WO2011153959A1 - 流感病毒m2或ha片段与蛋白载体的缀合物及其制药应用 - Google Patents

流感病毒m2或ha片段与蛋白载体的缀合物及其制药应用 Download PDF

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WO2011153959A1
WO2011153959A1 PCT/CN2011/075585 CN2011075585W WO2011153959A1 WO 2011153959 A1 WO2011153959 A1 WO 2011153959A1 CN 2011075585 W CN2011075585 W CN 2011075585W WO 2011153959 A1 WO2011153959 A1 WO 2011153959A1
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protein
amino acid
acid sequence
carrier protein
seq
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PCT/CN2011/075585
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French (fr)
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李先钟
张琪
王鑫
孙宇石
刘方杰
胡品良
程虹
杨思仪
阚伟
白先宏
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北京精益泰翔技术发展有限公司
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    • 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
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza 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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • 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
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to protein conjugates and methods for their preparation, and to broad-spectrum influenza vaccines comprising the protein conjugates, in particular to M2e/HA2 18 _ 72 -carrier protein conjugates and methods for their preparation, and to the A broad spectrum influenza vaccine of at least one of two carrier protein conjugates. Background technique
  • Influenza virus strains are highly variable. Therefore, it is necessary to develop prevention and protection against influenza viruses of different serotypes (such as influenza A virus H1N1, influenza A virus H3N2, influenza A virus H5N1 (also known as avian influenza), etc.)
  • influenza viruses of different serotypes such as influenza A virus H1N1, influenza A virus H3N2, influenza A virus H5N1 (also known as avian influenza), etc.
  • Current influenza vaccines generally include highly conserved antigenic fragments, such as:
  • M2e-HBc VLPs such as US 7361352B2 and CN1913920A, which also utilize the M2e fragment of influenza virus to chemically couple with hepatitis B core antigen to form chimeric molecular particles against influenza virus.
  • the object of the present invention is to overcome the defects of the poor spectral spectrum of the existing influenza vaccine, and to provide a utilization flow.
  • the highly conserved, preventive and protective antigenic fragment M2e or HA2 18 _ 72 is chemically coupled to the carrier protein P64K to develop a broad-spectrum influenza vaccine that can effectively respond to various influenza viruses.
  • the broad-spectrum vaccine prepared by the combination is easy to prepare for large-scale production, and has a long effective period and can be stored in a large amount.
  • the present invention provides a M2e-carrier protein conjugate for preventing influenza, wherein the protein conjugate is chemically coupled with a carrier protein by an extracellular domain M2e of an influenza A virus M2 protein, wherein The M2e and the carrier protein are covalently linked by a thioether bond, and the amino acid sequence of the M2e is an amino acid sequence as shown in SEQ ID NO: 2, an amino acid sequence as shown in SEQ ID NO: 7, or a SEQ ID NO: : Amino acid sequence shown in 8:
  • Xa is threonine, isoleucine or serine
  • Xaa 2 is serine, aspartic acid or cysteine
  • Xaa 3 is cysteine, alanine or serine
  • the carrier protein is a Neisseria meningitidis outer membrane protein P64K; the amino acid sequence of the P64K is an amino acid sequence as shown in SEQ ID NO: 5 in the Sequence Listing.
  • the invention also provides a preparation method of the M2e-carrier protein conjugate described above, which comprises first activating the carrier protein with an activator, and then mixing with M2e to obtain a protein conjugate; wherein, M2e and the carrier
  • the molar ratio of protein is 10: 1 to 20: 1;
  • the pH of the coupling reaction is 6.5-7.5;
  • the coupling reaction time is 18-36 hours
  • the activator is selected from any one of SMCC, MBS and sulfo-MBS.
  • the present invention also provides a broad spectrum influenza vaccine, wherein the vaccine comprises the M2e-carrier protein conjugate of the present invention and an adjuvant.
  • the present invention also provides a HA2 18 _ 72 -carrier protein conjugate, wherein the protein conjugate is chemically coupled with avian influenza H5N1 virus HA2 18 _ 72 polypeptide and a carrier protein, wherein The HA2 18 _ 72 polypeptide is covalently linked to the carrier protein via a thioether bond, the ammonia of the HA2 18 _ 72 polypeptide
  • the acid sequence is the amino acid sequence shown in SEQ ID NO: 3; the carrier protein is selected from the group consisting of N.
  • meningitidis outer membrane protein P64K tetanus toxoid
  • hepatitis B core antigen keyhole limpet hemocyanin
  • the invention also provides a preparation method of the above-mentioned ⁇ 2 18 _ 72 -carrier protein conjugate, which is to activate the carrier protein with an activator and then mix with an appropriate ratio of ⁇ 2 18 _ 72 polypeptide to obtain protein coupling.
  • ⁇ 2 18 _ 72 wherein, the molar ratio of the ⁇ 2 18 _ 72 polypeptide to the carrier protein is 10: 1 to 20: 1;
  • the coupling reaction has a pH value of 6.5-7.5;
  • the coupling reaction time is 18-36 hours
  • the activator is selected from any one of SMCC, MBS, and sulfo-MBS.
  • the present invention further provides a broad spectrum influenza vaccine, wherein said vaccine comprises HA2 18 _ 72 according to the present invention a carrier protein conjugate and an adjuvant.
  • the present invention further provides a broad spectrum influenza vaccine, wherein said vaccine comprising a carrier protein conjugates M2e- according to the present invention containing HA2 18 according to the present invention _ 72-- carrier protein conjugate and the adjuvant Agent.
  • the broad-spectrum influenza vaccine comprising the M2e-carrier protein conjugate of the present invention and/or the HA2 18 _ 72 -carrier protein conjugate has small toxic and side effects, high safety, good tolerance, and can effectively prevent the H1N1 , Avian influenza H5N1 influenza virus infection, and even effective infection of other virus subtypes, broad spectrum. Immunization of mice with the vaccine of the present invention induces the production of high levels of antibodies in mice and protects the lethal dose of the virus from immunized mice. DRAWINGS
  • Figure 1 is an agarose gel electrophoresis pattern of the PCR product of the outer membrane protein P64K of N. meningitidis;
  • Figure 2 is a reversed phase chromatogram of the outer membrane protein P64K of N. meningitidis
  • Figure 3 is a peptide spectrum (trypsin) of the outer membrane protein P64K of N. meningitidis;
  • Figure 4 is an electropherogram of the outer membrane protein P64K of N. meningitidis
  • Figure 5 is a mass spectrometric analysis of the outer membrane protein P64K of N. meningitidis
  • Figure 6 is a reversed-phase high performance liquid chromatogram of M2e
  • Figure 7 is a mass spectrometry spectrum of M2e
  • Figure 8 is a SDS gel electrophoresis pattern of the M2e-P64K conjugate
  • Figure 9 is a gel chromatogram of the M2e-P64K conjugate
  • Figure 10 is the result of agarose gel electrophoresis of HA2 (18 _ 72 ) PCR amplification product
  • Figure 11 shows the vector construction of HA2 ( 18 _ 72 );
  • Figure 12 is a map of the Mono Q purification process of HA2 18 _ 72 ;
  • Figure 13 is a map of the SOURCE purification process of HA2 18 _ 72 ;
  • Figure 14 is an SDS-PAGE map of HA2 18 _ 72 ;
  • Figure 15 is a reversed-phase high performance liquid chromatogram of HA2 18 _ 72 ;
  • Figure 16 is an SDS-PAGE map of HA2 18 _ 72 -P64K
  • Figure 17 is a gel chromatogram of HA2 18 _ 72 -P64K
  • Figure 18 is an immunogenicity analysis of the M2e-P64K conjugate: antibody titer map;
  • Figure 19 is an immunogenicity analysis of the M2e-P64K conjugate: antibody subtype identification map
  • Figure 20 is an immunogenicity analysis of the M2e-P64K conjugate: serum cross-reactivity
  • Figure 21 is an immunogenicity analysis of the HA2 18 _ 72 - P64K conjugate: antibody titer map;
  • Figure 22 is a graph showing the protective effect of the M2e-P64K conjugate on the mouse PR8 strain influenza virus;
  • Figure 23 is M2e-P64K The effect of the conjugate on the protection of avian H5N1 attack. detailed description
  • the present invention provides a M2e-carrier protein conjugate for preventing influenza, wherein the protein conjugate is chemically coupled with a carrier protein by an extracellular domain M2e of an influenza A virus M2 protein, wherein The M2e and the carrier protein are covalently linked by a thioether bond, and the amino acid sequence of the M2e is an amino acid sequence as shown in SEQ ID NO: 2, an amino acid sequence as shown in SEQ ID NO: 7, or a SEQ ID NO: : Amino acid sequence shown in 8:
  • Xa is threonine, isoleucine or serine
  • Xaa 2 is serine, aspartic acid or cysteine
  • Xaa 3 is cysteine, alanine or serine;
  • the carrier protein is a Neisseria meningitidis outer membrane protein P64K;
  • the amino acid sequence of the P64K is an amino acid sequence as shown in SEQ ID NO: 5 of the Sequence Listing.
  • a nucleotide sequence encoding P64K is preferably shown as SEQ ID NO: 6.
  • the M2e is derived from influenza A virus H1N1, has the amino acid sequence shown as SEQ ID NO: 1: SLLTEVETPTRSEWECRCSDSSDC [SEQ ID NO: 1], and influenza A virus H3N2 has SEQ ID NO: 7
  • influenza A virus H5N1 ie avian influenza
  • the M2e is derived from influenza A virus H3N2 having the amino acid sequence set forth in SEQ ID NO: 7: SLLTEVETPIRNEWGCRCNDSSDC [SEQ ID NO: 7].
  • the M2e according to the invention is preferably obtained by solid phase synthesis.
  • the invention also provides a preparation method of the M2e-carrier protein conjugate described above, which comprises first activating the carrier protein with an activator, and then mixing with M2e to obtain a protein conjugate; wherein, M2e and the carrier
  • the molar ratio of protein is 10: 1 to 20: 1;
  • the pH of the coupling reaction is 6.5-7.5;
  • the coupling reaction time is 18-36 hours
  • the activator is selected from any one of SMCC, MBS and sulfo-MBS.
  • SMCC is called Succinimidyl-4-(N-maleimidomethyl) cy dohexane- 1 -carboxylate
  • MBS is called m-maleimidobenzoyl-N-hydroxy succinimide ester
  • sulfo-MBC is called m -maleimidobenzoyl- N-hydroxysulfosuccinimide ester.
  • the ratio of the M2e to the carrier protein is from 12:1 to 18:1, more preferably 15:1; the pH of the coupling reaction is 6.8-7.2; the time of the coupling reaction is 24 hours.
  • the activator is SMCC.
  • the present invention also provides a broad spectrum influenza vaccine, wherein the vaccine comprises the M2e-carrier protein conjugate of the present invention and an adjuvant.
  • the present invention also provides a HA2 18 _ 72 -carrier protein conjugate, wherein the protein conjugate is chemically coupled with avian influenza H5N1 virus HA2 18 _ 72 polypeptide and a carrier protein, wherein The HA2 18 _ 72 polypeptide is covalently linked to the carrier protein via a thioether bond, the ammonia of the HA2 18 _ 72 polypeptide
  • the acid sequence is the amino acid sequence shown in SEQ ID NO: 3 (MVDGWYGYHHSNEQGSG YAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFC [SEQ ID NO: 3]);
  • the carrier protein is selected from the group consisting of Neisseria meningitidis outer membrane protein P64K, tetanus toxoid, hepatitis B Any of the core antigen, keyhole limpet hemocyanin, rotavirus capsid protein, and L1 protein of bovine or human papillom
  • the carrier protein is Neisseria meningitidis outer membrane protein ⁇ 64 ⁇ , and the amino acid sequence of ⁇ 64 ⁇ is the amino acid sequence shown in SEQ ID NO: 5.
