4° C. for 1 hour.
  • extract solution B (1.5M NaCl, 100mM CaCl 2 , 100mM MgCl2, 0.002% DNase I). For every 1000 mL of the above lysate, add 100 mL of extraction solution B.
  • the precipitated inclusion bodies were collected and evenly suspended in 50 mM phosphate buffer (containing 0.15 M sodium chloride and 4 M urea, pH 7.0). Add 10 mL of buffer solution to 1 g of precipitate, centrifuge at 10,000 g at 4°C for 10 min, the precipitate is insoluble inclusion body, which can be collected and stored at -80°C for half a year.
  • 50 mM phosphate buffer containing 0.15 M sodium chloride and 4 M urea, pH 7.0.
  • the supernatant is concentrated by an ultrafiltration concentrator, and every 2000 mL is concentrated to 300 mL.
  • the sample was purified by AKTA protein purification system Sephadex G-75 chromatography.
  • the length of the column is 1.2 meters, the diameter is 4cm, and the flow rate is 1mL/min.
  • the purified protein is passed through a Polymyxin (Bio-rad) chromatography column to remove endotoxin.
  • the present invention combined with the previous research of our laboratory, starting from the structural and functional analysis of the S protein of ⁇ -coronavirus, carried out the regionalization and linearization of amino acid sequences of various coronavirus proteins. Origin matching analysis, segmented expression through S protein homology structure, establishment of a recombinant protein fragment library covering the entire region of S protein, and research on the cross-reaction of serum antibody library obtained from immunized mice with various S protein fragments, etc.
  • the S protein fragment COVID19-SF5 of SARS-CoV-2 which has a general cross-reaction with each fragment of the SARS-CoV S protein and each fragment of the SARS-CoV-2 S protein, was found.
  • the COVID19-SF5 protein fragment with general cross-reactivity and the COVID19-SF2 protein fragment containing the virus receptor binding domain (RBD) are connected through a flexible linking peptide Gly4Ser to form a fusion protein with multifunctional effects, and through The fusion protein was immunized into mice to obtain serum IgG antibody. After testing, the ability of the fusion protein to bind to cells is significantly higher than that of individual COVID19-SF2 or COVID19-SF5 protein fragments, and its serum IgG antibody can cross-react with various S protein fragments, and can significantly inhibit pseudovirus infection of cells.
  • the universal fusion protein vaccine has obvious advantages:
  • the product of the present invention is a fusion protein after the connection of two targeted specific fragments of the viral S protein, and does not involve genes, other viral vectors, or inactivated viruses entering the human body, product quality control and quality assurance system Clearly, to ensure the safety of the product;
  • Figure 1 is a schematic diagram of double enzyme digestion verification plasmid construction
  • Fig. 2 is the expression of SDS-PAGE identification recombinant fusion protein, wherein, Lane M: protein marker; Lane 1: COVID19-SF2+COVID19-SF5 fusion protein;
  • Figure 3 shows the binding ability of three protein fragments to Vero-E6 cells.
  • Example 1 Construction of expression strain and protein expression and purification of fusion protein "COVID19SF-2+5" of COVID19-SF2 protein fragment and COVID19-SF5 protein fragment of SARS-CoV-2.
  • the amplification conditions are: 94°C, 30s; 56°C, 1min; 72°C, 1min30s; 35 cycles.
  • PCR products were verified by 2% agarose gel electrophoresis. PCR products were purified using a PCR product purification kit. Among them, the COVID19-SF2 gene sequence is shown in SEQ ID NO.7, and the COVID19-SF5 gene sequence is shown in SEQ ID NO.8.
  • the amplification conditions are: 94°C, 30s; 56°C, 1min; 72°C, 2min40s; 30 cycles.
  • PCR products were verified by 1% agarose gel electrophoresis. PCR products were purified using a PCR product purification kit.
  • Enzyme digestion The target gene (SEQ ID NO.2) was connected to the expression vector pQE-3 through the BamH I and Hind III restriction sites at the 5' end and the 3' end of the sequence.
  • the enzyme digestion system is as follows:
  • the digestion conditions are as follows: digestion in a water bath at 37°C for 0.5 hours. 1% agarose gel to verify the digested product. The vector and the target gene were recovered and purified using gel recovery and purification kits to recover and purify the digested products. After purification, one drop was used to detect the nucleic acid concentration.
  • the enzyme-linked system is as follows:
  • the obtained enzyme-linked product is the expression vector containing the fusion protein gene.
  • the expression vector containing the fusion protein gene was transformed into Escherichia coli M15 strain by competent method.
  • Ni-NTA affinity column purification Pack the column according to the steps recommended by the Ni-NTA affinity column manufacturer, and then equilibrate the affinity column with 8M urea (5 column volumes, dissolved in phosphate buffer, pH8.0) , Loading the inclusion body solution dissolved in guanidine hydrochloride at a speed of 5ml/min, after loading, the impurity protein was eluted with sodium phosphate (5 column volumes) of pH 6.0, and then collected with sodium acetate of pH 4.5 target protein.
  • 8M urea 5 column volumes, dissolved in phosphate buffer, pH8.0
  • Loading the inclusion body solution dissolved in guanidine hydrochloride at a speed of 5ml/min after loading, the impurity protein was eluted with sodium phosphate (5 column volumes) of pH 6.0, and then collected with sodium acetate of pH 4.5 target protein.
  • Refolding step using urea gradient solution dialysis Dilute the above purified protein solution to 0.3mg/ml with 3M urea (included in sodium acetate buffer, pH4.5), and then dialyze with different concentrations of urea at 4°C Each solution was dialyzed once for 24 hours each time.
  • the ratio of the inner and outer fluids of the dialysis bag was 1:5, the inner fluid was 3.5M urea-sodium acetate buffer, and the outer fluids were 3M, 2.5M, 1.5M, 1M, and 0.5M in sequence. , 0M and 0M urea in dialysis buffer.
  • the target protein solution was centrifuged at 15,000 rpm for 20 minutes in a low-temperature centrifuge, and the protein concentration was determined by the Braford method. It was sterilized by filtration through a 0.22 ⁇ m filter membrane, added mannitol, and stored in a -80°C refrigerator.
