WO2016004904A2 - 一种流感血凝素糖蛋白多聚物纳米颗粒的制备方法 - Google Patents

一种流感血凝素糖蛋白多聚物纳米颗粒的制备方法 Download PDF

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WO2016004904A2
WO2016004904A2 PCT/CN2015/088748 CN2015088748W WO2016004904A2 WO 2016004904 A2 WO2016004904 A2 WO 2016004904A2 CN 2015088748 W CN2015088748 W CN 2015088748W WO 2016004904 A2 WO2016004904 A2 WO 2016004904A2
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influenza virus
hemagglutinin
influenza
signal peptide
transmembrane region
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WO2016004904A3 (zh
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吴军
刘波
唱韶红
巩新
王莎
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中国人民解放军军事医学科学院生物工程研究所
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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
    • 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
    • C07K14/08—RNA viruses
    • C07K14/11—Orthomyxoviridae, e.g. influenza virus
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

Definitions

  • the invention relates to a method for preparing influenza hemagglutinin glycoprotein multi-particle nanoparticles, belonging to the field of biotechnology.
  • Influenza referred to as influenza
  • influenza is an acute respiratory infection caused by influenza A, B, and C viruses.
  • Influenza vaccination is an important means of preventing influenza.
  • Influenza hemagglutinin is one of the main proteins on the surface of influenza virus. It exists in the form of a trimer. It is an important component of the surface of influenza virus involved in host cell adsorption and invasion. The induced neutralizing antibody can block the virus on the surface of host cells. The adsorption and invasion are therefore the main components of the flu vaccine.
  • Influenza hemagglutinin variability, reassortment is fast, and various new types of influenza occur frequently.
  • H5N1 highly pathogenic avian influenza, H1N1 influenza A, and H7N9 highly pathogenic avian influenza have caused serious problems in recent years. After the outbreak of the new epidemic, rapid development and production of vaccines is the key to epidemic control.
  • the existing influenza vaccines are mainly whole virus inactivated vaccines, split vaccines or subunit vaccines obtained by purifying the vaccines. These vaccines are obtained by virus culture of chicken attenuated strains or reassortant strains. Purified, inactivated or further lysed and purified.
  • the main problem with chicken embryo flu vaccine production technology is that virus production is limited by the supply of qualified chicken embryos.
  • the reassortment, attenuation and chicken embryo adaptation of virus strains require a lot of time and there is uncertainty.
  • the prepared vaccine contains, in addition to influenza hemagglutinin which is a main active ingredient, other proteins of the virus, and chicken embryo-derived proteins. The protein purity of the vaccine is low, and it is easy to cause side effects such as allergies.
  • influenza virus hemagglutinin HA protein is derived from the fact that the viral particles bind to the specific sialic acid-containing receptor through the HA protein to agglutinate the red blood cells. Its synthesis is to first synthesize and synthesize a HA protein precursor (HA0) containing 562-566 amino acids in the endoplasmic reticulum of the cell, ie hemagglutinin precursor; hemagglutinin (HA) mature protein encoded by influenza virus RNA Containing about 550 amino acid residues, including heavy chain (HA1) and light chain (HA2), the basic amino acid position between the two is hydrolyzed by cell-specific protease when the mature virus particles are released from extracellular sprouts.
  • HA0 HA protein precursor
  • HA1 hemagglutinin precursor
  • HA hemagglutinin mature protein encoded by influenza virus RNA Containing about 550 amino acid residues, including heavy chain (HA1) and light chain (HA2),
  • Influenza hemagglutinin before proteolytic cleavage is also known as influenza hemagglutinin precursor (HA0). This specific cleavage is necessary for the fusion of influenza virus and host cell membrane, but it is not related to the binding of influenza hemagglutinin to the receptor.
  • the influenza hemagglutinin before and after specific protease cleavage has the same antigenicity and receptor binding activity, HA0 Hydrolysis of the molecules to HA1 and HA2 is a prerequisite for viral infectivity.
  • influenza hemagglutinin or HA of the present invention includes influenza hemagglutinin precursor (HA0) and disulfide-bonded HA1 and HA2 formed by cleavage of a specific protease, unless otherwise specified.
  • the hemagglutinin HA monomer has a molecular weight of about 60 kD, and the HA on the surface of the influenza virus forms a spike in the form of a HA-trimer, which is required for its binding to the sialic acid receptor.
  • a HA-trimer which is required for its binding to the sialic acid receptor.
  • the erythrocyte surface of some animals such as chickens, guinea pigs and the like has a sialylated glycosyl group capable of binding to the influenza virus HA trimer. Multiple HA spikes on the surface of the virus bind to multiple sialylated glycosyl groups on the surface of different red blood cells.
  • hemagglutination activity is an important method for examining the activity of HA receptor binding
  • hemagglutination inhibition assay is an important method for studying whether an antibody has a neutralizing activity for blocking the binding of influenza virus HA trimer to a receptor.
  • Recombinant HA obtained by different expression systems and preparation methods differs significantly in structure, glycosylation, ability to induce neutralizing antibodies, and the like.
  • Athmaram, TN and other yeasts were successfully secreted and expressed by yeast to obtain HA0 of the 2009 new H1N1 influenza, but the prepared HA0 was mainly purified as a monomer and a small amount of trimer when purified by FPLC, at a dose of 10 ⁇ g/only and 50 ⁇ g/dose.
  • the hemagglutination inhibitory activity was only 1:32 after two immunizations of mice. No reports of yeast preparation of influenza hemagglutinin glycoprotein polymer nanoparticles have been reported.
  • the invention provides a preparation method of influenza hemagglutinin glycoprotein polymer nanoparticles.
  • the present invention provides a method for preparing influenza virus hemagglutinin glycoprotein polymer nanoparticles, comprising the steps of: hemagglutinin of an influenza virus containing a signal peptide sequence upstream of the N-terminus and comprising a C-terminal transmembrane region sequence
  • the coding gene of HA (HA gene) is expressed in yeast; the yeast is subjected to cell disruption, and a detergent is added to obtain a solution containing influenza virus hemagglutinin glycoprotein; the solution is purified to prepare blood coagulation Active influenza virus hemagglutinin glycoprotein polymer nanoparticles.
  • the hemagglutinin HA of the influenza virus containing the signal peptide sequence upstream of the N-terminus and comprising the C-terminal transmembrane region sequence further contains a Kozak sequence before the signal peptide sequence, and the Kozak sequence is 5'-aaacg-3'.
  • the cell disruption and the addition of the detergent further comprise the step of centrifuging the precipitate.
  • the step of adding the detergent and the solution containing the influenza virus hemagglutinin glycoprotein specifically further comprises the step of centrifuging the supernatant.
  • influenza virus hemagglutinin glycoprotein polymer nanoparticles have a molecular weight greater than 670 KD.
  • the hemagglutinin-influenza influenza hemagglutinin glycoprotein polymer nanoparticle exhibits a rosette structure under electron microscope, which proves that it is formed by at least three trimers, and three influenza virus hemagglutinin protein precursors
  • the HA0 trimer composed of HA0 has a molecular weight of about 180 KD, thus demonstrating that the influenza virus hemagglutinin glycoprotein polymer nanoparticles are involved in at least 9 influenza virus hemagglutinin protein precursors HA0 to form polymer nanoparticles, wherein influenza virus blood
  • the clotting protein precursor HA0 forms a HA0 trimer.
  • the gene encoding the hemagglutinin HA of the influenza virus containing the signal peptide sequence upstream of the N-terminal and comprising the C-terminal transmembrane region sequence is expressed in yeast: the N-terminal upstream contains a signal peptide
  • the recombinant expression vector encoding the gene encoding the hemagglutinin HA of the influenza virus comprising the sequence of the C-terminal transmembrane region is transformed into yeast, and the transformed yeast is cultured to induce gene expression.
  • the yeast may be Pichia pastoris, Hansenula or Kluyveromyces lactis.
  • the step of inducing gene expression is specifically: culturing the transformed yeast and inducing expression of the gene.
  • the culture is specifically a shake flask culture or a fermentor culture.
  • the method of first culturing the transformed yeast to a certain density and then inducing gene expression may be employed.
  • the signal peptide is a signal peptide of the HA gene itself or other signal peptide that can function in the corresponding yeast.
  • the other signal peptides which may function in the corresponding yeast are the S. cerevisiae alpha mating factor signal peptide, the alpha amylase signal peptide or the signal peptide of albumin.
  • the signal peptide of the albumin is specifically a signal peptide of serum albumin.
  • the hemagglutinin HA of the influenza virus is HA of a H1, H3, H5 or H7 serotype influenza virus.
  • the HA of the H1, H3, H5 or H7 serotype influenza virus is HA of H1N1, H3N2, H5N1 or H7N9 influenza virus, respectively.
  • the recombinant expression vector is a gene encoding a hemagglutinin HA of an influenza virus containing a signal peptide sequence upstream of the N-terminus and comprising a C-terminal transmembrane region sequence, comprising AOX promoter.
  • the vector of the sub is obtained.
  • the recombinant expression vector inserts the gene encoding the hemagglutinin HA of the influenza virus containing the signal peptide sequence and the C-terminal transmembrane region into the NotI and NspV cleavage sites of pPICZ ⁇ , and linearizes BglII.
  • the yeast is Pichia pastoris.
  • the influenza virus is specifically an H7N9 avian influenza virus, and the gene encoding the hemagglutinin HA of the influenza virus containing the signal peptide sequence upstream of the N-terminal and comprising the C-terminal transmembrane region sequence is specifically as shown in SEQ ID No. 4. .
  • the H7N9 avian influenza virus was A/Hongzhou/1/2013 (H7N9).
  • the influenza virus is specifically an influenza A H1N1 influenza virus, and the gene encoding the hemagglutinin HA of the influenza virus containing the signal peptide sequence upstream of the N-terminal and comprising the C-terminal transmembrane region sequence is specifically as shown in SEQ ID No. 14. Show.
  • the influenza A H1N1 influenza virus is specifically A/FortMonmouth/1/47 (H1N1).
  • the influenza virus is specifically an influenza A H3N2 influenza virus, and the gene encoding the hemagglutinin HA of the influenza virus containing the signal peptide sequence upstream of the N-terminal and comprising the C-terminal transmembrane region sequence is specifically as shown in SEQ ID No. 21. Show.
  • the influenza A H3N2 influenza virus is specifically A/reassortant/NYMC X-223A (Texas/50/2012x PuertoRico/8/1934) (H3N2).
  • the recombinant expression vector is a gene encoding a hemagglutinin HA of an influenza virus containing a signal peptide sequence upstream of the N-terminus and comprising a C-terminal transmembrane region sequence, comprising a MOX promoter.
  • the vector of the sub is obtained.
  • the recombinant expression vector specifically inserts the gene encoding the hemagglutinin HA of the influenza virus containing the signal peptide sequence upstream of the N-terminus and comprising the C-terminal transmembrane region sequence into the NotI and BstB I cleavage sites of pPICZ ⁇ .