  • the HA2 18 _ 72 polypeptide is expressed by a genetically engineered bacterium; the nucleotide sequence of the HA2 18 _ 72 polypeptide encoding gene is as shown in SEQ ID NO: 4 in the sequence listing; the genetically engineered bacterium is the large intestine Bacillus Ecoli.BL21.
  • the invention also provides a preparation method of the above-mentioned HA2 18 _ 72 -carrier protein conjugate, which is to activate the carrier protein with an activator and then mix with an appropriate ratio of the HA2 18 _ 72 polypeptide to obtain a protein coupling.
  • HA2 18 _ 72 wherein the molar ratio of the HA2 18 _ 72 polypeptide to a carrier protein is from 10: 1 to 20: 1; the coupling reaction pH of 6.5-7.5; the coupling reaction The time is 18-36 hours; the activator is selected from any one of SMCC, MBS, sulfo-MBS.
  • the molar ratio of the HA2 18 _ 72 polypeptide to the carrier protein is from 12:1 to 16:1, more preferably 15:1; the pH of the coupling reaction is preferably 6.8-7.2; The reaction time is preferably 24 hours; the activator is preferably SMCC.
  • the present invention further provides a broad spectrum influenza vaccine, wherein said vaccine comprises HA2 18 _ 72 according to the present invention a carrier protein conjugate and an adjuvant.
  • the present invention further provides a broad spectrum influenza vaccine, wherein said vaccine comprising a carrier protein conjugates M2e- according to the present invention containing HA2 18 according to the present invention _ 72-- carrier protein conjugate and the adjuvant Agent.
  • the broad spectrum influenza vaccine of the present invention may comprise various immunological adjuvants such as aluminum hydroxide and Freund's adjuvant which are commonly used in the art.
  • the experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials are commercially available unless otherwise specified.
  • the virus used in this example was purchased from the National Influenza Center.
  • Neisseria meningitidis of GenBank Accession No. X77920.1
  • Primer 5.0 software was used to design primers, upstream primer: 5-CATGCCATGGCTTTAGTTGAATTGAA-3, and downstream primer: 5-CCGGAATTCTTATTTTTTCTTTTGCGGAG-3, wherein the underlined parts were Ncol and EcoRI cleavage site.
  • Neisseria meningitidis CMCC 29336 purchased from China Medical Bacteria Collection, CMCC was boiled at 100 °C for 10 min, and 3 ⁇ l was used as a template for PCR amplification.
  • the PCR reaction system is: 3 ⁇ 1 template, lOxPCR buffer 5 ⁇ 1, 10mmol/L dNTP ⁇ , Pyrobest high-fidelity DNA polymerase (Shanghai Shenggong) ⁇ , 0.5 ⁇ l of the upstream and downstream primers with a final concentration of 0.5 mol/L, plus super Pure water to 50 ⁇ 1.
  • the PCR reaction conditions were as follows: pre-denaturation at 94 °C for 5 min; then denaturation at 94 °C for 45 s, annealing at 50 °C for 45 s, and extension at 72 °C for 2 min; a total of 30 cycles, and a final extension of 72 °C for 10 min.
  • lane 1 is the molecular weight standard of DL2000 DNA
  • lane 2 is the PCR amplification product of the gene encoding P64K.
  • the results showed that a coding gene of P800K of about 1800 bp was amplified.
  • the coding gene of P64K obtained above was sequenced, and the sequencing result showed that the nucleotide sequence thereof is shown as SEQ ID NO: 6 in the sequence listing, and the encoded amino acid sequence is shown in SEQ ID NO: 5 in the sequence listing.
  • the above-mentioned target fragment was recovered, and the recovered PCR product and the pET28a vector were double-digested with Ncol and EcoRI, respectively, and the product was subjected to 1% agarose gel electrophoresis, and the fragment was cut by a gel, and the enzyme was cleaved with the enzyme of the vector pET28a.
  • the cut products were mixed at a molar ratio of 1:3, and ligated overnight at room temperature by T4 DNA ligase.
  • the ligation product was transformed into JM109 competent cells by CaCl 2 method. After 12 hours, the monoclonal clones were picked and the positive clones were screened by colony PCR. An E. coli engineered strain expressing P64K was obtained. 3. Large-scale cultivation of engineering strains, purification and detection of Neisseria meningitidis outer membrane protein (P64K)
  • the P64K-expressing Escherichia coli engineering bacteria obtained in the above step 2 was amplified by a tertiary culture, and finally transferred to a 500 L broth medium, and the fermenter parameters were set as follows: stirring speed 350-400 rpm, temperature 35-37 ° C, dissolution The oxygen is controlled at 30-40%, and after 36-48 hours of culture, the culture is terminated.
  • the culture medium is subjected to continuous high-speed centrifugation for solid-liquid separation to collect the cells, and the collected cells are homogenized by high pressure, and then the bacterial fragments are removed by centrifugation, solid ammonium sulfate is added to the supernatant, and hydrophobic chromatography is sequentially performed.
  • Anion exchange chromatography (Q-sepharose FF GE ) and gel filtration ( sephadex s200 GE ) gave a stock solution of P64K.
  • Figure 1-5 shows the reversed-phase high performance liquid chromatogram of Neisseria meningitidis outer membrane protein (P64K) with a detection wavelength of 280 nm. The results show only one peak and the chromatographic retention time is 7.71 minutes. The purity is 100%;
  • Figure 3 is the outer membrane protein (P64K) peptide spectrum (trypsin) of N. meningitidis. The results show that the N.
  • FIG. 4 is the electrophoresis pattern of the outer membrane protein of Neisseria meningitidis (P64K), and P64K indicates the Neisseria meningitidis expressed by Escherichia coli engineering bacteria obtained above.
  • Outer membrane protein (P64K), LMWP is a low molecular weight protein standard (provided by GE).
  • the P64K obtained above has only one band with a purity of 100%.
  • Figure 5 shows the outer membrane of N. meningitidis.
  • the mass spectrogram of the protein P64K has a molecular weight of 61932.7 Da, which is consistent with the theoretical molecular weight estimated from the amino acid sequence.
  • the M2e peptide was purified by preparative high performance liquid chromatography with a stationary phase of 0.1% CF 3 COOH/H 2 0 and a mobile phase of 0.1% CF 3 COOH/CH 3 CN.
  • the specific detection results are shown in Fig. 6. -7 is shown.
  • Figure 6 is a reversed-phase high performance liquid chromatogram of the M2e peptide with a detection wavelength of 280 nm. The results show that the chromatographic retention time is 22.532 min and the chromatographic purity is 96.52%.
  • Figure 7 is a mass spectrometry diagram of the M2e peptide with a molecular mass of 2670.7 Da, which is consistent with the theoretical molecular weight estimated from the amino acid sequence.
  • SMCC succinimidyl-4-(N-maleimidomethyl)cydohexane- 1 -carboxylate
  • P64K:SMCC with a molar ratio of 1:20 was mixed in phosphate buffer pH 7.2, stirred at room temperature for 2 hours, added The reaction was terminated with an appropriate amount of glycine, and then ultra-filtered activated P64K with an ultrafiltration cup (purchased from Millipore (Shanghai) Trading Co., Ltd., MW: 30 KDa) to remove unreacted SMCC and excess glycine.
  • the activated P64K was reacted at a known concentration with M2e at a molar ratio of 1:15 at pH 6.5, 4 ° C for 24 h, and the reaction was terminated with mercaptoethanol.
  • the amount of mercaptoethanol used is 15 mmol/mol M2e.
  • the obtained conjugate was purified by an ultrafiltration cup (MW: 30 KDa), and M2e, mercaptoethanol, and the like which did not participate in the reaction were removed. The yield of the obtained M2e-P64K conjugate was 58%.
  • the activated P64K was reacted at a known concentration with M2e at a molar ratio of 1:12 at pH 7.2, 4 ° C for 24 h, and the reaction was terminated with mercaptoethanol.
  • the obtained conjugate was purified by an ultrafiltration cup (MW: 30 KDa), and M2e, mercaptoethanol, and the like which did not participate in the reaction were removed. The yield of the obtained M2e-P64K conjugate was 50%.
  • MBS ( m-maleimidobenzoyl-N-hydroxysuccinimide ester )
  • P64K:MBS with a molar ratio of 1:20 was mixed in phosphate buffer pH 7.2, stirred at room temperature for 2 hours, and the reaction was stopped by adding appropriate amount of glycine.
  • the activated P64K was then ultrafiltered with an ultrafiltration cup (MW: 30 KDa) to remove unreacted MBS and excess glycine.
  • the activated P64K was reacted at a known concentration with M2e at a molar ratio of 1:10 at pH 7.5, 4 ° C for 36 h, and the reaction was terminated with mercaptoethanol.
  • the obtained conjugate was purified by an ultrafiltration cup (MW: 30 KDa), and M2e, mercaptoethanol, and the like which did not participate in the reaction were removed. The yield of the obtained M2e-P64K conjugate was 47%.
  • sulfo-MBC m -maleimidobenzoyl- N-hydroxysulfosuccinimide ester
  • sulfo-MBC m -maleimidobenzoyl- N-hydroxysulfosuccinimide ester
  • the activated P64K was reacted at a known concentration with M2e at a molar ratio of 1:20 at pH 6.5, 4 ° C for 18 h, and the reaction was terminated with mercaptoethanol.
  • the obtained conjugate was purified by an ultrafiltration cup (MW: 30 KDa), and M2e, mercaptoethanol, and the like which did not participate in the reaction were removed. The yield of the obtained M2e-P64K conjugate was 55%.
  • SMCC succinimidyl-4-(N-maleimidomethyl)cydohexane- 1 -carboxylate
  • P64K:SMCC with a molar ratio of 1:20 was mixed in phosphate buffer pH 7.2, stirred at room temperature for 2 hours, and added. The reaction was stopped with an appropriate amount of glycine, and then activated P08K was ultrafiltered with an ultrafiltration cup (MW: 30 KDa) to remove unreacted SMCC and excess glycine.
  • the activated P64K was reacted at a known concentration with M2e at a molar ratio of 1:18 at pH 6.8, 4 ° C for 24 h, and the reaction was terminated with mercaptoethanol.
  • the obtained conjugate was purified by an ultrafiltration cup (MW: 30 KDa), and M2e, mercaptoethanol, and the like which did not participate in the reaction were removed.
  • the yield of the obtained M2e-P64K conjugate was 54%.
  • the degree of coupling of the carrier protein P64K to M2e was determined by SDS-PAGE. As shown in Fig. 8, lane 4 was a standard Marker, and lane 3 was the M2e-P64K conjugate obtained in Example 3.
  • primers were designed using Primer 5.0 software.
  • Downstream primer 5 'GCACGATCCGCTCGAGGC AGTTGTTAAACTCGCGGCCC ACG GCC3, underlined BamHI and Xhol restriction sites, respectively.
  • the HA2 18 _ 72 gene was amplified by PCR.
  • the PCR reaction system was: 5 lOx PCR buffer, 4 L 2.5 mmol/L dNTP, Pyrobest high-fidelity DNA polymerase 0.5 L, two primers at a final concentration of 0.5 mol/L, template 0.5 L, ultrapure water H 2 0 supplementation
  • the system to 50 ⁇ PCR reaction conditions were: pre-denaturation at 95 °C for 5 min and then into the PCR cycle.
  • the PCR parameters were: denaturation at 94 °C for 45 s, annealing at 55 °C for 45 s, extension at 72 °C for 45 s, and after 30 cycles, extension at 72 °C for 10 min.
  • the PCR amplification product was subjected to 1.5% agarose gel electrophoresis, and the target fragment was recovered by gelatinization.
  • Lane 1 is a digested product of the PCR product of the above step 1 which was digested with BamHI/XhoI, and the fragment was cut by a gel.