  • Plasmid construction was verified by double enzyme digestion, as shown in Figure 1. Use BamHI restriction endonuclease and Ncol restriction endonuclease 673bp away from the HindIII restriction endonuclease to perform double digestion. The two bands after digestion: the target gene plus the 673 bp between HindIII enzyme and Ncol enzyme is 1966 bp, and the remaining vector is 2723 bp, which is consistent with the theory.
  • Example 2 Identification of a universal fusion protein of ⁇ -coronavirus.
  • the specific and general cross-reactivity of the fusion protein antibody was tested by ELISA method.
  • the comprehensive antibody IgG (50 ⁇ g/mL) titer (see Table 2) after the first immunization of the fusion protein was detected by ELISA (see Table 2).
  • the mice had a good immune effect on the fusion protein fragment, and the antibody titer reached Still up to 1:1600.
  • the binding ability of the fusion protein comprehensive antibody IgG to each S protein fragment was detected by ELISA, and it was found that the antibody had a certain cross-reactivity with each S protein fragment, and the binding ability was high, even 6 months after the first immunization, although The reaction efficiency is weakened, but the antibody still has obvious cross-reaction with most of the protein fragments, suggesting that the fusion protein can not only produce highly specific neutralizing antibodies after immunizing mice, but also contain a variety of ⁇ -coronaviruses that are constantly conserved, Specific protein fragments, ELISA detection results are shown in Table 7-2 and 7-3.
  • Example 3 Detection of mouse safety and antibody response of universal specific coronavirus fusion protein vaccine.
  • mice 20 BALB/c mice were immunized with 0.20mg/ml COVID19-SF2+5 fusion protein, the safety of the mice during injection was observed, and the IgG response level was detected on day 28 and day 45.
  • mice Observe the safety and IgG response detection of mice 45 days after injection of COVID19-SF2+5 fusion protein.
  • mice inoculated with the COVID19-SF2+5 fusion protein were in good health, and all of them could produce effective IgG antibodies.
  • the results of safety testing and IgG response testing are shown in Table 8:
  • Example 4 Detection of the binding ability of the universal specific coronavirus fusion protein COVID19-SF2+5 to cells.
  • Example 5 Detection of the inhibitory ability of the synthetic antibody IgG corresponding to the universal fusion protein of ⁇ -coronavirus against pseudoviruses.
  • the expression of luciferase in the cells infected with the SARS-CoV-2 pseudovirus was detected by a multi-functional microplate reader, so as to judge the inhibitory ability of the comprehensive antibody corresponding to the universal fusion protein against the pseudovirus.
  • hACE2-293T cells were used as infected cells, and hACE2-293T cells were seeded in 96-well plates at 2 ⁇ 10 4 /well the night before. After 18 hours, 10 ⁇ g/mL of fusion protein antiserum IgG was mixed with 650 TCID 50 /well of pseudovirus. The mixture was then added to the cells and incubated for 48 hours. Measure the expression of luciferase by a multiplate reader using a luciferase detection kit according to the manufacturer's protocol to obtain the antiviral capacity of serum antibodies. Set up a cell control containing only cells and a virus control containing only virus and cells in each plate. Three parallel experiments were set up for each group. The inhibition rate of the serum antibody was calculated considering the inhibition rate of the cell control containing only cells as 100%, and the inhibition rate of the virus control containing both virus and cells as 0%.
  • Detect the inhibitory rate (see Table 10) of fusion protein antiserum to pseudovirus infected cells by pseudovirus neutralization experiment be the result value of three parallel experiments, as seen in the table, the serum that fusion protein COVID19-SF2+5 immune mice produces IgG antibody can inhibit the infection of cells by pseudovirus to a certain extent, and its inhibition rate is about 40%.
  • Fermentation tanks and pipelines are sterilized. Empty tanks need to be sterilized at 121°C for 30 minutes before each fermentation, and the prepared medium is put into the tank and sterilized again at 121°C for 30 minutes. After cooling to the required temperature of 37°C, inoculate the seed solution ( The dominant expression strains that were frozen before, the seed bacteria were amplified by night shaking); 40L fermenter was added with 500mL of seed liquid and 35000mL of medium (overnight bacteria were used as seed liquid, and the medium was 2 ⁇ YT medium), of which, 2 ⁇ YT Culture medium: 1L of culture medium contains 16g of tryptone, 10g of yeast extract, and 5g of sodium chloride. Stir evenly and then sterilize under high temperature and high pressure.
  • Fermentation conditions such as parameters such as temperature, pH, oxygen flow rate and fermentation time, are all controlled by the supporting computer operating system of the fermenter. Set the temperature at 37°C, pH 7.0, and ferment for about 7 hours. (Dissolved oxygen value or dissolved oxygen concentration: DO value 60%, temperature 37°C, pH 7.0. Add inducer IPTG when the bacterial concentration reaches the peak value, and the total incubation time is 7 hours.)
  • the bacterial cells were collected by an industrialized automatic continuous centrifuge at a centrifugation speed of 10,000 g and a temperature of 4° C. for 1 hour.
  • extract solution B (1.5M NaCl, 100mM CaCl 2 , 100mM MgCl2, 0.002% DNase I). For every 1000mL of the above lysate, add 100mL of extraction solution B.
  • the precipitated inclusion bodies were collected and evenly suspended in 50 mM phosphate buffer (containing 0.15 M sodium chloride and 4 M urea, pH 7.0). Add 10 mL of buffer solution to 1 g of precipitate, centrifuge at 10,000 g at 4°C for 10 min, the precipitate is insoluble inclusion body, which can be collected and stored at -80°C for half a year.
  • 50 mM phosphate buffer containing 0.15 M sodium chloride and 4 M urea, pH 7.0.
  • the supernatant is concentrated by an ultrafiltration concentrator, and every 2000 mL is concentrated to 300 mL.
  • the sample was purified by AKTA protein purification system Sephadex G-75 chromatography.
  • the length of the column is 1.2 meters, the diameter is 4cm, and the flow rate is 1mL/min.
  • the purified protein is passed through a Polymyxin (Bio-rad) chromatography column to remove endotoxin.
  • the present invention proposes an idea of fusion protein of ⁇ -coronavirus and its preparation method. There are many methods and approaches to realize the technical solution of the present invention. The above description is only a preferred embodiment of the present invention. Those of ordinary skill in the art can also make some improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. All components that are not specified in this embodiment can be realized by existing technologies.