  • the intermediate vector 1 was obtained; the AOX promoter of the intermediate vector 1 was replaced with the alcohol oxidase promoter MOX of Hansenula, and linearized with BglII.
  • the yeast is Hansenula, specifically Hansenula polymorpha.
  • the MOX promoter is a PCR-amplified DNA molecule represented by SEQ ID No. 9 and SEQ ID No. 10 using a genomic DNA of Hansenula polymorpha as a template to obtain a MOX promoter.
  • the method for replacing the AOX promoter of the intermediate vector 1 with the alcohol oxidase promoter MOX of Hansenula is specifically that the MOX promoter obtained by the PCR amplification is subjected to BglII digestion and phosphorylation to obtain 5'.
  • the 3'-terminally phosphorylated MOX promoter was ligated to the intermediate vector 1 excising the AOX promoter.
  • the influenza virus is specifically an H5N1 avian influenza virus, and the gene encoding the hemagglutinin HA of the influenza virus containing the signal peptide sequence upstream of the N-terminal and comprising the C-terminal transmembrane region sequence is specifically as shown in SEQ ID No. 8. .
  • the H5N1 avian influenza virus is specifically A/duck/Guangxi/27/2003 (H5N1).
  • the recombinant expression vector is a gene encoding a hemagglutinin HA of an influenza virus containing a signal peptide sequence upstream of the N-terminus and comprising a C-terminal transmembrane region sequence, which comprises a LAC4 promoter.
  • the vector of the sub is obtained.
  • the recombinant expression vector inserts a gene encoding a hemagglutinin HA of an influenza virus containing a signal peptide sequence upstream of the N-terminus and comprising a C-terminal transmembrane region sequence between the HindIII and NotI restriction sites of pKLAC1. It is then linearized with SacII.
  • the yeast is Kluyveromyces lactis.
  • the influenza virus is specifically an H7N9 avian influenza virus, and the gene encoding the hemagglutinin HA of the influenza virus containing the signal peptide sequence upstream of the N-terminal and comprising the C-terminal transmembrane region sequence is specifically as shown in SEQ ID No. 17. .
  • the H7N9 avian influenza virus is specifically A/Hongzhou/1/2013 (H7N9).
  • the cell disruption method is a physical method, a biological method or a chemical method.
  • the physical method is specifically a glass bead oscillation method, a high pressure homogenization method or a ball milling method.
  • the biological method is specifically an enzymatic cleavage method.
  • the chemical method is specifically an alkali cracking method.
  • the detergent is a non-ionic detergent or a weakly ionic detergent.
  • the nonionic detergent is specifically Triton, Tween or ethyl phenyl polyethylene glycol.
  • the weak ion type detergent is specifically deoxycholate or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.
  • the Triton is specifically Triton X-100.
  • the Tween is specifically Tween20.
  • the ethyl phenyl polyethylene glycol is specifically NP-40.
  • influenza virus hemagglutinin glycoprotein polymer nanoparticles are expressed on the cell membrane of the yeast.
  • the action of the detergent is to dissolve the influenza virus hemagglutinin glycoprotein polymer nanoparticles from the yeast cell membrane and to better maintain its structure.
  • the method of purifying the solution comprises cation exchange chromatography and/or anion exchange chromatography and/or gel exclusion chromatography.
  • the filler for the cation exchange chromatography is specifically Sepharose FF SP.
  • the filler for the anion exchange chromatography is specifically Source 30Q.
  • the filler of the gel exclusion chromatography is specifically Superdex 200.
  • the purified component has HA band and hemagglutination activity according to each step in the purification process to determine whether it is a component containing the influenza virus hemagglutinin glycoprotein polymer nanoparticle, if the component has a HA band And hemagglutination activity is a component containing the influenza virus hemagglutinin glycoprotein polymer nanoparticles.
  • the non-influenza virus hemagglutinin protein content of the influenza virus hemagglutinin glycoprotein polymer nanoparticle obtained after the purification is ⁇ 20%, specifically ⁇ 10%;
  • the HA comprises the influenza virus hemagglutinin precursor HA0 and the disulfide-linked HA1 and HA2 formed by cleavage of a specific protease.
  • influenza vaccine prepared by the influenza virus hemagglutinin glycoprotein polymer nanoparticle prepared by the method described in any of the above methods is also within the scope of protection of the present invention.
  • the vaccine specifically further comprises an adjuvant.
  • the adjuvant is specifically an aluminum adjuvant, such as an aluminum hydroxide adjuvant.
  • Figure 1 shows the results of cation exchange chromatography of H7N9 influenza hemagglutinin glycoprotein multiparticle nanoparticles prepared by recombinant Pichia pastoris.
  • Fig. 3 shows the results of gel exclusion chromatography of H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles prepared by recombinant Pichia pastoris.
  • Figure 4 shows the glycosylation assay of H7N9 influenza hemagglutinin glycoprotein multiparticle nanoparticles prepared by recombinant Pichia pastoris.
  • Figure 5 is a TPCK-treated trypsin digestion result of H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles prepared by recombinant Pichia pastoris.
  • Figure 6 is a graph showing the hemagglutination activity of H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles prepared by recombinant Pichia pastoris.
  • Figure 7 is a molecular exclusion chromatography analysis of H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles prepared by recombinant Pichia pastoris.
  • Figure 8 is an electron micrograph of H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles prepared by recombinant Pichia pastoris.
  • Figure 9 is an evaluation of the effect of influenza vaccine prepared from H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles prepared by recombinant Pichia pastoris.
  • Figure 10 is a diagram showing the glycosylation detection of H5N1 highly pathogenic avian influenza hemagglutinin glycoprotein multiparticle nanoparticles prepared by recombinant Hansenula.
  • Figure 11 is a molecular exclusion chromatography analysis of H5N1 highly pathogenic avian influenza hemagglutinin glycoprotein polymer nanoparticles prepared by recombinant Hansenula.
  • Figure 12 is a graph showing the hemagglutination activity of H5N1 highly pathogenic avian influenza hemagglutinin glycoprotein polymer nanoparticles prepared by recombinant Hansenula.
  • Figure 13 is a graph showing the effect of influenza vaccine effect of H5N1 highly pathogenic avian influenza virus hemagglutinin glycoprotein polymer prepared by recombinant Hansenula.
  • Figure 14 shows the glycosylation detection of H1N1 influenza hemagglutinin glycoprotein multiparticle nanoparticles prepared by recombinant Pichia pastoris.
  • Figure 15 is a molecular exclusion chromatography analysis of H1N1 influenza hemagglutinin glycoprotein multiparticle nanoparticles prepared by recombinant Pichia pastoris.
  • Figure 16 is a graph showing the detection of hemagglutination activity of H1N1 influenza hemagglutinin glycoprotein multiparticle nanoparticles prepared by recombinant Pichia pastoris.
  • Figure 17 is an evaluation of the effect of influenza vaccine obtained from H1N1 influenza hemagglutinin glycoprotein polymer nanoparticles prepared by recombinant Pichia pastoris.
  • Figure 18 is a diagram showing the glycosylation detection of H7N9 influenza hemagglutinin glycoprotein multiparticle nanoparticles prepared by recombinant K. lactis.
  • Figure 19 is a H7N9 influenza hemagglutinin glycoprotein nanoparticle prepared by recombinant Kluyveromyces lactis Molecular exclusion chromatography analysis.
  • Figure 20 is a graph showing the detection of hemagglutination activity of H7N9 influenza hemagglutinin glycoprotein multiparticle nanoparticles prepared by recombinant K. lactis.
  • Figure 21 is a graph showing the effect of influenza vaccine obtained from H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles prepared by recombinant K. lactis.
  • Figure 22 is a diagram showing the glycosylation detection of H3N2 influenza hemagglutinin glycoprotein multiparticle nanoparticles prepared by recombinant Pichia pastoris.
  • Figure 23 is a graph showing the detection of hemagglutination activity of H3N2 influenza hemagglutinin glycoprotein multiparticle nanoparticles prepared by recombinant Pichia pastoris.
  • Figure 24 is an evaluation of the effect of influenza vaccine obtained from H3N2 influenza hemagglutinin glycoprotein polymer nanoparticles prepared by recombinant Pichia pastoris.
  • Figure 25 is a graph showing the detection of hemagglutination activity in Example 7.
  • Figure 26 is a graph showing the detection of hemagglutination activity in Example 8.
  • Figure 27 is a graph showing the detection of hemagglutination activity in Example 9.
  • influenza hemagglutinin or HA of the present invention includes influenza hemagglutinin precursor (HA0) and disulfide-bonded HA1 and HA2 formed by cleavage of a specific protease, unless otherwise specified.
  • the pPICZ ⁇ vector was purchased from Invitrogen.
  • Pichia pastoris X-33 was purchased from Invitrogen.
  • TPCK-treated trypsin (TPCK-Trypsin) was purchased from Sigma.
  • Pyrobest DNA polymerase was purchased from Bao Bioengineering (Dalian) Co., Ltd.
  • pKLAC1 was purchased from NEB.
  • Kluyveromyces lactis is purchased from NEB.
  • the H7N9 influenza reconstituted vaccine strain (NIBRG-268) was published in the literature "Yang Juan, Zheng Yaming, Feng Zhuzhao, Yu Hongjie. Progress in the development of human H7N9 avian influenza vaccine, Chinese Journal of Preventive Medicine, 2014, 48 (2)", the public can Obtained from the Institute of Biotechnology of the Chinese Academy of Military Medical Sciences.
  • Example 1 H7N9 influenza hemagglutinin glycoprotein multiparticle nanoparticles prepared by recombinant Pichia pastoris
  • HA7-3 5'-ATC GCGGCCGC TTAAATACAGATAGTACATCTCAT-3' (SEQ ID No. 2)
  • the sequence underlined is the NotI digestion recognition site.
  • HA7-5 5'-ATC TTCGAA ACGATGAACACCCAAATACTGGTTTTC-3' (SEQ ID No. 3)
  • the sequence underlined is the NspV digestion recognition site.
  • SEQ ID No. 4 is from the 8th to the 12th position of the Kozak sequence from the 5' end, the signal peptide coding sequence is from the 13th to the 66th, and the HA gene is from the 67th to the 1695th.
  • the 1668th position is the C-terminal transmembrane region sequence.
  • NspV and NotI are digested with the DNA molecule shown in SEQ ID No. 4 to obtain a gene fragment; NspV and NotI are double-digested with the pPICZ ⁇ vector to obtain a large fragment of the vector; and the gene fragment is ligated with the large fragment of the vector to obtain a recombinant plasmid. It was named pPICZ ⁇ -HA7. The pPICZ ⁇ -HA7 was sequenced and the results were correct.