  • the PCR product was digested with pET28a at a ratio of 1:3 by T4 DNA ligase overnight, and the ToplO competent state was transformed by CaCl 2 method. After incubation at 37 ° C for 12 hours, monoclonal clones were selected and positive clones were screened by colony PCR.
  • Use buffer B (20 mmol/L Tris, 500 mmol/L NaCl, pH 6.5) To carry out the stage elution, first wash the protein with 20% buffer B, then elute the target protein with 30% buffer B, and collect the elution peak, see Figure 12.
  • the purified product exchange buffer from Sephadex G25 was buffered into buffer C (5% acetonitrile, 0.05% trifluoroacetic acid) for SOURCE reverse phase chromatography using buffer D (80% acetonitrile, 0.05% trifluoroacetic acid).
  • Stage elution first wash the protein with 25% buffer D, then elute the target protein with 35% buffer D, and collect the elution peak, see Figure 13.
  • the collected target protein was freeze-dried and stored at -20 °C.
  • the SDS-PAGE results of the purified product are shown in Fig. 14.
  • the purity of the target protein was determined by HPLC.
  • the color column was ZORBAX 300SB-C8, the solution A was ultrapure water containing 0.1% trifluoroacetic acid, and the solution B was ultrapure water containing 0.1% trifluoroacetic acid and 90% acetonitrile.
  • the degraded gradient process was 0 to 100% solution B for 30 min.
  • the test results showed that the purity of the target protein was 95.7% (Fig. 15).
  • SMCC succinimidyl-4-(N-maleimidomethyl)cydohexane- 1 -carboxylate
  • P64K:SMCC with a molar ratio of 1:20 was mixed in phosphate buffer pH 7.2, stirred at room temperature for 2 hours, added The reaction was terminated with an appropriate amount of glycine, and then ultra-filtered activated P64K with an ultrafiltration cup (purchased from Millipore (Shanghai) Trading Co., Ltd., MW: 30 KDa) to remove unreacted SMCC and excess glycine.
  • the activated P64K was reacted at a known concentration with HA2 18-72 at a molar ratio of 1:15 at pH 6.5, 4 ° C for 24 h, and the reaction was terminated with mercaptoethanol.
  • the amount of mercaptoethanol used is 15 mmol/mol HA2 18-72 .
  • the obtained conjugate was purified by an ultrafiltration cup (MW: 30 KDa), and M2e, mercaptoethanol, and the like which did not participate in the reaction were removed.
  • the yield of the resulting HA2 18 _ 72 -P64K conjugate was 62%.
  • SMCC succinimidyl-4-(N-maleimidomethyl)cydohexane- 1 - carboxylate
  • P64K:SMCC with a molar ratio of 1:20 was mixed in phosphate buffer pH 7.2, stirred at room temperature for 2 hours, and added. Stop the reaction with the appropriate amount of glycine, then use the ultrafiltration cup (MW: 30KDa) The activated P64K was ultrafiltered to remove unreacted SMCC and excess glycine.
  • the activated P64K was reacted with HA2 18-72 at a molar ratio of 1:12 at a known concentration for 24 h at pH 7.2, 4 ° C, and quenched with mercaptoethanol.
  • the obtained conjugate was purified by an ultrafiltration cup (MW: 30 KDa), and ⁇ 2 dish and mercaptoethanol which did not participate in the reaction were removed.
  • the yield of the resulting HA2 18-72 -P64K conjugate was 54%.
  • MBS ( m-maleimidobenzoyl-N-hydroxysuccinimide ester )
  • P64K:MBS with a molar ratio of 1:20 was mixed in phosphate buffer pH 7.2, stirred at room temperature for 2 hours, and the reaction was stopped by adding appropriate amount of glycine.
  • the activated P64K was then ultrafiltered with an ultrafiltration cup (MW: 30 KDa) to remove unreacted MBS and excess glycine.
  • the activated P64K was reacted with HA2 18-72 at a molar ratio of 1:10 at a known concentration for 36 h at pH 7.5, 4 ° C, and the reaction was terminated with mercaptoethanol.
  • the obtained conjugate was purified by an ultrafiltration cup (MW: 30 KDa), and ⁇ 2 dish and mercaptoethanol which did not participate in the reaction were removed.
  • the yield of the resulting HA2 18-72 -P64K conjugate was 47%.
  • sulfo-MBC m -maleimidobenzoyl- N-hydroxysulfosuccinimide ester
  • sulfo-MBC m -maleimidobenzoyl- N-hydroxysulfosuccinimide ester
  • the activated P64K was reacted at a known concentration with HA2 18-72 at a molar ratio of 1:20 at pH 6.5, 4 ° C for 18 h, and the reaction was terminated with mercaptoethanol.
  • the obtained conjugate was purified by an ultrafiltration cup (MW: 30 KDa), and ⁇ 2 dish and mercaptoethanol which did not participate in the reaction were removed.
  • the yield of the resulting HA2 18-72 -P64K conjugate was 57%.
  • SMCC succinimidyl-4-(N-maleimidomethyl)cydohexane- 1 -carboxylate
  • P64K:SMCC with a molar ratio of 1:20 was mixed in phosphate buffer pH 7.2, stirred at room temperature for 2 hours, and added. The reaction was stopped with an appropriate amount of glycine, and then activated P08K was ultrafiltered with an ultrafiltration cup (MW: 30 KDa) to remove unreacted SMCC and excess glycine.
  • the activated P64K was reacted with HA2 18-72 at a molar ratio of 1:16 at a known concentration for 24 h at pH 6.8, 4 ° C, and quenched with mercaptoethanol.
  • the obtained conjugate was purified by an ultrafiltration cup (MW: 30 KDa), and ⁇ 2 dish and mercaptoethanol which did not participate in the reaction were removed.
  • the yield of the resulting HA2 18-72 -P64K conjugate was 52%.
  • the degree of coupling of the carrier protein P64K to HA2 18 _ 72 was determined by SDS-PAGE. As shown in Fig. 16, lane 1 is the molecular weight standard Marker, and lane 4 is the HA2 18 _ 72 - P64K conjugate.
  • M2e-P64K conjugate is mixed with Freund's incomplete adjuvant and emulsified
  • immunization group 1 Freund's incomplete adjuvant conjugate group
  • immunization group 2 aluminum adjuvant conjugate group
  • immunization group 3 M2e group
  • immunization group 4 negative control group (saline group). 50 g of the above four groups of test substances were taken separately, and BALB/C mice were immunized by subcutaneous injection, immunized once every 14 days, and immunized 3 times. Blood samples were taken 14 days after the last immunization and serum was separated for antibody titer detection.
  • the titer of the specific anti-influenza antibody in the serum obtained in the above step 2 was determined by an ELISA method.
  • the porous plate is first coated with M2e, and the immune serum obtained in the above step 2 diluted in a certain ratio is added, incubated for 2 hours, then the enzyme-labeled antibody is added, and finally the substrate liquid is added for color development, and the OD value is measured: on the ELISA detector, At 450 nm, the OD value of each well was measured after zeroing with a blank control well. See Figure 18.
  • Immunization group 2 aluminum adjuvant conjugate group 1: 64000
  • the multi-well plate is first coated with the antigen M2e, and the immune serum is diluted in proportion to the above-mentioned closed In the wells, incubate at 37 ° C for 1 h, wash (simultaneous blank wells, negative dilution of the negative control wells). Goat anti-mouse IgA ⁇ IgM ⁇ IgG, IgG1, IgG2, IgG3 were added and reacted for 2 h, washed. The enzyme-labeled antibody rabbit anti-goat IgG was added to each reaction well, and incubated at 37 ° C for 1 h, and the substrate solution was colored. On the ELISA detector, the OD value of each well was measured at 450 nm with zero adjustment of the blank control well. See Figure 19.
  • the H1N1, H3N2, and H5N1 synthetic sequences were each diluted to 2 / ml with a coating solution.
  • the OD value of each well was measured at 450 nm with the blank control well adjusted to zero. (See Figure 20)
  • mice were divided into a model group, a test vaccine group, a test vaccine + Freund's adjuvant group, and 10 rats in each group.
  • Each experimental group was intramuscularly injected with 50 ⁇ (immunization dose), and the model group was intramuscularly injected with 50 ⁇ L of normal saline. 0, 14, 28 days immunization.
  • BALB/C mice were anesthetized and then intranasally infected with 10 times LD50 of H1N1 influenza virus. After 14 days of challenge, the effect of influenza vaccine on mouse attack protection is shown in Table 4.
  • the protection rate of the vaccine + Freund's adjuvant group was 100%, indicating that the conjugate vaccine has a good preventive effect and laid the foundation for the research of broad-spectrum influenza vaccine.
  • Example 10 Protective effect of M2e-P64K conjugate on mouse PR8 strain influenza virus
  • the vaccine is diluted with PBS to the appropriate concentration for use;
  • Freund's adjuvant emulsification method The same amount of Freund's adjuvant and antigen solution are separately inhaled into one syringe, and the other empty syringe is connected with a fine rubber tube, and the air is removed. Then push the needles alternately until a thick emulsion is formed.
  • When formulating the vaccine with Al(OH) 3 adjuvant add the Al(OH) 3 adjuvant to the equal volume of the protein solution and mix quickly.
  • Balb/c mice were immunized by intramuscular injection. Immune 3 needles, 2 weeks per needle interval, 14 days after the third needle immunization. Ten mice in each group were challenged with 10 times LD50 of PR8. The body weight and survival rate of the mice were recorded every day after challenge, and recorded continuously for 20 days.
  • the M2e-P64k (new H1N1) + Freund's adjuvant group had the best protective effect on mice, up to 100%, and the mildest in mice; M2e-P64k (H3N2) + Freund's group It has a good protective effect and the protection rate is 90%; the protection rate of M2e-P64k (new H1N1) + ⁇ 1 ( ⁇ ) 3 groups in mice is 70%.
  • Example 11 Protective effect of M2e-P64K conjugate on avian influenza H5N1 influenza virus protection
  • Sixty female 4 to 6 week old clean-grade BLAB/c mice were randomly divided into 6 groups according to body weight, 10 rats/group.
  • the vaccine immunization group 5 groups are: TA group (M2e-P64k (new H1N1 is SEQ ID No.l)) , TB (M2e-P64k (new HlNl is SEQ ID No.l) + Freund's), TC (M2e-P64k (new HlNl ie SEQ ID No.l) + Al(OH) 3 ), TD (M2e-P64k ( H3N2 is SEQ ID No.
  • TA, TB, TC, TD and TE were co-immunized 3 times on days 0, 14, and 28, respectively.
  • the immunization route was hind limb ⁇ meat injection.
  • the immunization dose was 50 g/head, and the volume was 0.1 ml/ only.
  • the CP group was immunized with an equal volume (0.1 ml/only) of Freund's adjuvant. Forty-two days after immunization, six groups of animals were infected with H5N1 avian influenza virus A/shenzhen/406h/06 strain of influenza virus by nasal instillation.
  • the infection dose was 102TCID50/only.
  • the infection volume is 50 ⁇ 1/only.
  • the clinical symptoms of the animals were observed, weighed daily, and the survival status was observed in detail, and the survival rate, average survival time, and prolonged life rate of the animals in each test group were calculated.
  • immunization group 1 Freund's adjuvant conjugate group
  • immunization group 2 aluminum adjuvant conjugate group
  • immunization group 3 HA2 18 _ 72
  • Immunization group 4 Negative control group (saline group). 50 g of the above four groups of test substances were taken separately, and BALB/C mice were immunized subcutaneously, immunized once every 14 days, and immunized 3 times. Blood samples were taken 14 days after the last immunization and serum was separated for antibody titer detection.
  • the titer of the antibody specific for the influenza virus in the serum obtained in the above step 2 was determined by an ELISA method.