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Abstract

本发明公开一种β属冠状病毒融合重组蛋白，包括新冠病毒COVID-19的S蛋白的RBD区域和COVID19-SF5片段，所述COVID19-SF5片段的氨基酸序列为新冠病毒COVID-19的S蛋白的第880位氨基酸至第1084位氨基酸。本发明通过将恒定保守片段(COVID19-SF5)与受体结合域(RBD)片段进行融合表达，提供更强效的、针对该类冠状病毒的恒定通sF用疫苗候选重组融合蛋白，从抑制受体识别和通用保护两个维度提供更广泛、更有利的保护措施。

Description

β属冠状病毒融合重组蛋白及其制备方法和应用 技术领域
本发明属于生物学领域，更具体地，本发明涉及β属冠状病毒融合重组蛋白及其应用。
背景技术
2019年底起，新型冠状病毒(SARS-CoV-2)感染性肺炎(COVID-19)从中国、世界各地逐渐流行，并迅速蔓延至国内及全球。除中国以外，在世界各地出现了严重疫情，造成了全球范围内严重卫生问题，截止目前，疫情的流行形势仍然十分严峻。新冠病毒与非典型性肺炎病毒(SARS-CoV)和中东呼吸综合征病毒(MERS-CoV)同属β属-冠状病毒，能够引起极为严重的呼吸系统综合征。虽然目前已有多款疫苗上市，但由于新冠病毒的高突变率，不同疫苗在预防感染方面的效力均出现不同程度的降低，特别是最近出现的Omicron毒株具有强烈的免疫逃逸能力，不仅可躲避大多数治疗性单克隆抗体，且在很大程度上避开了已上市疫苗产生的抗体免疫。最新的研究报道也有表明，Omicron毒株有新的侵入细胞途径。此外，未来仍存在爆发其他新型冠状病毒的风险，因此研发针对此类冠状病毒(包括正流行的新冠病毒、Delta毒株、Omicron毒株及未来可能发生的冠状病毒)的通用性预防性高效疫苗迫在眉睫。
目前在研的新冠病毒疫苗主要包括灭活疫苗、腺病毒载体疫苗、核酸疫苗(mRNA疫苗)、减毒活疫苗等，这些疫苗普遍存在特异免疫原性不足，保护效果在人群间差别较大，存在抗体依赖感染增强作用，安全性有待考量等缺点。此外，面对新冠病毒的快速变异，如目前广泛出现的Delta和Omicron等变体导致疫苗的特异性作用时间和作用效果大大受限。目前新冠确诊已超3亿，鉴于新冠病毒的高传染性、突变性，寻找应对多种冠状病毒及其变体的通用疫苗防治药物迫在眉睫。
S蛋白(Spike)在冠状病毒的结合和入侵中发挥着重要作用。S蛋白位于冠状病毒表面，组成了病毒表面上独特的穗状结构，S蛋白由S1和S2两个亚基组成，其中S1形成了刺突蛋白的球状头，包含S蛋白的大受体结合结构域(N端结构域NTD和受体结合结构域RBD)，负责识别宿主细胞受体，而S2形成了刺突蛋白的茎，参与膜融合过程。S2亚基包含三个功能域，融合肽(FP)和肽重复序列(HR1和HR2)，在S1尖端的RBD与受体结合后，S2中的FP插入宿主细胞膜而改变构象，刺激HR1和HR2形成六螺旋束(6HB)，导致病毒膜与细胞膜融合。
S蛋白具有与人上呼吸道细胞受体结合和膜融合活性，是介导这类病毒识别、感染人细胞的关键蛋白。CN113943375A公开了一类来源于新型冠状病毒S2蛋白HR区域的重组融合蛋白及其应用。该类新型冠状病毒重组融合蛋白是由新型冠状病毒膜蛋白S2蛋白的两个膜融合相关的保守氨基酸序 列HR1和HR2通过连接肽连接得到的重组融合蛋白。该重组融合蛋白可在大肠杆菌中诱导表达，表达量高，同时易于纯化。本发明提供的新型冠状病毒重组融合蛋白，可以形成并维持稳定的三聚体结构，模拟新型冠状病毒膜融合中间态的构象，可作为检测新型冠状病毒膜融合过程的检测原料；具有很好的抗新型冠状病毒活性和很好的免疫原性，在预防或治疗新型冠状病毒蛋白药物开发以及新型冠状病毒疫苗和抗新型冠状病毒抗体开发领域具有广阔的应用前景。
CN112409469B公开了一种跨膜表达新型冠状病毒抗原S2的融合蛋白、重组载体、重组树突状细胞及其应用，属于全细胞疫苗技术领域，所述融合蛋白，包括顺次链接的CD4信号肽、新型冠状病毒抗原S2蛋白、Flag标签序列和CD4跨膜结构域；该发明将S2单独进行跨膜的细胞表达，避免了其他S蛋白表位可能导致的ADE风险，本发明提供的融合蛋白构建的细胞疫苗在小鼠体内可以诱发出更高的中和抗体滴度。
然而，更强效的通用性预防性高效疫苗仍迫在眉睫。
发明内容
我们前期已通过该属S蛋白同源性结构与其生物学功能分析，对S蛋白进行分段重组表达，制备了综合血清IgG抗体库，进而筛选出与多种冠状病毒S蛋白能够交叉反应的抗体及对应的恒定保守区域蛋白片段，该片段为COVID19-SF5，序列为新冠病毒COVID-19的S蛋白的第880位氨基酸至第1084位氨基酸，具体地，该片段的氨基酸序列为(SEQ ID NO.SEQ ID NO.13)：
Figure PCTCN2023071585-appb-000001
本发明将该恒定保守片段(COVID19-SF5)与受体结合域(RBD)片段进行融合表达，得到β属冠状病毒融合重组蛋白，其氨基酸序列如SEQ ID NO.1，从而提供更强效的、针对该类冠状病毒的恒定通用疫苗候选重组融合蛋白，从抑制受体识别和通用保护两个维度提供更广泛、更有利的保护措施。
其中，所述新冠病毒COVID-19的S蛋白的RBD区域为COVID19-SF2片段(SEQ ID NO.10)，其氨基酸序列为新冠病毒COVID-19的S蛋白的第305位氨基酸至第525位氨基酸。本发明的RBD区域主要是根据参考文献Wrapp D,Wang N,Corbett KS,Goldsmith JA,Hsieh C-L,Abiona O,et al.Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.Science.2020；367(6483):1260-3中公开的RBD是第335位-第522位，本申请的COVID19-SF2包含了该RBD区域，同时与前后区域有一定交叉重叠的部分。