  • pPICZ ⁇ -HA7 plasmid About 10 ⁇ g of pPICZ ⁇ -HA7 plasmid was linearized with BglII, and the linearized plasmid was precipitated with 1/10 volume of 3 M aqueous sodium acetate solution and 3 volumes of absolute alcohol. The salt was washed twice with a 70% by volume aqueous solution of ethanol to remove the salt, air-dried, and about 30 ⁇ L of water was added to resuspend the precipitate to obtain a pPICZ ⁇ -HA7 linearized plasmid for transformation.
  • Pichia pastoris X-33 was isolated on a YPD plate (yeast extract 10 g/L, tryptone 20 g/L, glucose 20 g/L, agar 15 g/L) by scribing, and cultured in a 28 ° C incubator for 2 days. . Inoculate a single clone into a 50 mL flask containing 10 mL of YPD liquid medium (yeast extract 10 g/L, tryptone 20 g/L, glucose 20 g/L), and incubate overnight at 28 ° C to an OD 600 of about 2 Bacteria.
  • 0.1-0.5 mL of the bacterial solution was inoculated into a 3.5 L shake flask containing 500 ml of LYPD liquid medium, and cultured overnight to an OD 600 to 1.3-1.5.
  • the bacterial solution was transferred to a sterile centrifuge bottle and centrifuged at 1500 g for 10 minutes at 4 °C.
  • the cells were resuspended in 500 mL of pre-cooled sterile water, and the cells were harvested by centrifugation at 1500 g for 10 minutes at 4 ° C, and washed again with 250 mL of pre-cooled sterile water.
  • the cells were resuspended in 20 mL of pre-cooled sterile 1 M sorbitol, and the cells were harvested by centrifugation at 1500 g for 10 minutes at 4 ° C, and the cells were resuspended in pre-cooled 1 M sorbitol to a final volume of 1.5 mL to obtain a bacterial suspension.
  • the cells harvested by centrifugation per 1 ml of bacterial suspension were resuspended in 100 ⁇ l of PBS, and 1/4 volume of pickled glass beads (diameter 425-600 ⁇ m or 0.5 mm) were added, and each sample was vortexed at maximum speed for 1 minute, repeated six times. Every two vortex shakes the middle ice bath for two minutes to prevent protein degradation.
  • the glass beads and the unbroken cells were precipitated by centrifugation at 3500 g for 1 minute at 4 ° C in a low temperature microcentrifuge to obtain a supernatant.
  • the bacterial solution was first diluted with PBS at a volume ratio of 1:20, and then serially diluted with PBS at a volume ratio of 1:2, and analyzed for hemagglutination activity with 1% chicken red blood cells (for the method, see "Guo Yuanji et al.” Virus and its experimental techniques, Beijing, China Three Gorges Press, 1997). Pick a clone corresponding to the hemagglutination activity of the bacterial cell (recombinant Pichia pastoris monoclonal) for influenza hemagglutinin glycoprotein polymer Preparation of nanoparticles.
  • Seed culture The recombinant P. pastoris obtained by the second step was randomly inoculated into fresh MD plate (1.34 g/100 ml YNB, 4 ⁇ 10 -5 g/100 ml Biotin, 1 g/100 ml glucose, 1.5 g/100 ml agar). On the powder, the monoclonal colonies on which the cells were cultured and picked were inoculated into YPD liquid medium, and cultured at 24 ° C, 250 rpm for about 48 hours.
  • Fermentation culture Prepare a fermentation medium of 2.1 L (H 3 PO 4 3.5 mL/L, K 2 SO 4 2.4 g/L, KOH 0.65 g/L, CaSO 4 (anhydrous) 0.14 g/L, MgSO 4 ⁇ 7H 2 O 1.95g / L, glycerin 40.0g / L, PTM11.2mL / L, 0.02g / 100ml biotin 0.5mL / L, the balance is water.
  • the composition of PTM1 is: CuSO 4 ⁇ 5H 2 O 6.0g / L, MnSO 4 ⁇ H 2 O 3.0g/L, FeSO 4 ⁇ 7H 2 O 65g/L, ZnSO 4 ⁇ 7H 2 O 20g/L, CoCl 2 ⁇ 6H 2 O 0.5g/L, NaMoO 4 ⁇ 2H 2 O 0.2 g/L, KI 0.1 g/L, concentrated H 2 SO 4 5 mL/L), added to a 5 L fermentor, autoclaved at 121 ° C for 30 min. The fermenter was allowed to cool to room temperature, and pH 6.0 was adjusted with ammonia water.
  • the seed liquid was connected to the fermenter at a 10% inoculation amount, the ammonia water was controlled at pH 6.0, the temperature was 28 ° C, and the stirring speed and the aeration amount were maintained at 10% or more of dissolved oxygen.
  • the feed growth medium 50g/100ml of glycerol solution (containing 12mL/L PTM1, 2mL/L 500 ⁇ biotin (purchased from Beijing Xinjing Biotechnology Co., Ltd.)
  • 40ml / h adding 6-8h, stop feeding.
  • start methanol induction maintain the temperature at 24 ° C, adjust the pH to 6.4 with aqueous ammonia solution.
  • anhydrous methanol starts at 2.4mL / h Add, add 2.4mL of 100% methanol per hour, increase to 12mL / h after 5h, at this time for induction 0 hours, then every 12h sampling.
  • the fermentation is finished, the fermentation broth is centrifuged at 7000rpm/min for 20min at 4 ° C.
  • the suspension was suspended in 40 g/100 ml, and the high-pressure homogenizer was sterilized (1200 bar, sterilized 3 times) to obtain a homogenate for purification of influenza hemagglutinin glycoprotein multiparticle nanoparticles.
  • 1-6 represents the eluent of the 1-6 collection tube obtained by eluting 15% of the liquid C
  • 100% C represents the eluent of the 100% C solution
  • 3+endoh represents the eluent of the 3 collection tube.
  • the sample after digestion with EndoH represents the sample solution before the column
  • M represents the protein marker.
  • Figure 1 shows that the sample in which the HA component is located is a 15% C liquid eluent.
  • Q is a sample that is not bound by Source 30Q when loading; 10% C represents an eluent of 10% C solution; 100% C represents an eluate of 100% C solution; NaOH represents a 0.5 M aqueous solution of NaOH Eluent.
  • the sample before purification (the eluate of the 15% C solution obtained in the step (2)), the sample which was not adsorbed by the Q column (Source 30Q), the 10% C liquid eluate (the first tube collection solution, and The second tube collection solution, the third tube collection solution), the collection solution eluted with 100% C solution, and the collected solution eluted with a 0.5 M NaOH aqueous solution was subjected to reduction SDS-PAGE analysis, and the result was as shown in Fig. 2 Show. In Fig.
  • the Q column represents the sample before purification (the 15% C solution eluate obtained in step (2)), and the Q band represents the sample which is not adsorbed by the Q column (Source 30Q), and 1 represents 10
  • the %C solution elutes the first tube collection solution
  • 2 represents the 10% C solution elutes the second tube collection solution
  • 3 represents the 10% C solution elutes the third tube collection solution
  • 100% C represents the 100% C solution for elution.
  • the collection solution, NaOH represents a collection solution eluted with a 0.5 M aqueous NaOH solution
  • M represents a protein marker.
  • the arrow shows the HA strip.
  • Figure 2 shows that the purification of this step resulted in an HA sample of increased purity, with some of the heteroprotein removed, and the sample of the HA component was the eluate of the 10% C solution of this step.
  • the first eluting main peak indicated by the arrow is purified H7N9 influenza hemagglutinin glycoprotein multimeric nanoparticles, and the protein concentration thereof was determined to be 0.33 mg/ml.
  • Step (3) 10% C liquid elution fraction, EndoH digestion, gel exclusion chromatography
  • the H7N9 influenza hemagglutinin glycoprotein multiparticle nanoparticles and the H7N9 influenza hemagglutinin glycoprotein polymer nanoparticle purified by gel exclusion chromatography were subjected to reduction SDS-PAGE analysis, and the results are shown in B of FIG. .
  • the column front represents the step (3) 10% C liquid elution component
  • the HA+endoh represents the endoH digestion gel exclusion chromatography to obtain the H7N9 influenza hemagglutinin glycoprotein polymer nanoparticle.
  • HA represents H7N9 influenza hemagglutinin glycoprotein multimer nanoparticles purified by gel exclusion chromatography
  • M represents a protein marker.
  • the arrow shows the HA strip.
  • Figure 3 shows that this step was purified to obtain a pure HA with high purity and analyzed by EndoH glycosidase, indicating that the correct glycosylation modification occurred.
  • the N-glycosidase F (PNGF) (purchased from NEB) was used to analyze the molecular weight change before and after glycosylation, and the H7N9 influenza hemagglutinin glycoprotein nanoparticle prepared in step four was analyzed.
  • the specific steps were as follows: 20ul Step 4 purified H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles (concentration: 330 ug/ml), which were treated by N-glycosidase F (PNGF) digestion method, and no enzyme control group and No sample control was added. Each sample was subjected to reduction SDS-PAGE detection, and the results are shown in Fig. 4.
  • PNGF represents N-glycosidase F
  • HA+PNGF represents N7 glycosyl glycoprotein polymer nanoparticle prepared in step four
  • N-glycosidase F is added
  • HA represents H7N9 influenza blood prepared in step 4.
  • Condenin glycoprotein polymer nanoparticles, M stands for protein marker. The arrow shows the HA strip.
  • Figure 4 shows that H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles not treated with PNGF have a HA0 molecular weight of about 64 KD, and after PNGF treatment, the molecular weight decreases to about 60 KD, and the theoretical molecular weight of the unglycosylated HA0 mature protein. (60172Da) consistent.
  • the H7N9 influenza hemagglutinin glycoprotein polymer nanoparticle prepared in the fourth step is a glycoprotein.
  • the N-terminal 5 amino acid sequence analysis of the component HA0 constituting the H7N9 influenza hemagglutinin glycoprotein polymer nanoparticle is DKIXL, wherein X is not detected, which is related to the N-terminal theoretical sequence of the H7N9 virus HA mature protein (HA0). Consistent (the theoretical sequence is DKICL, where C is destroyed when it is sequenced by the Edman method and cannot be measured). It is indicated that the signal peptide of HA has been successfully removed.
  • H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles with a concentration of 330 ug/ml, 16 ug of TPCK-treated trypsin, and 1 hour of ice bath treatment were used as an experiment. group.
  • another 1 ml of the same H7N9 influenza hemagglutinin glycoprotein polymer nanoparticle was taken, denatured in a water bath at 100 ° C for 5 minutes, cooled in an ice bath, and 16 ug of trypsin treated with TPCK was treated in the same ice bath for 1 hour as a control group. .
  • Protease-specific cleavage can be used not only to prepare H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles, but also to analyze the correct structure of HA0.
  • the HA0 with influenza virus trimer advanced structure is only between HA1 and HA2.
  • the basic amino acid site is exposed to be specifically cleaved by trypsin to HA1 with a molecular weight of about 40 kD and HA2 with a molecular weight of about 25 kD.