  • the perforated plate was first coated with HA2 18 _ 72 , and the serum obtained in the above step 2 was added in a certain proportion, incubated for 2 h, then the enzyme-labeled antibody was added, and finally the substrate liquid was added for color development, and the OD value was measured: in the ELISA detector Above, at 450 nm, the OD value of each well was measured by zeroing the blank control well, as shown in Fig. 21.
  • Immunization group 2 aluminum adjuvant conjugate group 1: 64000
  • the conjugate HA2 18 _ 72 - P64K can induce high levels of antibodies in mice and mice. Compared with aluminum adjuvant, Freund's adjuvant has more obvious immune effect; HA2 18 _ alone 72 mice produce only very low body Antibodies.
  • Example 13 HA2 18 _ 72 - P64K conjugate against mouse H1N1 influenza virus attack protection effect
  • the vaccine was diluted with PBS to the corresponding concentration for use;
  • Freund's adjuvant emulsification method equal amount of Freund's adjuvant and antigen solution respectively Inhaled into one syringe, connected to another empty syringe with a thin rubber tube, pay attention to the air, and then push the needle alternately until a thick emulsion is formed.
  • Al ( OH ) 3 adjuvant firstly, Al Add ( OH ) 3 adjuvant to an equal volume of protein solution and mix quickly, as for ice.
  • Balb/c mice were immunized by intramuscular injection.
  • mice in each group were challenged with 10 times LD50 of H1N1. The body weight and survival rate of the mice were recorded every day after challenge, and recorded continuously for 20 days. The effect of influenza vaccine on mouse attack protection is shown in Table 8 below.
  • the HA2-P64k+ Freund's adjuvant group had the best protective effect on mice, up to 40%, and the mice had the mildest incidence.
  • Example 14 Protective effect of M2e-P64 conjugate and HA2 18 ⁇ - P64K conjugate on mouse H1N1 influenza virus
  • Each experimental group was intramuscularly injected with 50 ⁇ (immunization dose), and the model group was intramuscularly injected with 50 ⁇ L of normal saline. 0, 14, 28 days immunization. After 14 days of the last immunization, BALB/C mice were anesthetized and then intranasally infected with 10 times LD50 of H1N1 influenza virus. After challenge for 14 days, the effect of influenza vaccine on mouse attack protection is shown in the following table. Table 9 Attack protection effect
  • the protection rate is also 100%, indicating that the combination of the two vaccines can achieve the effect of the M2e-P64K+ Freund's adjuvant group, but the dosage of the HA2 18 _ 72 -P64K+ Freund's adjuvant group can be appropriately reduced.
  • Influenza vaccines can be used alone or in combination.

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Description

流感病毒 M2或 HA片段与蛋白载体的缀合物及其制药应用
技术领域
本发明涉及蛋白偶联物及其制备方法， 以及包括该蛋白偶联物的广谱型 流感疫苗， 特别是涉及 M2e/HA218_72-载体蛋白偶联物及其制备方法， 以及包 括所述两种载体蛋白偶联物至少之一的广谱型流感疫苗。 背景技术
流感病毒株具有高度变异性， 因此， 需要研制对不同血清型 （例如甲型 流感病毒 H1N1、 甲型流感病毒 H3N2、 甲型流感病毒 H5N1 (又称禽流感） 等） 的流感病毒均有预防保护效果的广谱型流感疫苗。 目前的流感疫苗一般 以包括高度保守的抗原片段， 例如：
Merck 公司开发出预防流感的偶联物， 如 US2004/0223976A1 和 CN1756562A利用流感病毒中高度保守的，具有预防保护效果的抗原片段 M2e 和 HA0蛋白质与载体蛋白 OMP偶联。
Acambis公司开发出 M2e-HBc VLPs,如 US 7361352B2和 CN1913920A, 同样利用流感病毒的 M2e片段， 与乙肝核心抗原通过化学偶联形成抗流感病 毒的嵌合体分子颗粒。
然而， 现有流感疫苗的广谱性仍然需要提高， 尤其是目前全球流感形势 严峻， 新的流感病毒株如禽流感病毒 (H5N1)和甲型流感病毒 (H1N1)不断出 现， 而且存在病毒重组产生新的、 高致病性二代病毒的危险， 已有的疫苗对 新出现的病毒没有预防保护效果。 因此， 急需采用全新技术路线， 研制广谱 型的、 对甲型 （H1N1 )流感、 禽流感（H5N1 )、 该两种病毒重组产生的二代 病毒和季节性流感病毒都具有预防保护效果的流感疫苗， 彻底控制、 消除流 感的危害。 发明内容
本发明的目的在于克服现有流感疫苗光谱性差的缺陷， 提供一种利用流 感病毒中高度保守、 具有预防保护效果的抗原片段 M2e或 HA218_72与载体蛋 白 P64K化学偶联， 开发出广谱型的流感疫苗， 该疫苗能够有效的应对各型 流感病毒， 由该偶联物制备的广谱型疫苗易于大规模生产制备， 且有效周期 长， 可以大量储备。 克服了传统流感疫苗研制、 生产技术路线的不足， 从而 实现对各种流感病毒引发的疫情及时、 有效地控制。
本发明的技术方案如下：
本发明提供了一种预防流感的 M2e-载体蛋白偶联物， 其中， 所述蛋白偶 联物由甲型流感病毒 M2蛋白质的胞外结构域 M2e与载体蛋白化学偶联而 成， 其中， 所述的 M2e与载体蛋白通过硫醚键共价连接， 所述的 M2e的氨基 酸序列是如 SEQ ID NO: 2所示的氨基酸序列、 如 SEQ ID NO: 7所示的氨 基酸序列或者如 SEQ ID NO: 8所示的氨基酸序列：
SLLTEVETPTRXaa!EWEXaazRXaagSDSSDC [SEQ ID NO: 2]
其中，
Xa 为苏氨酸、 异亮氨酸或丝氨酸；
Xaa2为丝氨酸、 天冬氨酸或半胱氨酸；
Xaa3为半胱氨酸、 丙氨酸或丝氨酸；
所述的载体蛋白为脑膜炎奈瑟氏菌外膜蛋白 P64K; 所述 P64K的氨基酸 序列是如序列表中 SEQ ID NO: 5所示的氨基酸序列。
本发明还提供了以上所述的 M2e-载体蛋白偶联物的制备方法， 所述方法 包括先将载体蛋白用活化剂活化， 再与 M2e混合， 反应得到蛋白偶联物； 其 中， M2e与载体蛋白的摩尔比例为 10: 1至 20: 1;
所述偶联反应的 pH值为 6.5-7.5;
所述偶联反应的时间为 18-36小时；
所述的活化剂选自 SMCC、 MBS和 sulfo-MBS的任意一种。
本发明还提供了一种广谱型流感疫苗， 其中， 所述的疫苗含有本发明所 述的 M2e-载体蛋白偶联物和佐剂。
本发明还提供了一种 HA218_72-载体蛋白偶联物， 其中， 所述蛋白偶联物 是由禽流感 H5N1病毒 HA218_72多肽与载体蛋白化学偶联而成， 其中， 所述 的 HA218_72多肽与载体蛋白通过硫醚键共价连接， 所述的 HA218_72多肽的氨 基酸序列为 SEQ ID NO: 3所示的氨基酸序列； 所述的载体蛋白选自脑膜炎 奈瑟氏菌外膜蛋白 P64K、 破伤风类毒素、 乙肝核心抗原、 匙孔血蓝蛋白、 轮 状病毒衣壳蛋白和牛或人乳头瘤病毒 VLP的 L1蛋白的任意一种。
本发明还提供了以上所述的 ΗΑ218_72-载体蛋白偶联物的制备方法， 是先 将载体蛋白用活化剂活化， 再与适当比例的 ΗΑ218_72多肽混合， 反应得到蛋 白偶联物 ΗΑ218_72; 其中， 所述的 ΗΑ218_72多肽与载体蛋白的摩尔比例为 10: 1至 20: 1;
所述偶联反应的 ρΗ值为 6.5-7.5;
所述偶联反应的时间为 18-36小时；
所述的活化剂选自 SMCC、 MBS, sulfo-MBS的任意一种。
本发明还提供了一种广谱型流感疫苗， 其中， 所述的疫苗含有本发明所 述的 HA218_72载体蛋白偶联物和佐剂。
本发明还提供了一种广谱型流感疫苗， 其中， 所述的疫苗含有本发明所 述的 M2e-载体蛋白偶联物和含有本发明所述的 HA218_72 -载体蛋白偶联物和 佐剂。