所述融合重组蛋白的氨基酸序列如SEQ ID NO.1所示。具体地，COVID19-SF2+5的氨基酸序列如下：
Figure PCTCN2023071585-appb-000002
本发明进一步提出了编码上述融合重组蛋白的基因。优选地，所述基因的核苷酸序列为SEQ ID NO.2。
本发明还提出了一种重组载体，其包含编码上述融合重组蛋白的基因和载体。其中，所示载体可以为pET系列载体，哺乳动物表达载体pcDNA3系列等。在具体的实施方式中，本申请采用表达载体pQE-3。
进一步地，本发明还提出了一种重组菌，其包含上述重组载体。宿主菌可以选用包含大肠杆菌BL21、M15，昆虫细胞sf9,哺乳动物细胞CHO，293等。
本发明还提出了上述融合重组蛋白、编码融合重组蛋白的基因、重组载体、重组菌在制备β属冠状病毒通用疫苗、通用抗体上的应用。
在一个具体的实施方式中，通过如下方法实现SARS-CoV-2的COVID19-SF2蛋白片段和COVID19-SF5蛋白片段融合蛋白的表达菌株构建及蛋白表达纯化：
1)以SARS-CoV-2全长DNA作为模板，设计针对COVID19-SF2蛋白片段和COVID19-SF5蛋白片段的不同的PCR引物，在COVID19-SF2蛋白片段的5’端引入BamH I酶切位点，3’端引入柔性连接肽的反向互补序列，在COVID19-SF5蛋白片段的5’端引入柔性连接肽序列，3’端引入HindⅢ酶切位点，C端引入6×His编码基因。首先分别进行COVID19-SF2蛋白片段和COVID19-SF5蛋白片段PCR，完成扩增后，2％琼脂糖凝胶电泳验证PCR产物。用PCR产物纯化试剂盒对PCR产物进行纯化。
2)将第一步中分别扩增得到的COVID19-SF2蛋白片段和COVID19-SF5蛋白片段的PCR产物进行重叠延伸PCR，通过连接得到融合蛋白表达基因，完成扩增后，1％琼脂糖凝胶电泳验证PCR产物。用PCR产物纯化试剂盒对PCR产物进行纯化。
3)将目的基因通过序列5’-端和3’-端的BamH I和HindⅢ两个酶切位点与表达载体pQE-3 连接。1％琼脂糖电泳验证酶切产物。载体和目的基因分别使用胶回收纯化试剂盒对酶切产物进行回收纯化。纯化后，用One drop分光光度仪检测核酸浓度。
4)按目的基因片段与pQE-3质粒载体摩尔比4:1计算酶连体系中目的基因片段和质粒的量。酶连条件：4℃，过夜。得到的酶连产物即含有融合蛋白基因的表达载体。
5)转化：将含有融合蛋白基因的表达载体用感受态法转化入大肠杆菌M15菌株中。
6)挑选阳性克隆：挑选在选择性平板上生长的菌株并进行菌落PCR，对PCR呈阳性的菌株进行蛋白表达诱导。
7)诱导表达：取菌落PCR呈阳性的克隆进行扩大培养，具体方法为：挑取平板上的阳性克隆过夜培养，取过夜菌，加新鲜培养基扩大培养，培养4h左右，添加终浓度为100mM的IPTG，诱导表达4h。离心收获菌体沉淀，SDS-PAGE验证蛋白表达情况。
8)收获包涵体并进行纯化复性：收获表达菌体并裂解收获重组融合蛋白包涵体，溶解于6M盐酸胍溶液(0.05mol/L tris，5mmol/L EDTA，6mol/L盐酸胍，1％β-巯基乙醇，pH 8.0)中，1g包涵体溶解于100ml 6M盐酸胍中。
Ni-NTA亲和柱纯化：按照Ni-NTA亲和柱制造商建议的步骤进行装柱，随后用8M的尿素(5个柱体积，溶于磷酸盐缓冲液，PH8.0)平衡亲和柱，以5ml/min的速度上样溶解在盐酸胍中的包涵体溶液，上样结束后以PH6.0的磷酸钠(5个柱体积)洗脱杂蛋白，随后以PH4.5的醋酸钠收集目的蛋白。
采用尿素梯度溶液透析的复性步骤：以3M尿素(包含于醋酸钠缓冲液中，15PH4.5)稀释上述经纯化的蛋白液至0.3mg/ml，在4℃下依次用不同浓度的尿素透析液各透析1次，每次24h，其中透析袋内外液的比例为1:5，内液为3.5M尿素-醋酸钠缓冲液，外液依次为3M、2.5M、1.5M、1M、0.5M、0M和0M尿素的透析缓冲液。
9)收获重组融合蛋白：透析后将目的融合蛋白液利用低温离心机15000rpm离心20min，通过Braford法测定蛋白浓度，并经0.22μm滤膜过滤灭菌，添加甘露醇后，存放于-80℃冰箱。
通过以上方法，获得高效通用性冠状病毒融合蛋白，命名为“COVID19-SF2+5”，其抗体与每个S蛋白片段均存在一定的交叉反应性，特别是与COVID19-SF2和COVID19-SF5的结合能力较高。提示该融合蛋白既保留了RBD区域，能够诱导产生特异性阻断病毒与受体结合的IgG抗体，又同时包括了恒定保守片段COVID19-SF5，能够诱导产生更广泛的，与多个S蛋白片段发生交叉反应性的广谱IgG抗体。
进一步，利用小鼠免疫获得“COVID19-SF2+5”融合蛋白的抗血清，纯化获得血清综合抗体IgG，制备新冠病毒SARS-CoV-2S蛋白COVID19-SF2+COVID19-SF5融合蛋白血清综合抗体IgG。
本发明进一步提出了工业化发酵制备重组融合蛋白的方法，包括如下步骤：
(1)以重组菌作为种子菌，种子菌经过夜摇菌放大作为种子液；
(2)将种子液在2×YT培养基中进行发酵培养，发酵后菌体通过工业化自动连续离心机收集，收集的菌体先用提取液A制成悬液，再用水性裂解酶裂解，然后用提取液B处理，离心后收取沉淀包涵体，用缓冲液稀释后，离心，沉淀物为不溶性包涵体；
(3)进一步将包涵体溶于缓冲液后，离心去上清超滤浓缩，浓缩样品经Ni-NTA亲和柱，在AKTA蛋白纯化系统上进行纯化，收集样品；