  • HA0 which does not have the correct high-level structure, can be trypsinized into various fragments of varying sizes.
  • H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles, experimental group and control group were subjected to reduction SDS-PAGE, The test results are shown in Figure 5.
  • HA represents H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles
  • HA + trypsin is an experimental group
  • HA denaturing + pancreatin is a control group.
  • the arrows represent the HA0 strips.
  • Figure 5 shows that after HA0 was trypsin-cleaved, the molecular weight of 64KD was similar to that of uncut HA0 by non-reduced SDS-PAGE electrophoresis.
  • the reduction electrophoresis analysis showed that HA0 had been specifically cleaved into two molecular weights of 40kD and 24kD. Fragments, consistent with the molecular weights of HA1 and HA2. It is indicated that the HA0 obtained by the present invention is specifically cleaved by trypsin to form HA composed of disulfide-bonded HA1 and HA2 subunits.
  • HA0 of the present invention has the same high-order structure as influenza virus hemagglutinin.
  • the H7N9 influenza hemagglutinin glycoprotein polymer nanoparticle prepared above was first diluted with PBS by a ratio of 1:10, and then diluted with PBS in a volume ratio of 1:2, using physiological saline as a control, using 1%.
  • Chicken red blood cells are analyzed for hemagglutination activity. For details, see “Guo Yuanji et al., "Influenza virus and its experimental techniques", Beijing, China Three Gorges Publishing House, 1997".
  • the first row from left to right is a 2-fold serial dilution of H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles starting at 1:20, and the second row is a saline control.
  • Figure 6 shows that the H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles have significant hemagglutination activity, and the blood coagulation titer of chicken blood reaches 1:8000. Therefore, the influenza hemagglutinin (H7N9 influenza hemagglutinin glycoprotein multimeric nanoparticle) of the present invention has sialic acid receptor binding activity.
  • the upper graph of A is a chromatogram of the molecular weight standard protein, and the retention times of each molecular weight are: 670 KD, 20.593 min; 150 KD, 22.478 min; 44 KD, 24.074 min; 17.6 KD, 25.363 min; 1.35 KD, 26.966 min. ).
  • the lower panel of A shows the chromatogram of H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles.
  • Figure 7B shows that the H7N9 influenza hemagglutinin glycoprotein polymer nanoparticle purity reached 99.69%.
  • Figure 7 shows that the H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles have a retention time of 16.971 min under the conditions of the analysis, indicating that the molecular weight is significantly greater than 670 KD, and the molecular weight of the trimer is due to the molecular weight of the HA0 monomer being 64 KD. It is about 180KD, indicating that H7N9 influenza hemagglutinin glycoprotein nanoparticle is a multimer composed of more than 9 HA0 monomers.
  • H7N9 influenza hemagglutinin glycoprotein is poly-aggregated.
  • the nanoparticle has a trimer high structure of influenza hemagglutinin, and therefore, the H7N9 influenza hemagglutinin glycoprotein polymer nanoparticle is a polymer formed of three or more HA0 trimers.
  • the prepared H7N9 influenza hemagglutinin glycoprotein multimer nanoparticles were negatively stained and photographed with a 50,000-fold electron microscope, and the results are shown in Fig. 8.
  • the scale in Figure 8 is 20 nanometers.
  • Figure 8 shows that the H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles are at least three hemagglutinin trimers (the hemagglutinin trimer here refers to the trimer formed by the polymerization of three HA0 monomers).
  • the tail is polymerized internally, and the head protrudes outward to form polymer particles having a diameter of about 20-50 nm.
  • Example 2 Preparation of influenza vaccine by H7N9 influenza hemagglutinin glycoprotein multiparticle nanoparticles prepared by recombinant Pichia pastoris
  • H7N9 influenza hemagglutinin glycoprotein polymer nanoparticles prepared in Example 1 were diluted to 7.5 ⁇ g/50 ⁇ l with PBS pH 7.4, and an equal volume of 1.2 mg/ml Al(OH) 3 adjuvant (purchased from GE) was added.
  • the company, trade name Rehydragel@LV) is made into a flu vaccine and is an injection for the experimental group.
  • a solution in which no H7N9 influenza hemagglutinin glycoprotein nanoparticle was added was used as a control injection.
  • mice in each group were injected with 100 ⁇ l of the corresponding group of injections into the hind leg muscle of each group. After three weeks of the first injection, boost the immunization once. After one week of booster immunization, the blood was collected for serum hemagglutination inhibition test.
  • the standard hemagglutinin of the hemagglutination inhibition test is the British National Biological Standard and Reference recommended by the WHO (World Health Organization).
  • H7N9 influenza reassortant vaccine strain (NIBRG-268) provided by NIBSC (National Institute for Biological Standards and Control, a centre of the Medical and Healthcare products Regulatory Agency (MHRA), United Kingdom of Great Britain and Northern Ireland)
  • the chicken embryo culture virus was prepared after inactivation of 1:2000 formaldehyde.
  • the preparation of standard hemagglutinin, dilution and hemagglutination inhibition of virus prepared by inactivated chicken embryo culture virus of H7N9 influenza reconstituted vaccine strain (NIBRG-268) after 1:2000 formaldehyde inactivation is described in "Guo Yuanji et al” influenza virus And its experimental techniques, Beijing, China Three Gorges Publishing House, 1997”.
  • Fig. 9 the ordinate is the hemagglutination inhibition titer (HI), the abscissa is the grouping, 0.0 is the control group, and 7.5 is the experimental group.
  • Figure 9 shows that the serum of the control mice did not produce hemagglutination inhibition.
  • the serum hemagglutination inhibition titer of the experimental group was greater than 1:40, and the average hemagglutination inhibition titer was 1:640. It is generally believed that the hemagglutination inhibition titer induced by the influenza vaccine is greater than 1:40 to provide effective immune protection to the body. Therefore, H7N9 influenza hemagglutinin glycoprotein multimeric nanoparticles prepared with recombinant yeast can be used to prepare influenza vaccines.
  • Example 3 H5N1 highly pathogenic avian influenza virus hemagglutinin glycoprotein polymer nanoparticle prepared by Hansenula
  • HA5-5 5'-ATC TTCGAA ACGatggagaaaatagtgcttc-3' (SEQ ID No. 6)
  • the sequence underlined is the BstB I restriction recognition site.
  • HA5-3 5'-ATC GCGGCCGC ttaaatgcaaattctgcattg-3'. (SEQ ID No. 7)
  • the sequence underlined is the NotI digestion recognition site.
  • the sequence includes a Kozak sequence, a signal peptide coding sequence, and an HA gene (including a C-terminal transmembrane region sequence) in this order from the 5' end.
  • the DNA molecule shown by SEQ ID No. 8 was digested with NotI and BstB I to obtain a gene fragment; the pPICZ ⁇ vector was digested with NotI and BstB I to obtain a large fragment of the vector; the gene fragment was ligated with the large fragment of the vector to obtain a recombinant plasmid, and the recombinant plasmid was obtained. It is named pPICZ- (H5N1) HA.
  • the pPICZ- (H5N1) HA was sequenced and the results were correct.
  • the synthetic primer MOX5 5'-ATC AGATCT TCGACGCGGAGAACGATCT-3' (SEQ ID No. 9, underlined BglII restriction recognition site) and MOX3: 5'- TGTTTTTGTACTTTAGATTGATG-3' (SEQ ID No. 10).
  • PCR system 1 ⁇ g of genomic DNA, 4 ⁇ l of dNTP (2.5 mM), 5 ⁇ l of 10 ⁇ Pyrobest Buffer, 1 ⁇ l of 10 ⁇ M MOX5 primer, 1 ⁇ l of ⁇ M MOX3 primer, 0.5 ⁇ l of Pyrobest DNA polymerase, 37.5 ⁇ l of water, and a total volume of 50 ⁇ l.
  • PCR procedure pre-denaturation at 94 ° C for 5 min; denaturation at 94 ° C for 30 sec, annealing at 55 ° C for 30 sec, extension at 72 ° C for 2 min, 30 cycles; extension at 72 ° C for 10 min, storage at 4 ° C.
  • the PCR amplification product was a target fragment of about 1.5 kb, which was digested with BglII, and the restriction fragment was phosphorylated to obtain a 1.5 kb MOX start-up at the 5' end for BglII digestion and 3' phosphorylation. child.
  • the pPICZ- (H5N1) HA vector was digested with NspV and then filled in with Klenow fragment large fragment enzyme and dNTP. After electrophoresis recovery, the AOX promoter was excised by BglII digestion.
  • the vector for excising the AOX promoter fragment was recovered by electrophoresis, and the vector was ligated to the 1.5 kb MOX promoter which was digested with BglII at the 5' end and phosphorylated at the 3' end to obtain a Hansenula alcohol oxidase promoter.
  • a vector for expression of H5N1 influenza hemagglutinin glycoprotein nanoparticle gene by MOX designated as pMOXZ-HA5.
  • the pMOXZ-HA5 vector was linearized with BglII, and then electrotransformed into Hansenula polymorpha (ATCC 26012 (available from the American Type Culture Collection)), and the transformed cells were coated to 100 ⁇ g/mL Zeocin.
  • YPD plate yeast extract 10 g/L, tryptone 20 g/L, glucose 20 g/L, agar 15 g/L). Incubate in a 30 ° C incubator for 2-4 days to form a monoclonal.
  • the preparation of the bacteriostatic solution and the analysis of the hemagglutination activity were the same as those in the second step of Example 1.
  • the positive strain for screening for high hemagglutination activity was named Hans (pMOX-HA5).
  • Single colonies of Hans (pMOX-HA5) were inoculated into 3 ml of YPD liquid medium, grown at 30 ° C, 250 rpm for 1-2 days, until the bacterial density OD600 was greater than 10, and transferred to a 100 ml YPD liquid medium at a volume ratio of 2%.
  • Incubate in a 1L shake flask at 30 ° C, 250 rpm for 24 hours transfer to 10 1L shake flasks containing 150 ml of BMGY liquid medium at a volume ratio of 5%, and incubate at 30 ° C, 250 rpm for 24 hours, add 0.5 by volume.
  • % methanol was induced to express, and methanol was added once every 12 hours.
  • the method of the second step of the first embodiment was used to sterilize the blood coagulation activity, and the clone corresponding to the hemagglutination activity was picked to carry out the engineering yeast fermentation by the method of the third step in the first embodiment.
  • the method of step 4 was purified to obtain H5N1 highly pathogenic avian influenza hemagglutinin glycoprotein multimer nanoparticles, which were designated as (H5N1) HA (Hans) .
  • H5N1 The molecular weight of HA (Hans) is about 66KD (the molecular weight of about 66KD here is the HA0 group of H5N1 highly pathogenic avian influenza hemagglutinin glycoprotein polymer nanoparticles). Minute).