包括本发明的 M2e-载体蛋白偶联物和 /或 HA218_72-载体蛋白偶联物的广 谱型流感疫苗的毒副作用小，安全性高，耐受性好，能够有效预防甲型 H1N1 , 禽流感 H5N1 流感病毒的感染， 甚至还能有效其他病毒亚型的感染， 广谱性 好。 用本发明的疫苗免疫小鼠， 能够诱导小鼠产生高水平的抗体， 并能保护 免疫小鼠对病毒的致死剂量攻击。 附图说明
图 1为脑膜炎奈瑟氏菌外膜蛋白 P64K PCR扩增产物的琼脂糖凝胶电泳 图；
图 2为脑膜炎奈瑟氏菌外膜蛋白 P64K的反相色谱图谱；
图 3为脑膜炎奈瑟氏菌外膜蛋白 P64K的肽谱（胰蛋白酶）；
图 4为脑膜炎奈瑟氏菌外膜蛋白 P64K的电泳图谱；
图 5为脑膜炎奈瑟氏菌外膜蛋白 P64K的质谱分析图谱；
图 6为 M2e的反相高效液相色谱图谱； 图 7为 M2e的质谱分析图谱；
图 8为 M2e-P64K偶联物的 SDS凝胶电泳图；
图 9为 M2e-P64K偶联物的凝胶色谱图；
图 10为 HA2 ( 18_72) PCR扩增产物的琼脂糖凝胶电泳结果；
图 11为 HA2 ( 18_72)的载体构建；
图 12为 HA218_72的 Mono Q纯化过程图谱；
图 13为 HA218_72的 SOURCE纯化过程图谱；
图 14为 HA218_72的 SDS-PAGE图谱；
图 15为 HA218_72的反相高效液相色谱图；
图 16为 HA218_72-P64K的 SDS-PAGE图谱；
图 17为 HA218_72-P64K的凝胶色谱图；
图 18为 M2e-P64K偶联物的免疫原性分析： 抗体效价图；
图 19为 M2e-P64K偶联物的免疫原性分析： 抗体亚型鉴定图； 图 20为 M2e-P64K偶联物的免疫原性分析： 血清交叉反应；
图 21为 HA218_72- P64K偶联物的免疫原性分析： 抗体效价图； 图 22为 M2e- P64K偶联物对小鼠 PR8株流感病毒攻击保护效果图； 图 23为 M2e- P64K偶联物对禽 H5N1攻击保护效果图。 具体实施方式
本发明提供了一种预防流感的 M2e-载体蛋白偶联物， 其中， 所述蛋白偶 联物由甲型流感病毒 M2蛋白质的胞外结构域 M2e与载体蛋白化学偶联而 成， 其中， 所述的 M2e与载体蛋白通过硫醚键共价连接， 所述的 M2e的氨基 酸序列是如 SEQ ID NO: 2所示的氨基酸序列、 如 SEQ ID NO: 7所示的氨 基酸序列或者如 SEQ ID NO: 8所示的氨基酸序列：
SLLTEVETPTRXaa!EWEXaa2RXaa3SDSSDC [SEQ ID NO: 2]
其中，
Xa 为苏氨酸、 异亮氨酸或丝氨酸；
Xaa2为丝氨酸、 天冬氨酸或半胱氨酸；
Xaa3为半胱氨酸、 丙氨酸或丝氨酸； 所述的载体蛋白为脑膜炎奈瑟氏菌外膜蛋白 P64K; 所述 P64K的氨基酸 序列是如序列表中 SEQ ID NO: 5所示的氨基酸序列。 其中， 优选编码 P64K 的核苷酸序列如 SEQ ID NO: 6所示。
进一步优选地， 所述的 M2e来源于甲型流感病毒 H1N1 , 具有如 SEQ ID NO: 1所示的氨基酸序列： SLLTEVETPTRSEWECRCSDSSDC [SEQ ID NO: 1] , 甲型流感病毒 H3N2 具有如 SEQ ID NO : 7 所示的氨基酸序列： SLLTEVETPIRNEWGCRCNDSSDC [SEQ ID NO: 7] , 或者甲型流感病毒 H5N1 ( 即禽流感） 具有如 SEQ ID NO : 8 所示的氨基酸序列： SLLTEVETPTRNEWECRCSDSSDC [SEQ ID NO: 8]。 最优选地， 所述的 M2e 来源于甲型流感病毒 H3N2 具有如 SEQ ID NO: 7 所示的氨基酸序列： SLLTEVETPIRNEWGCRCNDSSDC [SEQ ID NO: 7]。
优选地， 本发明所述的 M2e优选由固相合成法得到。
本发明还提供了以上所述的 M2e-载体蛋白偶联物的制备方法， 所述方法 包括先将载体蛋白用活化剂活化， 再与 M2e混合， 反应得到蛋白偶联物； 其 中， M2e与载体蛋白的摩尔比例为 10: 1至 20: 1;
所述偶联反应的 pH值为 6.5-7.5;
所述偶联反应的时间为 18-36小时；
所述的活化剂选自 SMCC、 MBS和 sulfo-MBS的任意一种。 其中 SMCC 全称为 Succinimidyl-4-(N-maleimidomethyl) cy dohexane- 1 -carboxylate , MBS 全称为 m-maleimidobenzoyl-N-hydroxy succinimide ester , sulfo-MBC全称为 m -maleimidobenzoyl- N-hydroxysulfosuccinimide ester。
优选地， 所述的 M2e与载体蛋白的比例为 12: 1至 18: 1 , 更优选 15:1; 所述偶联反应的 pH值为 6.8-7.2; 所述偶联反应的时间为 24小时； 所述的活 化剂为 SMCC。
本发明还提供了一种广谱型流感疫苗， 其中， 所述的疫苗含有本发明所 述的 M2e-载体蛋白偶联物和佐剂。
本发明还提供了一种 HA218_72-载体蛋白偶联物， 其中， 所述蛋白偶联物 是由禽流感 H5N1病毒 HA218_72多肽与载体蛋白化学偶联而成， 其中， 所述 的 HA218_72多肽与载体蛋白通过硫醚键共价连接， 所述的 HA218_72多肽的氨 基酸序列为 SEQ ID NO: 3所示的氨基酸序列 （ MVDGWYGYHHSNEQGSG YAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFC[SEQ ID NO: 3] ); 所述的载体蛋白选自脑膜炎奈瑟氏菌外膜蛋白 P64K、破伤风类毒素、 乙肝核 心抗原、 匙孔血蓝蛋白、 轮状病毒衣壳蛋白和牛或人乳头瘤病毒 VLP的 L1 蛋白的任意一种。
优选地， 所述的载体蛋白为脑膜炎奈瑟氏菌外膜蛋白 Ρ64Κ, 所述 Ρ64Κ 的氨基酸序列为 SEQ ID NO: 5所示的氨基酸序列。 所述的 HA218_72多肽是 经基因工程菌表达而来； 所述 HA218_72多肽编码基因的核苷酸序列如序列表 中 SEQ ID NO: 4所示； 所述基因工程菌为大肠杆菌 Ecoli.BL21。
本发明还提供了以上所述的 HA218_72-载体蛋白偶联物的制备方法， 是先 将载体蛋白用活化剂活化， 再与适当比例的 HA218_72多肽混合， 反应得到蛋 白偶联物 HA218_72; 其中， 所述的 HA218_72多肽与载体蛋白的摩尔比例为 10: 1至 20: 1; 所述偶联反应的 pH值为 6.5-7.5; 所述偶联反应的时间为 18-36 小时； 所述的活化剂选自 SMCC、 MBS, sulfo-MBS的任意一种。
优选地， 所述的 HA218_72多肽与载体蛋白的摩尔比例为 12: 1至 16: 1 , 更优选 15： 1； 所述偶联反应的 pH值优选为 6.8-7.2; 所述偶联反应的时间优 选为 24小时； 所述的活化剂优选为 SMCC。
本发明还提供了一种广谱型流感疫苗， 其中， 所述的疫苗含有本发明所 述的 HA218_72载体蛋白偶联物和佐剂。
本发明还提供了一种广谱型流感疫苗， 其中， 所述的疫苗含有本发明所 述的 M2e-载体蛋白偶联物和含有本发明所述的 HA218_72 -载体蛋白偶联物和 佐剂。
本发明所述广谱型流感疫苗，可以包括本领域常用的各种例如氢氧化铝、 弗氏佐剂的免疫佐剂。
实施例
本发明的实施方案通过下列实施例举例说明。 然而， 应当理解， 本发明 的实施方案不限于这些实施例的特定细节， 因为对于本领域的普通技术人员 来说， 其其他的变化是已知的， 或根据直接公开的内容和附属的权利要求是 显而易见的。 因此， 凡基于本发明上述内容所实现的技术均属于本发明的范 围。 本文引用的参考文献以其全文通过引用并入本文。
下述实施例中所述实验方法， 如无特殊说明， 均为常规方法； 所述试剂 和生物材料， 如无特殊说明， 均可从商业途径获得。 例如， 本实施例所用病 毒购自国家流感中心。
实施例 1、 脑膜炎奈瑟氏菌外膜蛋白 （P64K ) 的制备
1、 脑膜炎奈瑟氏菌外膜蛋白 （P64K )编码基因的克隆
根据 GenBank Accession No.X77920.1 的脑膜炎奈瑟氏菌的序列， 采用 Primer 5.0 软 件 设 计 引 物 ， 上 游 引 物 ： 5-CATGCCATGGCTTTAGTTGAATTGAA-3 , 下 游 引 物 ： 5-CCGGAATTCTTATTTTTTCTTTTGCGGAG-3，其中下划线部分分别为 Ncol 和 EcoRI的酶切位点。 将脑膜炎奈瑟氏菌 CMCC 29336 (购自中国医学细菌 保藏中心， 筒称 CMCC )于 100°C条件下煮沸 lOmin, 吸取 3μ1作为模板， 进 行 PCR扩增。 PCR反应体系为： 3μ1模板， lOxPCR緩沖液 5μ1, 10mmol/L dNTP Ιμΐ, Pyrobest高保真 DNA聚合酶（上海生工产品） Ιμΐ, 终浓度为 0.5 mol/L 的上下游引物各 0.5μ1, 加超纯水至 50μ1。 PCR反应条件为： 先 94°C预变性 5min; 然后 94°C变性 45s, 50°C退火 45s, 72°C延伸 2min; 共 30个循环， 最 后 72°C延伸 10min。 将 PCR扩增产物进行 1%琼脂糖凝胶电泳， 结果如图 1 所示。 其中， 泳道 1为 DL2000 DNA分子量标准， 泳道 2为 P64K编码基因 的 PCR扩增产物。 结果表明， 扩增得到 1800bp左右的 P64K的编码基因。 对 上述获得的 P64K的编码基因进行测序， 测序结果表明， 其核苷酸序列如序 列表中 SEQ ID NO: 6所示， 其编码的氨基酸序列如序列表中 SEQ ID NO: 5 所示。
2、 载体和工程菌的构建
回收上述目的片段， 将回收的 PCR产物和 pET28a载体分别用 Ncol和 EcoRI进行双酶切， 产物进行 1%琼脂糖凝胶电泳检测， 切胶回收酶切片段， 将酶切产物与载体 pET28a的酶切产物以 1 : 3的摩尔比进行混合，经 T4 DNA 连接酶室温过夜连接， 连接产物用 CaCl2法转化 JM109感受态细胞， 12小时 后， 挑取单克隆， 用菌落 PCR法筛选阳性克隆， 得到表达 P64K的大肠杆菌 工程菌。 3、 工程菌株大规模培养、 脑膜炎奈瑟氏菌外膜蛋白 （P64K ) 的纯化和 检测
将上述步骤 2获得的表达 P64K的大肠杆菌工程菌经三级培养放大， 最 后转至 500L的肉汤培养基中， 发酵罐参数设置为： 搅拌速度 350-400rpm, 温度 35-37°C , 溶氧控制在 30-40%, 培养 36-48小时后， 终止培养。
将培养液经连续高速离心进行固液分离以收集菌体， 将收集到的菌体经 高压匀浆后， 离心去除菌体碎片， 在上清液中加入固体硫酸铵， 依次进行疏 水层析、阴离子交换层析（ Q-sepharose FF GE )和凝胶过滤（ sephadex s200 GE ), 得到 P64K的原液。