(4)收集样品在4℃层析柜中透析，透析后的样品经离心取上清，上清液经超滤浓缩器浓缩，后再经AKTA蛋白纯化系统Sephadex G-75层析纯化；根据AKTA蛋白纯化系统的蛋白峰收集样品，即为精纯后的融合蛋白。
在一个具体的实施例中，包括如下步骤：
1)发酵接种前进行清洁，注意无菌操作，在同一发酵时间内不得进行其他菌种发酵；
2)发酵罐及管道灭菌，每次发酵前需进行空罐灭菌121℃30min，配制培养基装入罐内再次灭菌121℃30min，待冷却至所需温度37℃时接种种子液(之前冻存的优势表达菌株，种子菌经过夜摇菌放大)；40L发酵罐加种子液500mL，培养基35000mL(过夜菌作为种子液，培养基为2×YT培养基)，其中，2×YT培养基：1L培养液中含胰蛋白胨16g，酵母提取物10g，氯化钠5g，搅拌均匀后高温高压灭菌。
3)发酵条件，如温度、pH、氧流量及发酵时间等参数均由发酵罐配套电脑操作系统控制。设定温度37℃，pH7.0，发酵时间约7小时。(溶氧值或溶氧浓度：DO值60％，温度37℃，pH7.0.在细菌浓度量达峰值时加入诱导剂IPTG，总培养时间为7小时。)
4)发酵后菌体通过工业化自动连续离心机收集，离心速度10000g，温度4℃，离心1小时。
5)收获的菌体每40g加入提取液A(50mM Tris,pH8.0,含1.5mM EDTA)1000mL制成悬液，再用水性裂解酶(Lysozyme)250mg裂解。
6)用提取液B(1.5M NaCl，100mM CaCl 2，100mM MgCl2，0.002％DNase I)处理。每1000mL上述裂解液加100mL提取液B。
7)经4℃，10000g离心10min后，收取沉淀包涵体，均匀地悬浮于50mM的磷酸缓冲液(含0.15M的氯化钠和4M尿素，pH7.0)。1g沉淀加10mL缓冲液，10000g 4℃离心10min，沉淀物为不溶性包涵体，可收集贮存于-80℃，保存半年。
蛋白的复性与精纯化：
1)每10g粗纯化的包涵体溶于2000mL缓冲液中(0.1M Tris-HCl，pH7.5含6M盐酸胍，20mM DTT，20mM EDTA)，20℃搅拌1h。
2)10000g 4℃离心30min后取上清超滤浓缩。每2000mL浓缩至约200mL。
3)浓缩样品经Ni-NTA亲和柱，在AKTA蛋白纯化系统上进行纯化。具体操作步骤与前述相同。
4)收集的样品按照前述方法在4℃层析柜中透析。
5)透析后的样品经10000g，4℃离心30min，取上清。
6)上清液经超滤浓缩器浓缩，每2000mL浓缩到300mL。
7)样品再经AKTA蛋白纯化系统Sephadex G-75层析纯化。柱长1.2米，直径4cm，流速1mL/min。
8)根据AKTA蛋白纯化系统的蛋白峰收集样品，即为精纯后的融合蛋白。
蛋白的除菌和去内毒素
1)纯化的蛋白通过Polymyxin(Bio-rad)层析柱去除内毒素。
2)去内毒素的样品经0.22μm的无菌滤器过滤除菌，分装保存于4℃。
有益效果：不同于以往的研究手段，本发明结合本实验室前期研究，从β属冠状病毒S蛋白的结构和功能分析作为切入点，进行了多种冠状病毒蛋白的氨基酸序列区域化、线性同源性匹配分析，通过S蛋白同源性结构进行分段表达，建立覆盖S蛋白全区域的重组蛋白片段库，并通过免疫小鼠获得的血清抗体库与多种S蛋白片段的交叉反应等研究找到了与SARS-CoV S蛋白各片段及SARS-CoV-2S蛋白各片段有通用性交叉反应的SARS-CoV-2的S蛋白片段COVID19-SF5。本发明将有通用交叉反应性的COVID19-SF5蛋白片段和包含病毒受体结合域(RBD)的COVID19-SF2蛋白片段通过柔性连接肽Gly4Ser进行连接，形成了具有多功能效应的融合蛋白，并通过将该融合蛋白免疫小鼠，获得血清IgG抗体。经测试，该融合蛋白与细胞结合的能力较单独COVID19-SF2或COVID19-SF5蛋白片段显著提高，其血清IgG抗体能与多种S蛋白片段的发生交叉反应，并能显著抑制假病毒感染细胞。综上，通用融合蛋白疫苗优势明显：
(1)利用基因重组技术，本发明的产品是病毒S蛋白的两个靶向特异片段连接后的融合蛋白，不涉及基因、其它病毒载体、失活病毒进入人体，产品质量控制和质量保证体系明确，保证产品的安全性；
(2)从研究S蛋白的结构和功能切入，将β冠状病毒属的恒定保守、通用蛋白区域和受体结合域连接，能强烈激发人体免疫系统的应答，从而产生既可以高效预防病毒感染的IgG中和抗体又可以识别β冠状病毒属通用表位的特异性抗体；
(3)任何病毒的自然进化、变异，都会帮助病毒更易侵染寄主，并和寄主长期共生，而其各种功能都是在病毒自身蛋白或通过寄主的蛋白调控表现出来，新冠病毒S蛋白的融合蛋白功能很多方面仍待研究、明确，本项目的融合蛋白片段在小鼠体内产生的有效抗体，具有在功能上的特异性。
附图说明
图1为双酶切验证质粒构建示意图；
图2为SDS-PAGE鉴定重组融合蛋白的表达，其中，Lane M:蛋白标志物；Lane 1:COVID19-SF2+COVID19-SF5融合蛋白；
图3为三种蛋白片段与Vero-E6细胞的结合能力。
具体实施方式
下面结合具体实施例对本发明做进一步详细说明，实施例将有助于理解本发明，但是本发明的保护范围不限于下述的实施例。
实施例1：SARS-CoV-2的COVID19-SF2蛋白片段与COVID19-SF5蛋白片段融合蛋白“COVID19SF-2+5”的表达菌株构建及蛋白表达纯化。
1)以SARS-CoV-2全长DNA作为模板，设计针对COVID19-SF2蛋白片段和COVID19-SF5蛋白片段的不同的PCR引物，在COVID19-SF2蛋白片段的5’端引入BamH I酶切位点，3’端引入柔性连接肽的反向互补序列，在COVID19-SF5蛋白片段的5’端引入柔性连接肽序列，柔性连接肽为Gly4Ser，3’端引入HindⅢ酶切位点，C端引入6×His编码基因。首先分别进行COVID19-SF2蛋白片段和COVID19-SF5蛋白片段的PCR，其中PCR体系和扩增条件如下：