  • H5N1 highly pathogenic avian influenza hemagglutinin glycoprotein polymer nanoparticles with N-glycosidase F (PNGF) by the same method as in step 5 of Example 1, analyzing molecular weight changes before and after glycosylation, and finding PNGF
  • the molecular weight of the H (N5) HA (Hans) (the H0 component of the H5N1 highly pathogenic avian influenza hemagglutinin glycoprotein multimer nanoparticle) was reduced to 60 kD, and the results are shown in FIG.
  • the molecular exclusion chromatography (SEC) analysis of (H5N1) HA (Hans) was carried out in the same manner as in the eighth step of Example 1, and the results are shown in FIG.
  • the upper panel is a chromatogram of the molecular weight standard protein
  • the lower panel is a chromatogram of (H5N1) HA (Hans) . It is found that the retention time of (H5N1) HA (Hans) is less than the retention time of the molecular weight standard of 670 kD.
  • the polymer nanoparticles are multimers having a molecular weight greater than 670 kD.
  • H5N1 highly pathogenic avian influenza hemagglutinin glycoprotein polymer nanoparticles prepared by Hansenula can be used to prepare influenza vaccines.
  • Example 4 H1N1 influenza hemagglutinin glycoprotein polymer nanoparticle prepared by Pichia pastoris
  • SEQ ID No. 11 derived from H1N1 influenza virus (A/FortMonmouth/1/47 (H1N1)) was synthesized.
  • HA1-5 5'-ATC TTCGAA ACGatgaaagcaaaactactgatc-3' (SEQ ID No. 12)
  • the sequence underlined is the NspV digestion recognition site.
  • HA1-3 5'-gat GCGGCCGC tcagatgcatattctgcattg-3' (SEQ ID No. 13)
  • the sequence underlined is the NotI digestion recognition site.
  • NspV and NotI were digested with the DNA molecule shown in SEQ ID No. 14 to obtain a gene fragment; NspV and NotI were digested with pPICZ ⁇ vector to obtain a large fragment of the vector; the gene fragment was ligated with a large fragment of the vector to obtain a recombinant plasmid, which was named For pPICZ ⁇ -HA1.
  • the pPICZ ⁇ -HA1 was sequenced and the results were correct.
  • the engineering yeast fermentation is the same as step three in the first embodiment.
  • H1N1 influenza hemagglutinin glycoprotein multimer nanoparticles Purification of H1N1 influenza hemagglutinin glycoprotein multimer nanoparticles is the same as step 4 in Example 1, which is designated as (H1N1) HA.
  • H1N1 influenza virus hemagglutinin glycoprotein polymer (H1N1) HA was treated with N-glycosidase F (PNGF) in the same manner as in step 5 of Example 1, and the molecular weight change before and after glycosylation was analyzed, and it was found that the glycosyl group was excised by PNGF.
  • the molecular weight of the post-hemagglutinin glycoprotein (the HA0 component of the H1N1 influenza hemagglutinin glycoprotein nanoparticle) was decreased to 61 kD, and the results are shown in FIG.
  • H1N1 influenza hemagglutinin glycoprotein nanoparticle (H1N1) HA was subjected to size exclusion chromatography (SEC) analysis in the same manner as in the eighth step of Example 1, and the results are shown in FIG.
  • the upper panel is a chromatogram of the molecular weight standard protein
  • the lower panel is a chromatogram of (H1N1) HA. It is found that the retention time of (H1N1) HA is less than the retention time of the molecular weight standard of 670 kD, and the influenza hemagglutinin glycoprotein is more
  • the polymer nanoparticles are multimers having a molecular weight greater than 670 kD.
  • H1N1 influenza hemagglutinin glycoprotein nanoparticle (H1N1) HA was subjected to chicken red blood cell hemagglutination activity analysis in the same manner as in the first step of Example 1, and the results are shown in Fig. 16, in Fig. 16, the first row from From left to right, 2-fold serial dilutions of (H1N1) HA were started from 1:20, and the second row was physiological saline control, and (H1N1) HA was found to have good hemagglutination activity.
  • Electron micrographs show that (H1N1) HA forms 20-50 nm multimeric particles.
  • H1N1 influenza hemagglutinin glycoprotein nanoparticle (H1N1) HA influenza vaccine was prepared, and the mice were immunized, and the blood was collected for one week after the booster to perform hemagglutination inhibition test (see " Guo Yuanji et al., "Influenza virus and its experimental techniques", Beijing, China Three Gorges Publishing House, 1997"), the results are shown in Figure 17, in Figure 17, the ordinate is hemagglutination inhibition titer (HI), abscissa For grouping, 0.0 was the control group and 7.5 was the experimental group. The average hemagglutination inhibition titer of the serum of the experimental group was found to be 1:320.
  • H1N1 influenza hemagglutinin glycoprotein nanoparticle (H1N1) HA prepared by Pichia pastoris can be used to prepare influenza vaccine.
  • HA7-HindIII-5 5'-ATC AAGCTT ACGATGAACACCCAAATACTGGTTTTC-3' (SEQ ID No. 15)
  • the sequence underlined is the HindIII digestion recognition site.
  • HA7-3 5'-ATC GCGGCCGC TTAAATACAGATAGTACATCT-3' (SEQ ID No. 16).
  • the sequence underlined is the NotI digestion recognition site.
  • the molecule comprises, in order from the 5' end, a Kozak sequence, a signal peptide coding sequence, and an HA gene (containing a C-terminal transmembrane region sequence).
  • HindIII and NotI are digested with the DNA molecule shown in SEQ ID No. 17 to obtain a gene fragment; HindIII and NotI are double-digested with the K. lactis expression vector pKLAC1 to obtain a large fragment of the vector; the gene fragment and the vector large fragment are obtained. After ligation, a recombinant plasmid was obtained, which was named pKLAC1- (H7N9) HA7.
  • pKLAC1- (H7N9) HA7 plasmid was linearized with SacII, and the linearized plasmid was precipitated with 1/10 volume of 3 M sodium acetate and 3 volumes of absolute alcohol.
  • the salt was washed twice with a 70% by volume aqueous solution of ethanol to remove the salt, air-dried, and resuspended in about 30 ⁇ L of water to obtain a pKLAC1- (H7N9) HA7 linearized plasmid for transformation.
  • the method for preparing K. lactis electroporation competent cells is the same as that in Example 1.
  • the seed culture method and the fermentation culture method were the same as those in the third step of Example 1, except that the inducer was replaced by galactose instead of methanol.
  • H7N9 influenza hemagglutinin glycoprotein multiparticle nanoparticle is the same as that of the fourth step in the first embodiment, and is referred to as (H7N9) HA (K. lactis) .
  • the structural analysis method of the influenza hemagglutinin glycoprotein multimer nanoparticle is the same as that in the first step of the first embodiment.
  • HA K. lactis
  • HA0 monomer had a molecular weight of about 66 KD, and the molecular weight decreased by 61 KD after excision of the glycosyl group by PNGF, and the results are shown in Fig. 18.
  • H7N9 influenza hemagglutinin glycoprotein nanoparticle (H7N9) HA (K. lactis) was subjected to size exclusion chromatography (SEC) analysis in the same manner as in the first step of Example 1, and the results are shown in FIG. .
  • the upper panel is a chromatogram of the molecular weight standard protein
  • the lower panel is a chromatogram of (H7N9) HA (K.lactis) . It is found that the retention of (H7N9) HA (K.lactis) has a retention time of less than 670 kD.
  • influenza hemagglutinin glycoprotein multimer nanoparticle is a multimer having a molecular weight greater than 670 kD. Since the molecular weight of the HA0 monomer is 64 KD, the molecular weight of the trimer is about 180 KD, indicating (H7N9) HA (K. lactis ) is a polymer formed of 9 or more HA0 monomers.
  • H7N9 influenza hemagglutinin glycoprotein nanoparticle H7N9 HA (K. lactis) was negatively stained and photographed by a 50,000-fold electron microscope to find that the influenza hemagglutinin was at least 3 HA0 trimers. Multimer particles formed.
  • H7N9 influenza hemagglutinin glycoprotein nanoparticle (H7N9) HA K. lactis
  • the method is the same as step 7 in the first embodiment, the result is shown in FIG. In 20, the first row from left to right is 2 times serial dilution (H7N9) HA (K.lactis) starting from 1:20, and the second row is saline control, and it is found that (H7N9) HA (K.lactis) has Good hemagglutination activity.
  • H7N9 influenza hemagglutinin glycoprotein nanoparticle (H7N9) HA (K.lactis) prepared by recombinant K. lactis
  • H7N9 HA (K.lactis) was diluted with PBS of pH 7.4 to three different dose groups of 0 ⁇ g, 6 ⁇ g, and 12 ⁇ g/50 ⁇ l, respectively, and an equal volume of 1.2 mg/ml Al(OH) 3 was added to each group.
  • the adjuvant was prepared into a flu vaccine and was injected into the experimental group. Meanwhile, a solution in which no (H7N9) HA (K. lactis) was added to the above solution was used as a control injection.
  • Figure 21 shows that the hemagglutination inhibitory activity of the serum 7 days after booster immunization showed that the average hemagglutination inhibition titer of the experimental group reached 1:320.
  • H7N9 influenza hemagglutinin glycoprotein nanoparticle (H7N9) HA (K. lactis) prepared by recombinant K. lactis can be used to prepare influenza vaccine.
  • Example 6 H3N2 influenza hemagglutinin glycoprotein multiparticle nanoparticles prepared by Pichia pastoris
  • HA3-5 5'-ATC TTCGAA ACGatgaagactatcattgcttt-3' (SEQ ID No. 19)
  • the sequence underlined is the NspV digestion recognition site.
  • HA3-3 5'-gat gcggccgc tcaaatgcaaatgttgcacctaatgttgccctt-3' (SEQ ID No. 20)
  • the sequence underlined is the NotI digestion recognition site.
  • the DNA molecule shown by SEQ ID No. 21 contains a Kozak sequence, a signal peptide, and an HA gene sequence (containing a C-terminal transmembrane region sequence) from the N-terminus to the C-terminus.
  • the 8th to 12th positions from the 5' end are Kozak sequences
  • the 13th to 60th positions are signal peptide sequences
  • the 61st to 1713th positions are HA genes
  • the 1600th to 1674th positions are C-terminal transmembrane region sequence.
  • NspV and NotI are digested with the DNA molecule shown in SEQ ID No. 21 to obtain a gene fragment; NspV and NotI are double-digested with the pPICZ ⁇ vector to obtain a large fragment of the vector; the gene fragment is ligated to the large fragment of the vector to obtain a heavy
  • the plasmid was designated as pPICZ ⁇ -HA3, and pPICZ ⁇ -HA3 was sequenced, and the result was correct.
  • the engineering yeast fermentation is the same as step three in the first embodiment.
  • H3N2 influenza hemagglutinin glycoprotein multimer nanoparticles Purification of H3N2 influenza hemagglutinin glycoprotein multimer nanoparticles is the same as step 4 in Example 1, which is designated as (H3N2) HA.