按照 《中国人民共和国药典》 2005年版第三部要求和质量标准对上述获 得的 P64K原液进行纯度、 残留杂质和结构鉴别， 具体的检测结果如图 1-5 所示。 其中， 图 2为脑膜炎奈瑟氏菌外膜蛋白 （P64K ) 的反相高效液相色谱 图，检测波长为 280nm,结果如图中所示只有一个峰，其色谱保留时间为 7.71 分钟， 色谱纯度为 100%; 图 3为脑膜炎奈瑟氏菌外膜蛋白（P64K )肽谱（胰 蛋白酶）， 结果显示， 上述获得的脑膜炎奈瑟氏菌外膜蛋白 （P64K )与 P64K 标准品 （古巴分子免疫中心提供） 的谱图完全一致； 图 4为脑膜炎奈瑟氏菌 外膜蛋白 （P64K ) 的电泳图语， P64K表示上述获得的由大肠杆菌工程菌表 达的脑膜炎奈瑟氏菌外膜蛋白（ P64K ), LMWP表示低分子量蛋白标准品（ GE 公司提供）， 在电泳谱图上， 上述获得的 P64K只有一条带， 纯度为 100%; 图 5为脑膜炎奈瑟氏菌外膜蛋白 P64K的质谱分析图， 分子量为 61932.7Da, 与氨基酸序列推算的理论分子量一致。
实施例 2、 M2e肽的制备
1、 M2e肽的固相合成： 的氨基酸 Cys为起始反应物，将其上的 NH2用 Fmoc保护，与载体 Trityl chloride resin (也可为 Rink Amide resin或 Wang resin )反应, 摩尔比为 1: 1.5, 以 DCM或 DMF为反应溶剂， 碱性条件下混合， 反应 2小时左右； 用碱性溶剂 哌啶脱去连接在载体上第一个氨基酸氨基上的保护基 Fmoc, 用 DMF沖洗， 去除过量的氨基酸， Fmoc等小分子物质； 再加入活化剂 DIC/HOBT混合物， 以活化 Cys N端的自由氨基， 15分钟活化完毕后，加入第二个氨基酸（见 SEQ ID NO: 1 ), 依次循环， 完成 M2e肽上所有氨基酸的连接。 合成完毕后， 利 用三氟乙酸切除载体 Trityl chloride resin , 以及 M2e肽侧链上的保护基团， 以 得到完整的无保护基的 M2e肽链。
2、 M2e肽的纯化和结构确证：
合成完毕后， 利用制备高效液相色谱法纯化 M2e肽， 固定相为 0.1%的 CF3COOH/H20, 流动相为 0.1%的 CF3COOH/CH3CN, 具体的检测结果如图 6-7所示。 图 6为 M2e肽的反相高效液相色谱图， 检测波长为 280nm, 结果 显示， 色谱保留时间为 22.532min, 色谱纯度为 96.52%。 图 7为 M2e肽的质 谱分析图， 质谱分子量为 2670.7Da, 与氨基酸序列推算的理论分子量一致。
实施例 3、 M2e肽- P64K偶联物的制备
A组：
1、 P64K的活化：
以 SMCC( Succinimidyl-4-(N-maleimidomethyl )cydohexane- 1 -carboxylate ) 为活化剂， 将摩尔比为 1: 20的 P64K:SMCC在 pH 7.2的磷酸盐緩沖液里混 合， 室温搅拌 2小时， 加入适量甘氨酸终止反应， 然后用超滤杯（购自密理 博（上海）贸易有限公司， MW:30KDa )超滤活化的 P64K, 以除去未反应的 SMCC和过量的甘氨酸。
2、 偶联物的制备与纯化
活化的 P64K在已知浓度的情况下与 M2e以摩尔比 1: 15的比例在 pH6.5, 4°C的条件下反应 24h,用巯基乙醇终止反应。其中巯基乙醇的用量为 15mmol/ mol M2e。 用超滤杯（MW:30KDa ) 纯化制得的偶联物， 除去未参加反应的 M2e和巯基乙醇等。 所得 M2e- P64K偶联物的产率为 58%。
B组：
1、 P64K的活化：
以 SMCC( Succinimidyl-4-(N-maleimidomethyl )cydohexane- 1 - carboxylate ) 为活化剂， 将摩尔比为 1: 20的 P64K:SMCC在 pH 7.2的磷酸盐緩沖液里混 合，室温搅拌 2小时，加入适量甘氨酸终止反应，然后用超滤杯（MW:30KDa ) 超滤活化的 P64K, 以除去未反应的 SMCC和过量的甘氨酸。 2、 偶联物的制备与纯化
活化的 P64K在已知浓度的情况下与 M2e以摩尔比 1： 12的比例在 pH7.2, 4°C的条件下反应 24h, 用巯基乙醇终止反应。 用超滤杯（MW:30KDa ) 纯化 制得的偶联物， 除去未参加反应的 M2e和巯基乙醇等。 所得 M2e- P64K偶联 物的产率为 50%。
C组：
1、 P64K的活化：
以 MBS ( ( m-maleimidobenzoyl-N-hydroxysuccinimide ester ) 为活化剂 , 将摩尔比为 1 : 20的 P64K:MBS在 pH 7.2的磷酸盐緩沖液里混合， 室温搅拌 2 小时， 加入适量甘氨酸终止反应， 然后用超滤杯（MW:30KDa )超滤活化 的 P64K, 以除去未反应的 MBS和过量的甘氨酸。
2、 偶联物的制备与纯化
活化的 P64K在已知浓度的情况下与 M2e以摩尔比 1 : 10的比例在 pH7.5, 4°C的条件下反应 36h, 用巯基乙醇终止反应。 用超滤杯（MW:30KDa ) 纯化 制得的偶联物， 除去未参加反应的 M2e和巯基乙醇等。 所得 M2e- P64K偶联 物的产率为 47%。
D组：
1、 P64K的活化：
以 sulfo-MBC ( m -maleimidobenzoyl- N-hydroxysulfosuccinimide ester ) 为活化剂， 将摩尔比为 1 : 20的 Ρ64Κ: sulfo-MBC在 pH 7.2的磷酸盐緩沖液 里混合， 室温搅拌 2 小时， 加入适量甘氨酸终止反应， 然后用超滤杯 ( MW:30KDa )超滤活化的 P64K, 以除去未反应的 sulfo-MBC和过量的甘氨 酸。
2、 偶联物的制备与纯化
活化的 P64K在已知浓度的情况下与 M2e以摩尔比 1 : 20的比例在 pH6.5, 4°C的条件下反应 18h, 用巯基乙醇终止反应。 用超滤杯（MW:30KDa ) 纯化 制得的偶联物， 除去未参加反应的 M2e和巯基乙醇等。 所得 M2e- P64K偶联 物的产率为 55%。
E组： 1、 P64K的活化：
以 SMCC( Succinimidyl-4-(N-maleimidomethyl )cydohexane- 1 -carboxylate ) 为活化剂， 将摩尔比为 1: 20的 P64K:SMCC在 pH 7.2的磷酸盐緩沖液里混 合，室温搅拌 2小时，加入适量甘氨酸终止反应，然后用超滤杯（MW:30KDa ) 超滤活化的 P64K, 以除去未反应的 SMCC和过量的甘氨酸。
2、 偶联物的制备与纯化
活化的 P64K在已知浓度的情况下与 M2e以摩尔比 1: 18的比例在 pH6.8, 4°C的条件下反应 24h, 用巯基乙醇终止反应。 用超滤杯（MW:30KDa ) 纯化 制得的偶联物， 除去未参加反应的 M2e和巯基乙醇等。 所得 M2e- P64K偶联 物的产率为 54%。
实施例 4、 M2e- P64K偶联物的结构分析
SDS-PAGE检测载体蛋白 P64K与 M2e的偶联度， 如图 8所示， 泳道 4 为标准 Marker, 泳道 3为实施例 3所得的 M2e- P64K偶联物。
利用凝胶色谱 Superdex200, 根据被检测分子的大小差异分离不同组分， 分离条件： 流速： 0.5ml/min; 时间: 55min; 检测波长： 280nm; 温度： 室温； 流动相： 磷酸盐緩沖液 pH6.5。 得到 M2e- P64K的凝胶色谱图如图 9所示， 黑色曲线为 P64K , 灰色曲线为 P64K-M2e偶联物。
根据如下表 1的方法计算分子量 P64K与 M2e的偶联比例：
表 1
Figure imgf000012_0001
由上表可以得出， P64K与 M2e的偶联比例为 1: 9, 即 1个 P64K分子 可以偶联 9个 M2e分子。
实施例 5、 HA218_72多肽的制备
1、 HA218_72表达载体的构建
根据大肠杆菌的密码子偏性， 人工合成编码成熟的 HA218_72多肽的全长 DNA序列， 其核苷酸序列如序列表中 SEQ ID NO: 4所示。
以上述合成的重组序列为模板， 采用 Primer 5.0软件设计引物， 上游引 下游引物： 5 ' GCACGATCCGCTCGAGGC AGTTGTTAAACTCGCGGCCC ACG GCC3，， 下划线分别为 BamHI和 Xhol酶切位点。 采用 PCR的方法， 扩 增 HA218_72基因。 PCR反应体系为： 5 lOxPCR緩沖液，4 L 2.5mmol/L dNTP, Pyrobest高保真 DNA聚合酶 0.5 L,两条引物终浓度为 0.5 mol/L,模板 0.5 L, 超纯水 H20补充反应体系至 50μ PCR反应条件为： 95 °C预变性 5 min后 进入 PCR循环。 PCR反应参数为： 94 °C变性 45 s, 55 °C退火 45 s, 72 °〇延 伸 45 s; 30 个循环后， 72 °C延伸 10 min。 将 PCR扩增产物进行 1.5%琼脂糖 凝胶电泳， 切胶回收目的片段。
将 PCR回收产物与 pET28a载体分别进行 BamHI/XhoI双酶切， 经 1.5 % 琼脂糖凝胶电泳， 结果如图 10所示。 其中， 泳道 1为上述步骤 1的 PCR产 物经 BamHI/XhoI双酶切的酶切产物， 切胶回收酶切片段。 PCR酶切产物与 pET28a酶切产物按 1 : 3比例经 T4 DNA连接酶连接过夜， CaCl2法转化 ToplO 感受态。 37°C培养 12小时后， 挑选单克隆， 用菌落 PCR法筛选阳性克隆。
2、 HA218_72小量表达
将测序结果正确的表达载体 pet28a-HA218_72用 CaCl2法转化 Ecoli.BL21 感受态细胞。 如图 11所示。 挑选单菌落接种于 5ml LB培养基中 （卡那霉素 60 g/ml ), 37 °C培养过夜。将过夜菌按 1 : 100接种至 10 ml LB培养基中（卡 那霉素 60 g/ml ), 37 °C摇菌至 OD6。。=0.4-1.0,留取 1ml培养物后，加入 IPTG 至终浓度 lmmol/L, 37°C诱导过夜。 取 lml样品， 离心， 收集菌体， 超声破 碎， 然后 4°C , 12000 rpm, 离心 10 min。 10% SDS-PAGE检测上清和沉淀中 HA218_72的表达状况。
3、 HA218_72的纯化和检测
挑取高表达菌株单菌落至 5mL LLB培养基中培养过夜作为种子。将种子 按 1 : 100接种到 2xYT培养基中培养 3h,加入 IPTG至终浓度为 lmmol/L诱 导表达 6h后离心收集菌体。每克湿重的菌体加入 5mL緩沖液 A(20m mmol/L Tris, pH 6.5)重悬， 超声破碎 20min后离心取上清液经 0.22μιη过滤后进行 Mono Q离子交换层析。使用緩沖液 B(20mmol/L Tris, 500mmol/L NaCl, pH 6.5) 进行阶段洗脱， 先用 20%的緩沖液 B洗杂蛋白， 再用 30%的緩沖液 B洗脱目 的蛋白， 收集洗脱峰， 见图 12。 通过 Sephadex G25将上一步纯化产物更换緩 沖液至緩沖液 C(5%乙腈， 0.05%三氟乙酸)中进行 SOURCE反相层析， 使用 緩沖液 D(80%乙腈， 0.05%三氟乙酸)进行阶段洗脱， 首先用 25%的緩沖液 D 洗杂蛋白， 接着用 35%的緩沖液 D洗脱目的蛋白， 收集洗脱峰， 见图 13。 将 收集到的目的蛋白冷冻干燥后置 -20 °C保存。纯化产物的 SDS-PAGE结果如图 14所示。
使用 HPLC检测目的蛋白的纯度， 色语柱为 ZORBAX 300SB-C8 , 溶液 A为含 0.1 %三氟乙酸的超纯水， 溶液 B为含 0.1%三氟乙酸和 90%乙腈的超 纯水， 洗脱的梯度过程为 0至 100%的溶液 B 用时 30min。 检测结果显示目 的蛋白的纯度在 95.7% (图 15 )。
实施例 6、 HA218_79- P64K偶联物的制备
A组：
1、 P64K的活化：