表1-1 PCR引物序列
Figure PCTCN2023071585-appb-000003
表1-2 PCR体系
Figure PCTCN2023071585-appb-000004
Figure PCTCN2023071585-appb-000005
扩增条件为：94℃，30s；56℃，1min；72℃，1min30s；35个循环。
完成扩增后，2％琼脂糖凝胶电泳验证PCR产物。用PCR产物纯化试剂盒对PCR产物进行纯化。其中，COVID19-SF2基因序列如SEQ ID NO.7所示，COVID19-SF5基因序列如SEQ ID NO.8所示。
2)将第一步中分别扩增得到的COVID19-SF2蛋白片段和COVID19-SF5蛋白片段的PCR产物进行重叠延伸PCR，通过连接得到融合蛋白表达基因，其中PCR体系和扩增条件如下：
表2 PCR体系
Figure PCTCN2023071585-appb-000006
扩增条件为：94℃，30s；56℃，1min；72℃，2min40s；30个循环。
完成扩增后，1％琼脂糖凝胶电泳验证PCR产物。用PCR产物纯化试剂盒对PCR产物进行纯化。
3)酶切：将目的基因(SEQ ID NO.2)通过序列5’端和3’端的BamH I和HindⅢ两个酶切位点与表达载体pQE-3连接。酶切体系如下：
表3载体
Figure PCTCN2023071585-appb-000007
Figure PCTCN2023071585-appb-000008
表4目的基因
Figure PCTCN2023071585-appb-000009
酶切条件为：37℃水浴中酶切0.5小时。1％agarose gel验证酶切产物。载体和目的基因分别使用胶回收纯化试剂盒对酶切产物进行回收纯化。纯化后，用One drop检测核酸浓度。
4)酶连
按目的基因片段与pQE-3质粒载体摩尔比4:1计算酶连体系中目的基因片段和质粒的量。酶连体系如下：
表5
Figure PCTCN2023071585-appb-000010
酶连条件：4℃，过夜。
得到的酶连产物即含有融合蛋白基因的表达载体。
5)转化
将含有融合蛋白基因的表达载体用感受态法转化入大肠杆菌M15菌株中。
6)挑选阳性克隆
挑选在选择性平板上生长的菌株并进行菌落PCR，对PCR呈阳性的菌株进行蛋白表达诱导。
7)诱导表达
取菌落PCR呈阳性的克隆进行扩大培养，具体方法为：挑取平板上的阳性克隆过夜培养，取过夜菌，加新鲜培养基扩大培养，培养4h左右，添加终浓度为100mM的IPTG，诱导表达4h。离心收获菌体沉淀，SDS-PAGE验证蛋白表达情况。
8)收获包涵体并进行纯化复性
收获表达菌体并裂解收获重组融合蛋白包涵体，溶解于6M盐酸胍溶液(0.05mol/L tris，5mmol/L EDTA，6mol/L盐酸胍，1％β-巯基乙醇，pH 8.0)中，1g包涵体溶解于100ml 6M盐酸胍中。
Ni-NTA亲和柱纯化：按照Ni-NTA亲和柱制造商建议的步骤进行装柱，随后用8M的尿素(5个柱体积，溶于磷酸盐缓冲液，PH8.0)平衡亲和柱，以5ml/min的速度上样溶解在盐酸胍中的包涵体溶液，上样结束后以PH6.0的磷酸钠(5个柱体积)洗脱杂蛋白，随后以PH4.5的醋酸钠收集目的蛋白。
采用尿素梯度溶液透析的复性步骤：以3M尿素(包含于醋酸钠缓冲液中，PH4.5)稀释上述经纯化的蛋白液至0.3mg/ml，在4℃下依次用不同浓度的尿素透析液各透析1次，每次24h，其中透析袋内外液的比例为1:5，内液为3.5M尿素-醋酸钠缓冲液，外液依次为3M，2.5M，1.5M，1M,0.5M，0M和0M尿素的透析缓冲液。
9)收获融合蛋白
透析后将目的蛋白液利用低温离心机15000rpm离心20min，通过Braford法测定蛋白浓度，并经0.22μm滤膜过滤灭菌，添加甘露醇后，存放于-80℃冰箱。
通过双酶切验证质粒构建见图1。使用BamHⅠ限制性内切酶和距离HindⅢ酶切位点673bp的Ncol限制性内切酶进行双酶切，酶切结果见下图，其中全长质粒4689bp，在琼脂糖凝胶电泳中显示有双酶切后的两条带：目的基因加上HindⅢ酶与Ncol酶之间的673bp为1966bp，剩余载体2723bp，与理论相符。
通过SDS-PAGE验证蛋白表达见下图2。图中可见融合蛋白的条带位置与理论条带位置48.2kD一致，且经Ni柱纯化后蛋白条带单一，表达和复性效果良好。
实施例2：β属冠状病毒通用融合蛋白的鉴定。
通过ELISA方法检测融合蛋白抗体的特异性和通用交叉反应性。
1)测定抗体效价：使用进行免疫的重组融合蛋白作为抗原，将纯化后的血清IgG抗体进行倍比稀释并设置复孔，之后进行ELISA测定，于450nm处检测OD值，分析结果。
通过ELISA检测融合蛋白首次免疫6个月后的综合抗体IgG(50μg/mL)滴度(见表2)，小鼠对融合蛋白片段产生良好免疫效果，首次免疫6个月后的抗体滴度达仍可达1：1600。
表6.ELISA检测融合蛋白血清综合抗体IgG滴度
重组蛋白 抗体滴度
COVID-SF2+5融合蛋白 1：1600
2)测定通用交叉反应性：使用12个重组蛋白片段作为抗原，将融合蛋白抗体与其进行反应，每 组设置3个复孔，进行ELISA测定，于450nm处检测OD值，分析结果。其中，12个重组蛋白片段的起始-终止氨基酸位置以及对应的氨基酸序列如表7-1所示：
表7-1 12个包含重叠结构域的S蛋白片段
Figure PCTCN2023071585-appb-000011