  • Reduction SDS-PAGE analysis (H3N2) HA molecular weight is about 66KD, which is the HA0 component of H3N2 influenza hemagglutinin glycoprotein polymer nanoparticles.
  • H3N2 influenza hemagglutinin glycoprotein nanoparticle (H3N2) HA was subjected to size exclusion chromatography (SEC) analysis in the same manner as in the first step of Example 1, and the results showed that H3N2 influenza hemagglutinin glycoprotein was aggregated.
  • Nanoparticles (H3N2) HA is a multimer with a molecular weight greater than 670 kD.
  • H3N2 influenza hemagglutinin glycoprotein nanoparticle (H3N2) HA Analysis of chicken red blood cell hemagglutination activity of H3N2 influenza hemagglutinin glycoprotein nanoparticle (H3N2) HA, the method is the same as step 7 in the first embodiment, the result is shown in Fig. 23, in Fig. 23, the first From left to right, 2 times serial dilution ((H3N2) HA was started from 1:20, and the second row was saline control, and (H3N2) HA was found to have good hemagglutination activity.
  • H3N2 influenza hemagglutinin glycoprotein nanoparticle (H3N2) HA influenza vaccine was prepared, and the mice were immunized, and blood was collected for one week after boosting to inhibit hemagglutination.
  • H3N2 influenza hemagglutinin glycoprotein nanoparticle (H3N2) HA influenza vaccine was prepared, and the mice were immunized, and blood was collected for one week after boosting to inhibit hemagglutination.
  • “Guo Yuanji and other "Influenza virus and its experimental techniques", Beijing, China Three Gorges Publishing House, 1997” the results are shown in Figure 24.
  • Fig. 24 the ordinate is the hemagglutination inhibition titer (HI), the abscissa is the grouping, 0.0 is the control group, and 7.5 is the experimental group.
  • Figure 24 shows that the mean hemagglutination inhibition titer of the serum of the experimental group reached 1:320.
  • H3N2 influenza hemagglutinin glycoprotein nanoparticle (H3N2) HA prepared by Pichia pastoris can be used to prepare influenza vaccine.
  • Example 7 influenza hemagglutinin glycoprotein nanoparticle cation exchange chromatography
  • the sample liquid obtained in the first step (1) of the first embodiment is purified by the cation chromatography column of the step (2) to obtain a 15% C liquid elution peak, and the component is subjected to hemagglutination activity detection, and the method is the same.
  • Step 7 in Example 1 the results are shown in Figure 25.
  • Figure 25 the first row from left to right is a 15% C liquid elution peak from 2:20 serial dilution, and the second row is a saline control.
  • Figure 25 shows that this component contains influenza hemagglutinin glycoprotein polymer nanoparticles and has hemagglutination activity.
  • Example 8 influenza virus hemagglutinin protein polymer nanoparticle anion exchange chromatography
  • the loading solution obtained in (1) of the fourth step in the first embodiment is directly subjected to the step (3) anion exchange chromatography to obtain a 10% C liquid elution peak, which is subjected to hemagglutination activity detection in the same manner as in the first embodiment. Step seven, the result is shown in Figure 26.
  • Figure 26 the first row from left to right is a 10% C liquid elution peak from 2:20 serial dilution, and the second row is a saline control.
  • Figure 26 shows that this component contains influenza hemagglutinin glycoprotein polymer nanoparticles and has hemagglutination activity.
  • Example 9 Influenza virus hemagglutinin protein polymer nanoparticle gel exclusion chromatography
  • the sample solution obtained in the first step (4) of the first embodiment is subjected to the gel exclusion chromatography of the step (4), and the first eluting main peak is collected, and the hemagglutinin activity is detected, and the method is the same.
  • Step 7 in Example 1 and the results are shown in FIG.
  • Figure 27 the first row from left to right is the elution main peak from 2:20 serial dilution from 1:20, and the second row is the saline control.
  • Figure 27 shows that this component contains influenza hemagglutinin glycoprotein polymer nanoparticles and has hemagglutination activity.
  • Example 10 ELISA method for measuring the content of influenza virus hemagglutinin protein polymer nanoparticles in the supernatant after dissolving different detergents
  • the fermentation broth was centrifuged at 7000 rpm for 20 min at 4 °C. Resuspend with water to 40g/100ml suspension, high-pressure homogenizer to break bacteria (1200bar, 3 times of bacteria), obtain homogenate slurry, add 50g PEG2000 to yeast 500ml homogenate, stir and dissolve for 0.5h, centrifuge at 7000rpm for 20min, collect Precipitate, discard the supernatant; precipitate and add 500ml volume of solution (the solution contains 10mM Tris-HCl, detergent (Traton, Tween, ethyl phenyl polyethylene glycol or weaker ionic detergent deoxidation) Cholate, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 5g/100ml glycerol, balance water), resuspend, stir and dissolve for 2h . After ultracentrifugation for 2
  • the ELISA method was used to determine the content of hemagglutinin in the supernatant.
  • Sample coating with antigen (0.15g/100ml Na 2 ) CO 3, 0.293g / 100ml NaHCO 3 , pH9.6, balance water) diluted 100 times, and then from each sample was added to the ELISA plate in 100 ⁇ L, 4 °C coated overnight as a primary antibody rabbit anti HA7 (1: 500 (diluted with PBS) (this antibody was purchased from Beijing Bofikang Biotechnology Co., Ltd.), and the secondary antibody was goat anti-rabbit IgG-HRP (1:1000) (diluted with PBS) (The antibody was purchased from Huamei Bioengineering Co., Ltd.) ), add 100 ⁇ L of coloring solution to each well (1.84g/100ml Na 2 HPO 4 ⁇ 12H 2 O, 0.5g/100ml citric acid, add
  • the positive clones showed a certain color change, reflecting the content of HA, blank. (The hole in the sample without hemagglutinin is blank) and there is no obvious color change in the negative hole.
  • the data was read on the enzyme analyzer at 492 nm, and the results are shown in Table 1.