以 SMCC( Succinimidyl-4-(N-maleimidomethyl )cydohexane- 1 -carboxylate ) 为活化剂， 将摩尔比为 1 : 20的 P64K:SMCC在 pH 7.2的磷酸盐緩沖液里混 合， 室温搅拌 2小时， 加入适量甘氨酸终止反应， 然后用超滤杯（购自密理 博（上海）贸易有限公司， MW:30KDa )超滤活化的 P64K, 以除去未反应的 SMCC和过量的甘氨酸。
2、 偶联物的制备与纯化
活化的 P64K在已知浓度的情况下与 HA218-72以摩尔比 1 : 15的比例在 pH6.5, 4°C的条件下反应 24h, 用巯基乙醇终止反应。 其中巯基乙醇的用量为 15mmol/ mol HA218-72。 用超滤杯（ MW:30KDa ) 纯化制得的偶联物， 除去未 参加反应的 M2e和巯基乙醇等。 所得 HA218_72- P64K偶联物的产率为 62%。
B组：
1、 P64K的活化：
以 SMCC( Succinimidyl-4-(N-maleimidomethyl )cydohexane- 1 - carboxylate ) 为活化剂， 将摩尔比为 1 : 20的 P64K:SMCC在 pH 7.2的磷酸盐緩沖液里混 合，室温搅拌 2小时，加入适量甘氨酸终止反应，然后用超滤杯（MW:30KDa ) 超滤活化的 P64K, 以除去未反应的 SMCC和过量的甘氨酸。
2、 偶联物的制备与纯化
活化的 P64K在已知浓度的情况下与 HA218-72以摩尔比 1 : 12的比例在 pH7.2, 4°C的条件下反应 24h, 用巯基乙醇终止反应。 用超滤杯（ MW:30KDa ) 纯化制得的偶联物，除去未参加反应的 ΗΔ2皿和巯基乙醇等。所得 HA218-72- P64K偶联物的产率为 54%。
C组：
1、 P64K的活化：
以 MBS ( ( m-maleimidobenzoyl-N-hydroxysuccinimide ester ) 为活化剂 , 将摩尔比为 1 : 20的 P64K:MBS在 pH 7.2的磷酸盐緩沖液里混合， 室温搅拌 2 小时， 加入适量甘氨酸终止反应， 然后用超滤杯（MW:30KDa )超滤活化 的 P64K, 以除去未反应的 MBS和过量的甘氨酸。
2、 偶联物的制备与纯化
活化的 P64K在已知浓度的情况下与 HA218-72以摩尔比 1 : 10的比例在 pH7.5, 4°C的条件下反应 36h, 用巯基乙醇终止反应。 用超滤杯（ MW:30KDa ) 纯化制得的偶联物，除去未参加反应的 ΗΔ2皿和巯基乙醇等。所得 HA218-72- P64K偶联物的产率为 47%。
D组：
1、 P64K的活化：
以 sulfo-MBC ( m -maleimidobenzoyl- N-hydroxysulfosuccinimide ester ) 为活化剂， 将摩尔比为 1 : 20的 Ρ64Κ: sulfo-MBC在 pH 7.2的磷酸盐緩沖液 里混合， 室温搅拌 2 小时， 加入适量甘氨酸终止反应， 然后用超滤杯 ( MW:30KDa )超滤活化的 P64K, 以除去未反应的 sulfo-MBC和过量的甘氨 酸。
2、 偶联物的制备与纯化
活化的 P64K在已知浓度的情况下与 HA218-72以摩尔比 1 : 20的比例在 pH6.5, 4°C的条件下反应 18h, 用巯基乙醇终止反应。 用超滤杯（ MW:30KDa ) 纯化制得的偶联物，除去未参加反应的 ΗΔ2皿和巯基乙醇等。所得 HA218-72- P64K偶联物的产率为 57%。 E组：
1、 P64K的活化：
以 SMCC( Succinimidyl-4-(N-maleimidomethyl )cydohexane- 1 -carboxylate ) 为活化剂， 将摩尔比为 1: 20的 P64K:SMCC在 pH 7.2的磷酸盐緩沖液里混 合，室温搅拌 2小时，加入适量甘氨酸终止反应，然后用超滤杯（MW:30KDa ) 超滤活化的 P64K, 以除去未反应的 SMCC和过量的甘氨酸。
2、 偶联物的制备与纯化
活化的 P64K在已知浓度的情况下与 HA218-72以摩尔比 1: 16的比例在 pH6.8, 4°C的条件下反应 24h, 用巯基乙醇终止反应。 用超滤杯（ MW:30KDa ) 纯化制得的偶联物，除去未参加反应的 ΗΔ2皿和巯基乙醇等。所得 HA218-72- P64K偶联物的产率为 52%。
实施例 7、 HA218_72- P64K偶联物的结构表征
SDS-PAGE检测载体蛋白 P64K与 HA218_72的偶联程度， 如图 16所示， 泳道 1为分子量标准 Marker, 泳道 4为 HA218_72- P64K偶联物。
利用凝胶色谱 Superdex200, 根据被检测分子的大小差异分离不同组分。 分离条件为： 流速： 0.5ml/min; 时间: 55min; 检测波长： 280nm; 温度： 室 温； 流动相：磷酸盐緩沖液 pH6.5。检测结果如图 17所示，灰色曲线为 P64K; 黑色曲线为 HA218_72- P64K偶联物。
根据如下表 2的方法计算分子量 P64K与 HA218_72的偶联比例:
表 2
Figure imgf000016_0001
由上表可以得出， P64K与 HA218_72的偶联比例为 1: 4, 即 1个 P64K 子可以偶联 4个 HA218_72分子。
实施例 8、 M2e- P64K偶联物在小鼠中的免疫原性分析
1、 M2e-P64K偶联物与佐剂混合
( 1 ) M2e- P64K偶联物与弗氏不完全佐剂混合、 乳化
用一次性无菌无热原注射器取 l.OmL弗氏不完全佐剂， 分别与等体积的 上述实施例 3制备的偶联物 M2e- P64K混合于样品瓶中，反复吹打 20次，使 之形成油包水的混合物， 将反复吹打的混合物滴加到双蒸水中， 乳化物呈白 色液滴， 且不分散。
( 2 ) M2e- P64K偶联物与铝佐剂的混合、 吸附
分别取 1.0mg/mL上述实施例 3制备的偶联物 M2e- P64K于无菌无热原 容器中， 加入等体积的铝佐剂， 室温搅拌 1小时， 即得。
2、 免疫程序
取 40只 10周龄的 BALB/C小鼠随机分为四组， 分别为， 免疫组 1: 弗 氏不完全佐剂偶联物组； 免疫组 2: 铝佐剂偶联物组； 免疫组 3: M2e组； 免 疫组 4: 阴性对照组（生理盐水组）。 分别取 50 g上述四组供试物， 皮下注 射免疫 BALB/C小鼠， 每 14天免疫一次， 共免疫 3次。 最后一次免疫 14天 后采集血液样本， 分离血清用于抗体滴度检测。
3、 抗体滴度检测
采用 ELISA方法测定上述步骤 2中获得的血清中特异性抗流感病毒的抗 体的滴度。 多孔板先用 M2e包被， 加入按一定比例稀释的上述步骤 2获得的 免疫血清， 孵育 2h, 然后再加入酶标抗体， 最后加入底物液显色， 测 OD值： 在 ELISA检测仪上， 于 450nm处， 以空白对照孔调零后测各孔 OD值。 见图 18。
结果表明， 偶联组第三次免疫后的血清滴度达到 1: 128000。
表 3 抗体滴度测定结果
^ . 抗体滴度
免疫组 1 弗氏佐剂偶联物组 1: 128000
免疫组 2 铝佐剂偶联物组 1: 64000
免疫组 3 M2e 1: 1000
免疫组 4 生理盐水组 0
结果表明， 所有佐剂组中， 偶联物 M2e- P64K都能诱导小鼠体内高水平 的抗体，弗氏佐剂和铝佐剂在小鼠体内有明显的佐剂效应差异，而单独的 M2e 在小鼠体内仅有较低的免疫原性。
4、 抗体亚型鉴定
多孔板先用抗原 M2e包被， 将免疫血清按比例稀释， 加入上述已封闭的 反应孔中， 置 37°C孵育 lh, 洗涤（同时做空白孔， 阴性对照孔同步稀释）。 加入山羊抗鼠 IgA\IgM\IgG、 IgGl、 IgG2、 IgG3 , 反应 2h, 洗涤。 于各反应 孔中加入酶标抗体兔抗山羊 IgG, 37°C孵育 lh, 加底物液显色。 在 ELISA检 测仪上， 于 450nm处， 以空白对照孔调零后测各孔 OD值。 见图 19所示。
结果表明， M2e-P64k偶联物主要诱导产生 IgGl亚型抗体。
5、 抗甲型 H1N1流感 M2抗血清与禽流感 H1N1、 季节性流感 (H1N1、 H3N2)交叉反应
用包被液将 H1N1、 H3N2、 H5N1合成序列分别稀释至 2 / ml。 分别 在三列孔中加入 lOOul, 4°C过夜； 封闭 lh; 将免疫血清按比例稀释后， 加入 上述已封闭的反应孔中， 置 37°C孵育 lh, 洗涤； 加酶标抗体， 37°C孵育 1.5h, 洗涤； 加底物液显色， 室温放置 10 ~ 20min; 终止反应； 在 ELISA检测仪上， 于 450nm处， 以空白对照孔调零后测各孔 OD值。 （见图 20 )
结果表明， 抗甲型 H1N1流感 M2e的抗血清与禽流感 H5N1、 季节性流 感 (H1N1、 H3N2) 的 M2产生显著的交叉反应。
实施例 9、 M2e- P64K偶联物对小鼠 H1N1流感病毒攻击保护效果
将实验小鼠分为模型组、 受试疫苗组、 受试疫苗 +弗氏佐剂组， 每组 10 只。各实验组肌肉注射 50μΙ 只（免疫剂量），模型组肌肉注射生理盐水 50μΙ 只。 0, 14, 28天免疫。 末次免疫后 14天攻毒， 将 BALB/C小鼠麻醉后滴鼻 感染 10倍 LD50的 H1N1流感病毒， 攻毒后观察 14天， 流感疫苗对小鼠攻 击保护的效果如下表 4所示。
表 4 攻击保护效果
动物数量 死亡只数 死亡率 （％) 保护率 （％ ) 模型 10 9 90
疫苗 10 8 80 20* 疫苗 +弗氏佐剂组 10 0 0 100
*与模型组比较 ρ < 0.05
疫苗 +弗氏佐剂组保护率为 100 % , 表明偶联疫苗有良好的预防效果， 为 广谱型流感疫苗研究奠定了基础。
实施例 10: M2e-P64K偶联物对小鼠 PR8株流感病毒攻击保护效果 疫苗用 PBS稀释至相应浓度备用； 弗氏佐剂乳化方法： 将等量的弗氏佐 剂和抗原溶液分别吸入 1个注射器内， 与另一个空注射器以一细胶管相连， 注意排净空气， 然后交替推动针管， 直至形成粘稠的乳剂为止； 配制疫苗与 Al ( OH ) 3佐剂时， 先将 Al ( OH ) 3佐剂加至等体积的蛋白溶液中迅速混匀， 至于冰上备用。 采用肌肉注射的方式免疫 Balb/c小鼠。 免疫 3针， 每针间隔 2周， 第三针免疫后 14天攻毒。每组小鼠 10只， 用 10倍 LD50的 PR8攻毒， 攻毒后每天记录小鼠的体重及存活率情况， 连续记录 20天。
如图 22所示， M2e-P64k (新 H1N1 ) +弗氏佐剂组对小鼠的保护效果最 好， 可达 100% , 小鼠发病最轻； M2e-P64k ( H3N2 ) +弗氏组也具有较好的 保护效果， 保护率为 90%; M2e-P64k (新 H1N1 ) +Α1(ΟΗ)3组对小鼠的保护 率为 70%。
实施例 11 : M2e- P64K偶联物对禽流感 H5N1流感病毒攻击保护效果 选取 60只 4 ~ 6周龄雌性清洁级 BLAB/c小鼠，按照体重随机分为 6组， 10只 /组， 分为病毒对照组 CP (弗氏佐剂）和 5个疫苗免疫组， 如表 5所示， 其中疫苗免疫组 5组分别为： TA组（ M2e-P64k (新 H1N1即 SEQ ID No.l ) )、 TB ( M2e-P64k (新 HlNl即 SEQ ID No.l ) +弗氏）、 TC ( M2e-P64k (新 HlNl 即 SEQ ID No.l ) +Al(OH)3 )、 TD ( M2e-P64k ( H3N2即 SEQ ID No.7 ) +弗氏）、 TE ( M2e-P64k (禽 H5N1即 SEQ ID No.8 ) +弗氏）。 各试验组 TA、 TB、 TC、 TD和 TE分别在第 0、 14、 28天共免疫 3次， 免疫途径为后肢 ^^肉注射， 免 疫剂量均为 50 g/只， 体积均为 0.1ml/只。 CP组免疫等体积（0.1ml/只） 的 弗氏佐剂。六组动物免疫后 42天，使用 H5N1禽流感病毒 A/shenzhen/406h/06 株流感病毒通过鼻腔滴注方式进行感染， 感染剂量为 102TCID50/只