通过ELISA检测融合蛋白综合抗体IgG与各S蛋白片段的结合能力，发现其抗体与每个S蛋白片段均存在一定的交叉反应性，且结合能力较高，甚至在首次免疫6个月后，虽然反应效力减弱，但其抗体仍与大部分蛋白片段具有明显的交叉反应，提示该融合蛋白免疫小鼠后不仅可产生高特异性的中和抗体，而且包含多种β-属冠状病毒恒定保守、特异性的蛋白片段，ELISA检测结果见表7-2和7-3。
表7-2 COVID19-SF2+5融合蛋白抗体与蛋白片段的交叉反应检测
Figure PCTCN2023071585-appb-000012
Figure PCTCN2023071585-appb-000013
表7-3 COVID19-SF2+5融合蛋白抗体与蛋白片段的交叉反应检测
Figure PCTCN2023071585-appb-000014
实施例3：通用特异性冠状病毒融合蛋白疫苗的小鼠安全性和抗体应答检测。
将0.20mg/ml的COVID19-SF2+5融合蛋白给20只BALB/c小鼠进行免疫，观察小鼠在注射期间的安全性，于28天及45天检测IgG应答水平。
观察注射COVID19-SF2+5融合蛋白后45天，小鼠的安全性和IgG应答检测。
接种COVID19-SF2+5融合蛋白的20只小鼠健康状况良好，且均能产生有效的IgG抗体。安全性检测及IgG应答检测结果如下表8：
表8.COVID19-SF2+5在小鼠体内安全性及IgG应答检测
Figure PCTCN2023071585-appb-000015
实施例4：通用特异性冠状病毒融合蛋白COVID19-SF2+5与细胞的结合能力检测。
将非洲绿猴肾细胞(Vero-E6)细胞处理计数后，用100μL细胞洗液(含1％BSA的PBS)重悬1.5×10 5个细胞；加入终浓度2μg/mL的通用特异性冠状病毒融合蛋白，同时对照管中加入相同摩尔量的COVID19-SF2和COVID19-SF5做对照研究，充分混匀，至37℃孵育1h，孵育期间每隔10min晃动一下反应管，使细胞和蛋白充分反应；加入适量细胞洗液，5000rpm离心2min，弃上清，洗涤2次；加入适量的荧光标记二抗(anti-His Tag PE，Abcam，以1:50稀释)，充分混匀，至4℃避光孵育1h，孵育期间每隔10min晃动一下反应管；加入适量细胞洗液，5000rpm离心2min，弃上清，洗涤2次；使用200μL细胞洗液重悬细胞，以流式细胞仪检测细胞表面的荧光信号。
通用特异性冠状病毒蛋白融合蛋白COVID19-SF2+5与Vero-E6细胞结合后存在很强的荧光偏移，如图3所示。融合蛋白COVID19-SF2+5与Vero-E6细胞的结合能力高于单纯的COVID19-SF2或者COVID19-SF5片段。具体的细胞与蛋白结合的比例见表9。
表9.Vero-E6细胞与COVID19-SF2蛋白、COVID19-SF5蛋白或COVID19-SF2+COVID19-SF5融合蛋白的结
合
蛋白片段 COVID19--SF2 COVID19-SF5 COVID19-SF2+5
结合能力(％) 11.7 67.5 77.5
实施例5：β属冠状病毒通用融合蛋白对应的综合抗体IgG对假病毒抑制能力检测。
通过多功能酶标仪检测SARS-CoV-2假病毒感染细胞后细胞中荧光素酶表达情况，从而判断通用融合蛋白对应的综合抗体对假病毒抑制能力。
以hACE2-293T细胞为感染细胞，前一晚将hACE2-293T细胞以2×10 4/孔接种在96孔板中。18小时后将10μg/mL的融合蛋白抗血清IgG与650TCID 50/孔的假病毒混合。然后将混合物加入细胞中并孵育48小时。根据制造商的方案，利用荧光素酶检测试剂盒通过多功能酶标仪测量荧光素酶的表达，以获得血清抗体的抗病毒能力。在每个板中设置仅含有细胞的细胞对照和只含有病毒和细胞的病毒对照。每组设三个平行实验。将仅含有细胞的细胞对照抑制率视为100％，将具有病毒和细胞的病毒对照的抑制率视为0％，计算血清抗体的抑制率。
通过假病毒中和实验检测融合蛋白抗血清对假病毒感染细胞的抑制率(见表10)，为三次平行实验的结果值，表中可见，融合蛋白COVID19-SF2+5免疫小鼠产生的血清IgG抗体能够在一定程度上抑制假病毒对细胞的侵染，其抑制率在40％左右。
表10 COVID19-SF2+5融合蛋白抗体对SARS-CoV-2假病毒的抑制作用
Figure PCTCN2023071585-appb-000016
实施例6工业化发酵制备重组融合蛋白。
1)发酵接种前进行清洁，注意无菌操作，在同一发酵时间内不得进行其他菌种发酵；
2)发酵罐及管道灭菌，每次发酵前需进行空罐灭菌121℃30min，配制培养基装入罐内再次灭菌121℃30min，待冷却至所需温度37℃时接种种子液(之前冻存的优势表达菌株，种子菌经过夜摇菌放大)；40L发酵罐加种子液500mL，培养基35000mL(过夜菌作为种子液，培养基为2×YT培养基)，其中，2×YT培养基：1L培养液中含胰蛋白胨16g，酵母提取物10g，氯化钠5g，搅拌均匀后高温高压灭菌。
3)发酵条件，如温度、pH、氧流量及发酵时间等参数均由发酵罐配套电脑操作系统控制。设定温度37℃，pH7.0，发酵时间约7小时。(件溶氧值或溶氧浓度：DO值60％，温度37℃，pH7.0.在细菌浓度量达峰值时加入诱导剂IPTG，总培养时间为7小时。)
4)发酵后菌体通过工业化自动连续离心机收集，离心速度10000g，温度4℃，离心1小时。
5)收获的菌体每40g加入提取液A(50mM Tris,pH8.0,含1.5mM EDTA)1000mL制成悬液，再用水性裂解酶(Lysozyme)250mg裂解。