  • Table 1 shows that various detergents have effects and can detect influenza virus hemagglutinin protein polymer nanoparticles in the supernatant, of which 2g/100ml TritonX-100 (Traton) works best, followed by 5g/ 100ml Tween20 (Tween) and 2g/100ml deoxycholate, again 2g/100ml NP-40 (ethyl phenyl polyethylene glycol) and 2g/100ml CHAPS, but no detergent treatment, almost no flu detected Viral hemagglutinin protein polymer nanoparticles, indicating that detergent treatment is one of the important steps in the present invention.
  • TritonX-100 Traton
  • Table 1 ELISA analysis method to determine the content of influenza virus hemagglutinin protein polymer nanoparticles in the supernatant after dissolving different detergents
  • the influenza hemagglutinin glycoprotein multimer nanoparticle prepared by the method of the invention has a molecular weight of more than 670 KD, and the monomer (HA0) is a glycoprotein having a molecular weight of about 60 KD, and the vaccine prepared therefrom can induce high titer. And antibodies.
  • the present invention discloses for the first time a method for preparing influenza hemagglutinin glycoprotein multimer nanoparticles by using yeast, the nanoparticle is not fused to an exogenous carrier protein, and there is no problem of repeated inoculation causing an immune reaction to a carrier protein, and the present invention provides
  • the recombinant yeast has the characteristics of short construction period, fast growth, easy mass production and high safety, which makes it very suitable for efficient research and development and large-scale production of vaccines under sudden infectious diseases such as influenza and other emergency conditions.

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Abstract

本发明公开了一种流感病毒血凝素糖蛋白聚合物纳米颗粒的制备方法，利用该方法可以制备出空间结构完整的流感病毒血凝素糖蛋白聚合物。本发明公开一种制备流感病毒血凝素糖蛋白聚合物纳米颗粒的方法，包括如下步骤：使N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因在酵母中表达；将所述酵母进行细胞破碎，加入去污剂，获得含流感病毒血凝素糖蛋白的溶液；将所述溶液进行纯化，制备得到具有血凝活性的流感病毒血凝素糖蛋白聚合物纳米颗粒。用重组酵母制备流感病毒血凝素糖蛋白聚合物纳米颗粒还具有工程菌株构建周期短、生长快、易于大规模生产、安全性高等特点。

Description

一种流感血凝素糖蛋白多聚物纳米颗粒的制备方法 技术领域
本发明涉及一种流感血凝素糖蛋白多聚物纳米颗粒的制备方法，属于生物技术领域。
背景技术
流行性感冒简称流感，是由甲、乙、丙三型流感病毒分别引起的急性呼吸道传染病。流感疫苗接种是预防流感的重要手段。流感血凝素是流感病毒表面的主要蛋白之一，以三聚体形式存在，是流感病毒表面参与宿主细胞吸附和入侵的重要成分，其诱发的中和性抗体可以阻断病毒在宿主细胞表面的吸附和入侵，因此是流感疫苗的主要成分。流感血凝素变异、重配快，各种新型流感频发，如近年来爆发的H5N1高致病性禽流感、H1N1甲型流感、H7N9高致病性禽流感等均引起了严重的问题。新疫情爆发后，疫苗的快速研发和生产是疫情控制的关键。
现有的流感疫苗主要是全病毒灭活疫苗、裂解疫苗或在此基础上纯化获得的亚单位疫苗，这些疫苗都是通过流感病毒减毒株或重配株的鸡胚培养获得病毒，经病毒纯化、灭活或近一步裂解、纯化制备的。鸡胚流感疫苗生产技术存在的主要问题是，病毒生产受限于合格鸡胚的供应，病毒毒株的重配、减毒和鸡胚适应需要大量时间，且存在不确定性。制备的疫苗除作为主要有效成分的流感血凝素外，还含有病毒的其它蛋白，及鸡胚来源蛋白。疫苗的蛋白纯度低，易引起过敏等副反应。且有些亚型的病毒经灭活剂甲醛等的灭活后，抗原性可能发生改变，导致疫苗部分无效。近年来哺乳动物细胞替代鸡胚培养流感病毒的技术取得发展，但该技术只解决了鸡胚供应受限和GMP生产的问题，上述其它问题依然存在。而且还受限于细胞规模化生产的能力。通过基因工程技术，重组表达和制备高度纯化的流感血凝素可望为这些问题的解决提供新途径。
流感病毒血凝素HA蛋白的命名来源于病毒颗粒通过HA蛋白与特异性含唾液酸的受体结合，凝集血红细胞。其合成是首先经转录、翻译后在细胞内质网合成含有562～566个氨基酸的HA蛋白前体(HA0)，即血凝素前体；流感病毒RNA编码的血凝素(HA)成熟蛋白约含550个氨基酸残基，包括重链(HA1)和轻链(HA2)两部分，两者中间的碱性氨基酸位点在成熟病毒颗粒向细胞外芽生释放时，受细胞特异蛋白酶水解切开，成为通过二硫键相连的HA1和HA2。而未经蛋白酶切割前的流感血凝素又被称为流感血凝素前体(HA0)。这种特异性切割是流感病毒与宿主细胞膜融合必须的，但与流感血凝素与受体的结合无关，特异蛋白酶切割前后的流感血凝素都具有相同的抗原性和受体结合活性，HA0分子水解为HA1和HA2，是病毒感染性的先决条件。为方便叙述，除特殊说明外，本发明所述流感血凝素或HA均包括流感血凝素前体(HA0)和特异蛋白酶切割后形成的二硫键相连的HA1和HA2。
流感血凝素HA单体的分子量约为60KD，流感病毒表面的HA以三聚体(HA-trimer)形式组成刺突，这种三聚体结构是其与唾液酸受体结合必需的。一 些动物如鸡、豚鼠等的红细胞表面具有能与流感病毒HA三聚体结合的唾液酸化糖基。病毒表面的多个HA刺突与不同红细胞表面的多个唾液酸化糖基结合，红细胞表面的多个唾液酸化糖基又与多个病毒表面的HA刺突结合，可形成病毒与红细胞的互相交联，形成血凝现象。由于流感病毒感染宿主细胞时的黏附过程也是通过流感病毒HA三聚体与宿主细胞表面的唾液酸化糖基结合实现的。因此血凝活性是检验HA受体结合活性的重要方法，血凝抑制实验是研究抗体是否具有阻断流感病毒HA三聚体与受体结合的中和活性的重要方法。不同表达系统和制备方法获得的重组HA在结构、糖基化、诱导中和抗体的能力等方面存在明显不同。Athmaram，TN等用酵母成功分泌表达并纯化获得了2009新型H1N1流感的HA0，但制备的HA0用FPLC纯化时主要以单体和少量三聚体形式存在，以10μg/只和50μg/只的剂量两次免疫小鼠后血凝抑制活性仅为1:32。未见酵母制备流感血凝素糖蛋白多聚物纳米颗粒的报道。
发明公开
本发明提供了一种流感血凝素糖蛋白多聚物纳米颗粒的制备方法。
本发明提供了一种制备流感病毒血凝素糖蛋白聚合物纳米颗粒的方法，包括如下步骤：使N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因(HA基因)在酵母中表达；将所述酵母进行细胞破碎，加入去污剂，获得含流感病毒血凝素糖蛋白的溶液；将所述溶液进行纯化，制备得到具有血凝活性的流感病毒血凝素糖蛋白聚合物纳米颗粒。
所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA在信号肽序列之前还含有Kozak序列，Kozak序列为5’-aaacg-3’。
所述细胞破碎和加入去污剂之间具体还含有离心得沉淀的步骤。
所述加入去污剂和获得含流感病毒血凝素糖蛋白的溶液之间具体还含有离心得上清的步骤。
所述流感病毒血凝素糖蛋白聚合物纳米颗粒的分子量大于670KD。
所述具有血凝活性的流感病毒血凝素糖蛋白聚合物纳米颗粒在电镜下呈现玫瑰花环结构，证明其至少是由3个以上的三聚体形成，3个流感病毒血凝素蛋白前体HA0组成的HA0三聚体分子量约为180KD，因而证明流感病毒血凝素糖蛋白聚合物纳米颗粒至少由9个以上流感病毒血凝素蛋白前体HA0参与形成聚合物纳米颗粒，其中流感病毒血凝素蛋白前体HA0形成HA0三聚体。
上述方法中，所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因在酵母中表达的方法为：将含有N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因的重组表达载体转化酵母，培养得到转化的酵母，再诱导使基因表达。
所述酵母可为毕赤酵母、汉逊酵母或乳酸克鲁维酵母。
所述诱导使基因表达的步骤具体为：将所述转化的酵母进行培养，并诱导基因的表达。
所述培养具体为摇瓶培养或发酵罐培养，根据控制所述编码基因表达的启动子的不同，可以采用先培养所述转化的酵母至一定密度，再诱导基因表达的方法，也可以采用在培养所述转化的酵母的同时诱导基因表达的方法，其中前一种方法更有利于提高酵母工程菌的稳定性。
所述信号肽为所述HA基因自身的信号肽或其它可在相应酵母中起作用的其它信号肽。所述其它可在相应酵母中起作用的其它信号肽为酿酒酵母α交配因子信号肽、α淀粉酶信号肽或白蛋白的信号肽。所述白蛋白的信号肽具体为血清白蛋白的信号肽。
上述任一所述的方法中，所述流感病毒的血凝素HA为H1、H3、H5或H7血清型流感病毒的HA。上述任一所述的方法中，所述H1、H3、H5或H7血清型流感病毒的HA分别为H1N1、H3N2、H5N1或H7N9流感病毒的HA。
上述任一所述的方法中，所述重组表达载体是将所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因插入含有AOX启动子的载体得到。所述重组表达载体具体是将所述含有信号肽序列和C-端穿膜区序列的流感病毒的血凝素HA的编码基因插入pPICZα的NotI和NspV酶切位点间，并BglII线性化得到。所述酵母为毕赤酵母。所述流感病毒具体为H7N9禽流感病毒，所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因具体如SEQ ID No.4所示。所述H7N9禽流感病毒为A/Hongzhou/1/2013(H7N9)。所述流感病毒具体为甲型H1N1流感病毒，所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因具体如SEQ ID No.14所示。所述甲型H1N1流感病毒具体为A/FortMonmouth/1/47(H1N1)。所述流感病毒具体为甲型H3N2流感病毒，所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因具体如SEQ ID No.21所示。所述甲型H3N2流感病毒具体为A/reassortant/NYMC X-223A(Texas/50/2012x PuertoRico/8/1934)(H3N2)。
上述任一所述的方法中，所述重组表达载体是将所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因插入含有MOX启动子的载体得到。所述重组表达载体具体是将所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因插入pPICZα的NotI和BstB I酶切位点间，得到中间载体1；再将中间载体1的AOX启动子替换为汉逊酵母的醇氧化酶启动子MOX，再用BglII线性化得到。所述酵母为汉逊酵母，具体为多型汉逊酵母。所述MOX启动子是以多型汉逊酵母的基因组DNA为模板，以SEQ ID No.9和SEQ ID No.10所示的DNA分子为引物进行PCR扩增得到MOX启动子。所述将中间载体1的AOX启动子替换为汉逊酵母的醇氧化酶启动子MOX的方法具体为将所述PCR扩增得到的MOX启动子进行BglII酶切，并进行磷酸化，获得5’端为BglII酶切粘性末端，3’端磷酸化的MOX启动子；将中间载体1用NspV单酶切后用Klenow fragment大片段酶和dNTP补平，得到片段，回收片段后，再用BglII酶切，切除AOX启动子，得到切除AOX启动子的中间载体1；将5’端为BglII酶切粘性末端， 3’端磷酸化的MOX启动子与切除AOX启动子的中间载体1连接。所述流感病毒具体为H5N1禽流感病毒，所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因具体如SEQ ID No.8所示。所述H5N1禽流感病毒具体为A/duck/Guangxi/27/2003(H5N1)。
上述任一所述的方法中，所述重组表达载体是将所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因插入含有LAC4启动子的载体得到。所述重组表达载体具体是将所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因插入pKLAC1的HindIII和NotI酶切位点间，再用SacII线性化得到。所述酵母为乳酸克鲁维酵母。所述流感病毒具体为H7N9禽流感病毒，所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因具体如SEQ ID No.17所示。所述H7N9禽流感病毒具体为A/Hongzhou/1/2013(H7N9)。