(10LD50), 感染体积为 50μ1/只。 在病毒感染后 14天， 观察动物临床症状， 每天称重， 并详细观察其存活状况， 计算各试验组动物的存活率、 平均存活 时间和延长生命率。
动物保护率结果： 免疫动物在攻毒后， 每天观察死亡情况， 连续观察 14 天， 根据动物存活情况计算其死亡保护率。 如图 23和表 6所示， 其中病毒对 照组动物在观察期结束前 10只全部死亡， 各疫苗试验组动物 ΤΑ组死亡率最 高为 100% , TD组最低为 20%。 TE、 TB和 TC分别为 90%、 80%和 70%。 与病毒对照组比较， TD组动物死亡率有显著性差异（P=0.01 < 0.05 )。 与病 毒对照组比较， 以 TD组疫苗的死亡保护率最高为 80%。
表 5
Figure imgf000020_0001
注： "弗氏" 为弗氏佐剂； Al(OH)3为氢氧化铝佐剂。 表 6疫苗免疫对小鼠感染 H5N1禽流感病毒 A/shenzhen/406h/06株存活 观察结果 单位： 只
动 0d 1 d 2 d 3 d 4d 5 d 6d 7d 8 9 10 11 12 13d 物 d d d d d 组 数
别 里
CP 10 10 10 10 10 10 10 9 4 0 - - - - -
TA 10 10 10 10 10 10 10 10 8 2 1 0 - - -
TB 10 10 10 10 10 10 9 9 7 4 3 2 2 2 2
TC 10 10 10 10 10 10 10 9 5 3 3 3 3 3 3
TD 10 10 10 10 10 10 10 10 10 8 8 8 8 8 8
TE 10 10 10 10 10 10 10 10 8 4 2 2 1 1 1 实施例 12、 HA21S_77- P64K偶联物的免疫原性分析
( 1 ) HA218_72- P64K偶联物与弗氏不完全佐剂混合、 乳化
用一次性无菌无热原注射器取 l.OmL弗氏完不全佐剂， 分别与等体积的 上述实施例 3制备的偶联物 HA218_72- P64K混合于样品瓶中，反复吹打 20次， 使之形成油包水的混合物， 将反复吹打的混合物滴加到双蒸水中， 乳化物呈 白色液滴， 且不分散。
( 2 ) HA218_72- P64K偶联物与铝佐剂的混合、 吸附
分别取 1.0mg/mL上述实施例 3制备的偶联物 HA218_72- P64K于无菌无热 原容器中， 加入等体积的铝佐剂， 室温搅拌 1小时， 即得。
2、 免疫程序
取 40只 10周龄的 BALB/C小鼠随机分为四组， 分别为， 免疫组 1: 弗 氏佐剂偶联物组； 免疫组 2: 铝佐剂偶联物组； 免疫组 3: HA218_72; 免疫组 4: 阴性对照组（生理盐水组）。 分别取 50 g上述四组供试物， 皮下注射免疫 BALB/C小鼠， 每 14天免疫一次， 共免疫 3次。 最后一次免疫 14天后采集 血液样本， 分离血清用于抗体滴度检测。
3、 抗体滴度检测
采用 ELISA方法测定上述步骤 2中获得的血清中特异性抗流感病毒的抗 体的滴度。 多孔板先用 HA218_72包被， 加入按一定比例稀释的上述步骤 2获 得的血清， 孵育 2h, 然后再加入酶标抗体， 最后加入底物液显色， 测 OD值： 在 ELISA检测仪上， 于 450nm处， 以空白对照孔调零后测各孔 OD值， 见图 21。
表 7 抗体滴度测定结果
抗体滴度（第三次血样） 免疫组 1 弗氏不完全佐剂偶联组 1: 256000
免疫组 2 铝佐剂偶联物组 1: 64000
免疫组 3 HA218_72 1: 2000
免疫组 4 生理盐水组 0
结果表明， 偶联组第三次免疫后的血清滴度达到 1: 256000。 佐剂组中， 偶联物 HA218_72- P64K均能诱导 d、鼠体内产生高水平的抗体， 与铝佐剂相比， 弗氏佐剂具有更明显的免疫效果； 单独免疫 HA218_72的小鼠体内仅产生极低 的抗体。
实施例 13、 HA218_72- P64K偶联物对小鼠 H1N1流感病毒攻击保护效果 疫苗用 PBS稀释至相应浓度备用； 弗氏佐剂乳化方法： 将等量的弗氏佐 剂和抗原溶液分别吸入 1个注射器内， 与另一个空注射器以一细胶管相连， 注意排净空气， 然后交替推动针管， 直至形成粘稠的乳剂为止； 配制疫苗与 Al ( OH ) 3佐剂时， 先将 Al ( OH ) 3佐剂加至等体积的蛋白溶液中迅速混匀， 至于冰上备用。 采用肌肉注射的方式免疫 Balb/c小鼠。 免疫 3针， 每针间隔 2周， 第三针免疫后 14天攻毒。 每组小鼠 10只， 用 10倍 LD50的 H1N1攻 毒， 攻毒后每天记录小鼠的体重及存活率情况， 连续记录 20天。 流感疫苗对 小鼠攻击保护的效果如下表 8所示。
表 8 攻击保护效果
动物数量 死亡只数 死亡率 （％) 保护率 （％) 模型 10 10 100
疫苗 10 8 80 20 疫苗 +弗氏佐剂组 10 6 60 40
HA2-P64k+弗氏佐剂组对小鼠的保护效果最好， 可达 40%, 小鼠发病最 轻。
实施例 14: M2e-P64偶联物和 HA218^- P64K偶联物联用对小鼠 H1N1 流感病毒攻击保护效果
选取 40只 4~6周龄雌性清洁级 BALB/C小鼠， 按照体重随机分为 4组， 10只 /组，分为模型组和 3个疫苗免疫组，其中，其中免疫组分别为 M2e-P64+ 弗氏佐剂， HA218_72- P64K+弗氏佐剂，联用组（ M2e-P64+弗氏佐剂： HA218_72- P64K+弗氏佐剂 =2: 1 )
各实验组肌肉注射 50μΙ 只（免疫剂量），模型组肌肉注射生理盐水 50μϋ 只。 0, 14, 28天免疫。 末次免疫后 14天攻毒， 将 BALB/C小鼠麻醉后滴鼻 感染 10倍 LD50的 H1N1流感病毒， 攻毒后观察 14天， 流感疫苗对小鼠攻 击保护的效果如下表所示。 表 9 攻击保护效果
动物数量 死亡 J t 死亡率（％) 保护率（％ ) 模型组 10 9 90
M2e-P64K+弗氏佐剂组 10 0 0 100
HA218_72-P64K+弗氏佐剂组 10 6 60 40
联用组 10 0 0 100
M2e-P64K+弗氏佐剂组保护率为 100 % , 表明偶联疫苗有良好的预防效 果； 联用组（M2e-P64+弗氏佐剂： HA218_72- P64K+弗氏佐剂 =2: 1 ) 的保护 率也为 100%, 表明两种疫苗联用可以达到 M2e-P64K+弗氏佐剂组的效果， 但可以适当减少 HA218_72-P64K+弗氏佐剂组的用量， 上述试验表明两种流感 疫苗可单独使用也可联合使用。

Claims

权利 要求 书
1. 一种预防流感的 M2e-载体蛋白偶联物， 其特征在于， 所述蛋白偶联 物由甲型流感病毒 M2蛋白质的胞外结构域 M2e与载体蛋白化学偶联而成， 其中，所述的 M2e与载体蛋白通过硫醚键共价连接，所述的 M2e的氨基酸序 列是如 SEQ ID NO: 2所示的氨基酸序列、 如 SEQ ID NO: 7所示的氨基酸 序列或者如 SEQ ID NO: 8所示的氨基酸序列：
SLLTEVETPTRXaa!EWEXaazRXaagSDSSDC [SEQ ID NO: 2]
其中，
Xa 为苏氨酸、 异亮氨酸或丝氨酸；
Xaa2为丝氨酸、 天冬氨酸或半胱氨酸；
Xaa3为半胱氨酸、 丙氨酸或丝氨酸；
所述的载体蛋白为脑膜炎奈瑟氏菌外膜蛋白 P64K; 所述 P64K的氨基酸 序列是如序列表中 SEQ ID NO: 5所示的氨基酸序列。
2. 根据权利要求 1所述的蛋白偶联物， 其特征在于， 所述的 M2e的氨 基酸序列是如 SEQ ID NO: 1所示的氨基酸序列、 如 SEQ ID NO: 7所示的 氨基酸序列或者如 SEQ ID NO: 8所示的氨基酸序列。
3. 根据权利要求 1所述的蛋白偶联物， 其特征在于， 所述的 M2e的氨 基酸序列是如 SEQ ID NO: 7所示的氨基酸序列。
4. 权利要求 1-3中任意一项所述的蛋白偶联物的制备方法， 所述方法包 括先将载体蛋白用活化剂活化，再与 M2e混合，反应得到蛋白偶联物； 其中， 所述的 M2e与载体蛋白的摩尔比例为 10: 1至 20: 1;
所述偶联反应的 pH值为 6.5-7.5;
所述偶联反应的时间为 18-36小时；
所述的活化剂选自 SMCC、 MBS和 sulfo-MBS的任意一种。
5. 根据权利要求 4所述的方法， 其特征在于， 所述的 M2e与载体蛋白 的比例为 12: 1至 18: 1;
所述偶联反应的 pH值为 6.8-7.2;
所述偶联反应的时间为 24小时； 所述的活化剂为 SMCC。
6. 一种广谱型流感疫苗， 其特征在于， 所述的疫苗含有权利要求 1-3中 任意一项所述的 M2e-载体蛋白偶联物和佐剂。
7. 一种 HA218_72-载体蛋白偶联物， 其特征在于， 所述蛋白偶联物是由禽 流感 H5N1病毒 HA218_72多肽与载体蛋白化学偶联而成，其中，所述的 HA218_72 多肽与载体蛋白通过硫醚键共价连接， 所述的 HA218_72多肽的氨基酸序列为 SEQ ID NO: 3所示的氨基酸序列； 所述的载体蛋白选自脑膜炎奈瑟氏菌外膜 蛋白 P64K、 破伤风类毒素、 乙肝核心抗原、 匙孔血蓝蛋白、 轮状病毒衣壳蛋 白和牛或人乳头瘤病毒 VLP的 L1蛋白的任意一种。
8. 根据权利要求 7所述的蛋白偶联物， 其特征在于， 所述的载体蛋白为 脑膜炎奈瑟氏菌外膜蛋白 Ρ64Κ, 所述 Ρ64Κ的氨基酸序列为 SEQ ID NO: 5 所示的氨基酸序列。
9. 根据权利要求 7所述的蛋白偶联物， 其特征在于， 所述的 HA218_72多 肽是经基因工程菌表达而来；
所述 HA218_72多肽编码基因的核苷酸序列如序列表中 SEQ ID NO: 4所 示；
所述基因工程菌为大肠杆菌 Ecoli.BL21。
10. 根据权利要求 7-9中任意一项所述的蛋白偶联物的制备方法， 是先 将载体蛋白用活化剂活化， 再与适当比例的 HA218_72多肽混合， 反应得到蛋 白偶联物； 其中， 所述的 HA218_72多肽与载体蛋白的摩尔比例为 10: 1至 20: 1;
所述偶联反应的 pH值为 6.5-7.5;
所述偶联反应的时间为 18-36小时；
所述的活化剂选自 SMCC、 MBS, sulfo-MBS的任意一种。
11. 根据权利要求 10所述的方法， 其特征在于， 所述的 HA218_72多肽与 载体蛋白的比例为为 12: 1至 16: 1;
所述偶联反应的 pH值为 6.8-7.2;
所述偶联反应的时间为 24小时；
所述的活化剂为 SMCC。
12. 一种广谱型流感疫苗， 其特征在于， 所述的疫苗含有权利要求 7-9 中任意一项所述的偶联物和佐剂。
13. 一种广谱型流感疫苗， 其特征在于， 所述的疫苗含有权利要求 1-3 中任意一项所述的 M2e-载体蛋白偶联物和含有权利要求 7-9中任意一项所述 的 HA218_72 -载体蛋白偶联物和佐剂。
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