6)用提取液B(1.5M NaCl，100mM CaCl 2，100mM MgCl2，0.002％DNase I)处理。每1000mL上述裂解液加100mL提取液B。
7)经4℃，10000g离心10min后，收取沉淀包涵体，均匀地悬浮于50mM的磷酸缓冲液(含0.15M的氯化钠和4M尿素，pH7.0)。1g沉淀加10mL缓冲液，10000g 4℃离心10min，沉淀物为不溶性包涵体，可收集贮存于-80℃，保存半年。
蛋白的复性与精纯化：
1)每10g粗纯化的包涵体溶于2000mL缓冲液中(0.1M Tris-HCl，pH7.5含6M盐酸胍，20mM DTT，20mM EDTA)，20℃搅拌1h。
2)10000g 4℃离心30min后取上清超滤浓缩。每2000mL浓缩至约200mL。
3)浓缩样品经Ni-NTA亲和柱，在AKTA蛋白纯化系统上进行纯化。具体操作步骤与前述相同。
4)收集的样品按照前述方法在4℃层析柜中透析。
5)透析后的样品经10000g，4℃离心30min，取上清。
6)上清液经超滤浓缩器浓缩，每2000mL浓缩到300mL。
7)样品再经AKTA蛋白纯化系统Sephadex G-75层析纯化。柱长1.2米，直径4cm，流速1mL/min。
8)根据AKTA蛋白纯化系统的蛋白峰收集样品，即为精纯后的融合蛋白。
蛋白的除菌和去内毒素
1)纯化的蛋白通过Polymyxin(Bio-rad)层析柱去除内毒素。
2)去内毒素的样品经0.22μm的无菌滤器过滤除菌，分装保存于4℃。
本发明提出了一种β属冠状病毒融合蛋白及其制备方法的思路，具体实现本发明技术方案的方法和途径很多，以上所述仅是本发明的优选实施方式，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，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。本实施例中未明确的各组成部分均可用现有技术加以实现。

Claims (9)

  1. 一种β属冠状病毒融合重组蛋白，其特征在于，所述融合重组蛋白的氨基酸序列如SEQ ID NO.1所示。
  2. 编码权利要求1所述的融合重组蛋白的基因。
  3. 一种重组表达载体，其特征在于，包含权利要求2所述的基因和表达载体。
  4. 一种重组菌，其特征在于，包含权利要求3所述的重组表达载体。
  5. 权利要求1所述的β属冠状病毒融合重组蛋白的制备方法，包括如下步骤：
    (1)以SARS-CoV-2全长DNA作为模板，设计针对COVID19-SF2蛋白片段和COVID19-SF5蛋白片段的不同的PCR引物，在COVID19-SF2蛋白片段的5’端引入BamH I酶切位点，3’端引入柔性连接肽的反向互补序列，在COVID19-SF5蛋白片段的5’端引入柔性连接肽序列，3’端引入Hind Ⅲ酶切位点，C端引入6×His编码基因，得到COVID19-SF2蛋白片段和COVID19-SF5蛋白片段的PCR产物；其中，COVID19-SF2片段氨基酸序列为新冠病毒COVID-19的S蛋白的第305位氨基酸至第525位氨基酸，COVID19-SF5片段的氨基酸序列为新冠病毒COVID-19的S蛋白的第880位氨基酸至第1084位氨基酸；
    (2)将第一步中分别扩增得到的COVID19-SF2蛋白片段和COVID19-SF5蛋白片段的PCR产物进行重叠延伸PCR，通过连接得到融合蛋白表达基因；
    (3)将融合蛋白表达基因通过序列5’-端和3’-端的BamH I和Hind Ⅲ两个酶切位点与表达载体pQE-3连接，得到含有融合蛋白基因的表达载体；
    (4)将含有融合蛋白基因的表达载体用感受态法转化入大肠杆菌M15菌株中；
    (5)挑选在选择性平板上生长的菌株并进行菌落PCR，对PCR呈阳性的菌株进行蛋白表达诱导；
    (6)取菌落PCR呈阳性的克隆进行扩大培养，使用IPTG进行诱导表达；
    (7)收获包涵体并进行纯化复性收获重组融合蛋白。
  6. 权利要求1所述的融合重组蛋白的工业化制备方法，其特征在于，包括如下步骤：
    (1)以权利要求4所述的重组菌作为种子菌，种子菌经过夜摇菌放大作为种子液；
    (2)将种子液在2×YT培养基中进行发酵培养，发酵后菌体通过工业化自动连续离心机收集，收集的菌体先用提取液A制成悬液，再用水性裂解酶裂解，然后用提取液 B处理，离心后收取沉淀包涵体，用缓冲液稀释后，离心，沉淀物为不溶性包涵体；
    (3)进一步将包涵体溶于缓冲液后，离心去上清超滤浓缩，浓缩样品经Ni-NTA亲和柱，在AKTA蛋白纯化系统上进行纯化，收集样品；
    (4)收集样品在4℃层析柜中透析，透析后的样品经离心取上清，上清液经超滤浓缩器浓缩，后再经AKTA蛋白纯化系统Sephadex G-75层析纯化；根据AKTA蛋白纯化系统的蛋白峰收集样品，即为精纯后的融合蛋白。
  7. 根据权利要求6所述的方法，其特征在于，发酵条件为:溶氧值或溶氧浓度DO值60％，温度37℃，pH7.0，在细菌浓度量达峰值时加入诱导剂IPTG，总培养时间为7小时。
  8. 一种融合蛋白血清综合抗体IgG，其特征在于，利用权利要求1所述的重组融合蛋白免疫小鼠，获得抗血清，纯化获得血清综合抗体IgG。
  9. 权利要求1所述的融合重组蛋白、权利要求所述的基因、权利要求3所述的重组表达载体、权利要求4所述的重组菌在制备β属冠状病毒通用疫苗、通用抗体上的应用。
PCT/CN2023/071585 2022-02-08 2023-01-10 β属冠状病毒融合重组蛋白及其制备方法和应用 WO2023151446A1 (zh)

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