上述任一所述的方法中，所述细胞破碎的方法为物理方法、生物方法或化学方法。所述物理方法具体为玻璃珠振荡法、高压匀浆法或球磨法。所述生物方法具体为酶裂解法。所述化学方法具体为碱裂解法。所述去污剂为非离子型去污剂或弱离子型去污剂。所述非离子型去污剂具体为曲拉通、吐温或乙基苯基聚乙二醇。所述弱离子型去污剂具体为脱氧胆酸盐或3-[(3-胆酰胺基丙基)二甲基铵基]-1-丙磺酸盐。所述曲拉通具体为TritonX-100。所述吐温具体为Tween20。所述乙基苯基聚乙二醇具体为NP-40。
所述流感病毒血凝素糖蛋白聚合物纳米颗粒表达后位于所述酵母的细胞膜上。所述去污剂的作用是从所述酵母细胞膜上溶解所述流感病毒血凝素糖蛋白聚合物纳米颗粒并较好地保持其结构。
上述任一所述的方法中，所述将所述溶液进行纯化的方法包括阳离子交换层析和/或阴离子交换层析和/或凝胶排阻层析。所述阳离子交换层析的填料具体为Sepharose FF SP。所述阴离子交换层析的填料具体为Source 30Q。所述凝胶排阻层析的填料具体为Superdex200。
所述纯化过程中根据各步骤纯化组分是否有HA条带和血凝活性判断其是否为含有所述流感病毒血凝素糖蛋白聚合物纳米颗粒的组分，如果该组分有HA条带和血凝活性就为含有所述流感病毒血凝素糖蛋白聚合物纳米颗粒的组分。
所述纯化后获得的流感病毒血凝素糖蛋白聚合物纳米颗粒中非流感病毒血凝素蛋白含量≤20％，具体为≤10％；
所述HA包含流感病毒血凝素前体HA0和特异蛋白酶切割后形成的二硫键相连的HA1和HA2。
由上述任一所述的方法制备得到的流感病毒血凝素糖蛋白聚合物纳米颗粒制备的流感疫苗也属于本发明的保护范围；
所述疫苗具体还包含佐剂。所述佐剂具体为铝佐剂，例如氢氧化铝佐剂。
附图说明
图1为重组毕赤酵母制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒的阳离子交换层析的检测结果。
图2为重组毕赤酵母制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒的阴离子交换层析的检测结果。
图3为重组毕赤酵母制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒的凝胶排阻色谱层析的检测结果。
图4为重组毕赤酵母制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒的糖基化检测。
图5为重组毕赤酵母制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒的TPCK处理的胰蛋白酶酶切结果。
图6为重组毕赤酵母制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒的血凝活性检测。
图7为重组毕赤酵母制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒的分子排阻色谱分析。
图8为重组毕赤酵母制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒的电子显微镜照相。
图9为用重组毕赤酵母制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒制备的流感疫苗效果评价。
图10为重组汉逊酵母制备的H5N1高致病性禽流感血凝素糖蛋白多聚物纳米颗粒的糖基化检测。
图11为重组汉逊酵母制备的H5N1高致病性禽流感血凝素糖蛋白多聚物纳米颗粒的分子排阻色谱分析。
图12为重组汉逊酵母制备的H5N1高致病性禽流感血凝素糖蛋白多聚物纳米颗粒的血凝活性检测。
图13为重组汉逊酵母制备的H5N1高致病性禽流感病毒血凝素糖蛋白聚合物的流感疫苗效果评价。
图14为重组毕赤酵母制备的H1N1流感血凝素糖蛋白多聚物纳米颗粒的糖基化检测。
图15为重组毕赤酵母制备的H1N1流感血凝素糖蛋白多聚物纳米颗粒的分子排阻色谱分析。
图16为重组毕赤酵母制备的H1N1流感血凝素糖蛋白多聚物纳米颗粒的血凝活性检测。
图17为用重组毕赤酵母制备的H1N1流感血凝素糖蛋白多聚物纳米颗粒得到的流感疫苗效果评价。
图18为重组乳酸克鲁维酵母制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒的糖基化检测。
图19为重组乳酸克鲁维酵母制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒的 分子排阻色谱分析。
图20为重组乳酸克鲁维酵母制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒的血凝活性检测。
图21为用重组乳酸克鲁维酵母制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒得到的流感疫苗效果评价。
图22为重组毕赤酵母制备的H3N2流感血凝素糖蛋白多聚物纳米颗粒的糖基化检测。
图23为重组毕赤酵母制备的H3N2流感血凝素糖蛋白多聚物纳米颗粒的血凝活性检测。
图24为用重组毕赤酵母制备的H3N2流感血凝素糖蛋白多聚物纳米颗粒得到的流感疫苗效果评价。
图25为实施例7中的血凝活性检测。
图26为实施例8中的血凝活性检测。
图27为实施例9中的血凝活性检测。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。为方便叙述，除特殊说明外，本发明所述流感血凝素或HA均包括流感血凝素前体(HA0)和特异蛋白酶切割后形成的二硫键相连的HA1和HA2。pPICZα载体购自Invitrogen公司。毕赤酵母X-33购自Invitrogen公司。TPCK处理胰蛋白酶(TPCK-Trypsin)购自Sigma公司。Pyrobest DNA聚合酶购自宝生物工程(大连)有限公司。pKLAC1购自NEB。乳酸克鲁维酵母购自NEB。H7N9流感重配疫苗株(NIBRG-268)在文献“杨娟，郑亚明，冯录召，余宏杰.人用H7N9禽流感疫苗研发进展，中华预防医学杂志，2014,48(2)”中公开过，公众可从中国人民解放军军事医学科学院生物工程研究所获得。
实施例1、重组毕赤酵母制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒
一、重组表达载体的构建
(一)根据Genbank(KC853766)(A/Hongzhou/1/2013(H7N9))的流感HA全长氨基酸序列，根据酵母偏爱密码子和基因高表达原则优化编码基因，合成SEQ ID No.1所示的DNA分子。
(二)设计并合成如下引物：
HA7-3：5’-ATCGCGGCCGCTTAAATACAGATAGTACATCTCAT-3’(SEQ ID No.2)
下划线所示序列为NotI酶切识别位点。
HA7-5：5’-ATCTTCGAAACGATGAACACCCAAATACTGGTTTTC-3’(SEQ ID No.3)
下划线所示序列为NspV酶切识别位点。
(三)以SEQ ID No.1所示的DNA分子为模板，以HA7-3和HA7-5为引物，进行PCR扩增，得到PCR扩增产物，该产物的序列如SEQ ID No.4所示，SEQ ID No.4中自5’末端起第8位至第12位为Kozak序列，第13至第66位为信号肽编码序列，第67位至第1695位为HA基因，第1585至第1668位为C端穿膜区序列。
(四)NspV和NotI双酶切SEQ ID No.4所示的DNA分子，得到基因片段；NspV和NotI双酶切pPICZα载体得到载体大片段；将基因片段与载体大片段连接，得到重组质粒，将其命名为pPICZα-HA7。将pPICZα-HA7测序，结果正确。
二、重组酵母的构建和筛选
将约10μg pPICZα-HA7质粒，用BglII线性化，用1/10体积的3M醋酸钠水溶液和3倍体积的无水酒精沉淀线性化的质粒。用体积百分含量为70％的乙醇水溶液洗两次以除去其中的盐，晾干，加入约30μL水重悬沉淀，获得用于转化的pPICZα-HA7线性化质粒。
以下步骤中制备酵母电转化感受态细胞的方法参照Invitrogen公司的相关手册和“Molecular Cloning，A laboratory Manual(Fourth Edition)”,2012Cold Spring Harbor Laboratory Press,Cold Spring Harbor,New YorK。
将毕赤酵母X-33在YPD平板(酵母提取物10g/L，胰蛋白胨20g/L，葡萄糖20g/L，琼脂15g/L)上用划线法分离单克隆，28℃温箱培养2天。接种一个单克隆至一个装有10mL YPD液体培养基(酵母提取物10g/L，胰蛋白胨20g/L，葡萄糖20g/L)的50mL三角瓶中，28℃过夜培养至OD600约为2，得到菌液。再将0.1-0.5mL菌液接种到含有500mLYPD液体培养基的3.5L摇瓶中，培养过夜至OD600至1.3-1.5之间。将菌液转移至无菌的离心瓶中，4℃，1500g离心10分钟。用500mL预冷的无菌水重悬菌体，4℃，1500g离心10分钟收获细胞，用250mL预冷的无菌水再洗一次。用20mL预冷的无菌1M山梨醇重悬菌体，4℃，1500g离心10分钟收获细胞，用预冷的1M山梨醇重悬菌体至终体积为1.5mL，得到菌悬液。
取80μL菌悬液与10μL用于转化的pPICZα-HA7线性化质粒，在微量离心管中混匀，得到混合物，将其置冰上5min，将混合物转移到一个冰冷的0.2cm电转杯中，电穿孔细胞(Bio-Rad Gene Pulser，2000V，25μF，200Ω)，再立即向电转杯中加入1mL冰冷的1M山梨醇，并小心地将混合物(转化细胞)转移至15mL培养管。
将培养管放在28℃温箱孵育1h，不要摇动。然后加入1mL YPD液体培养基后在28℃，250rpm的摇床中孵育3h。取200μL转化细胞涂布到含100μg/mL Zeocin的YPD平板上。28℃温箱培养2-5天，至形成单克隆。
随机挑取单克隆接种到2ml YPD液体培养基中，28℃培养48h，按体积比5％的接种量接种到BMGY培养基(酵母提取物10g/L，胰蛋白胨20g/L，pH6.0，100mmol/L磷酸缓冲液，1.34g/100ml的YNB，4×10-5g/100ml Biotin，1g/100ml的甘油)中，24h后加入体积百分含量为0.5％的甲醇诱导表达，每12h补加1次，诱导60h后离心收集菌体。每1ml菌液离心收获的菌体用100μl PBS重悬，加入1/4体积的酸洗玻璃珠(直径425-600μm或0.5mm)，每个样品以最大速度涡旋震荡1分钟， 重复六次，每两次涡旋震荡中间冰浴两分钟以防蛋白降解。用低温微量离心机4℃，3500g离心1分钟，沉淀玻璃珠和未破损的细胞，得到上清即为破菌液。取破菌液先用PBS按照1：20体积比稀释后，再用PBS按照1：2体积比系列稀释后，用1％鸡红细胞进行血凝活性分析(方法见“郭元吉等《流行性感冒病毒及其实验技术》，北京，中国三峡出版社，1997)。挑取具有血凝活性的破菌液对应的克隆(重组毕赤酵母单克隆)，用于流感血凝素糖蛋白多聚物纳米颗粒的制备。
三、工程酵母发酵
种子培养：将步骤二得到的血凝活性高的重组毕赤酵母单克隆接种到新鲜MD平板(1.34g/100mlYNB，4×10-5g/100ml Biotin，1g/100ml葡萄糖，1.5g/100ml琼脂粉)上，培养，并挑取其上的单克隆菌落接种于YPD液体培养基中，24℃,250rpm培养约48h。再转接于YPD液体摇瓶培养基300mL中，接种量为1％，25℃,250rpm培养，至菌密度OD600大于10，得到种子液。
发酵培养：配制发酵培养基2.1L(H3PO43.5mL/L，K2SO42.4g/L，KOH 0.65g/L，CaSO4(无水)0.14g/L，MgSO4·7H2O 1.95g/L，甘油40.0g/L，PTM11.2mL/L，0.02g/100ml的生物素0.5mL/L，余量为水。其中PTM1的组成为：CuSO4·5H2O 6.0g/L，MnSO4·H2O 3.0g/L，FeSO4·7H2O 65g/L，ZnSO4·7H2O 20g/L，CoCl2·6H2O 0.5g/L，NaMoO4·2H2O 0.2g/L，KI 0.1g/L，浓H2SO45mL/L)，加入到5L发酵罐，121℃，30min高压灭菌。待发酵罐降至室温，用氨水调节pH6.0。
以10％的接种量将种子液接入发酵罐，氨水控制pH6.0，温度为28℃，调节搅拌转速和通气量维持在溶氧10％以上。当甘油耗尽时，溶氧回升，开始流加补料生长培养基(50g/100ml的甘油水溶液(含12mL/L PTM1，2mL/L 500×生物素(购自北京欣经科生物技术有限公司))，40ml/h，流加6-8h,停止补料。开始甲醇诱导，温度维持在24℃，用氨水溶液将pH调为6.4。起始阶段，无水甲醇以2.4mL/h开始流加，每小时增加2.4mL100％甲醇，5h后增至12mL/h，此时为诱导0小时，之后每12h取样。诱导48h后结束发酵，发酵液于4℃，7000rpm/min离心20min。用水重悬为40g/100ml的悬液，高压匀浆仪破菌(1200bar，破菌3次),得匀浆液，该匀浆液用于流感血凝素糖蛋白多聚物纳米颗粒的纯化。
四、流感血凝素糖蛋白多聚物纳米颗粒的纯化
(一)500ml步骤三得到的匀浆液中加50g PEG2000，搅拌溶解0.5h,7000rpm离心20min，收集沉淀，弃上清；沉淀加500ml体积的溶液(该溶液中含10mM Tris-HCl，体积百分含量2％TritonX-100(曲拉通，非离子型去污剂),5g/100ml的甘油，余量为水)重悬，搅拌溶解2h。8000rpm离心20min，收集上清，用磷酸盐调节pH为6.0，用水稀释至电导低于2.5ms/cm，得上样液。
(二)阳离子色谱柱(色谱介质为Sepharose FF SP(购自GE)，床层为
Figure PCTCN2015088748-appb-000001
检测波长为280nm，室温)先用A液平衡(A液：含有1g/100ml的Tween20、5g/100ml甘油，余量为20mM pH6.0的PB)后，将步骤(一)的上样液以50ml/min流速上样，用A液同样流速冲洗至A280小于0.2，换用B液(B液：含有1g/100ml的Tween20、 5g/100ml甘油，余量为20mM pH6.86的PB)平衡，用15％C液(A液中添加终浓度为150mM的NaCl)、100％C液洗脱(A液中添加终浓度为1M的NaCl)，得到15％C液、100％C液的洗脱峰，结果如图1中A所示。
图1中，1-6代表15％C液洗脱得到的1-6收集管的洗脱液，100％C代表100％C液的洗脱液，3+endoh代表3收集管的洗脱液用EndoH酶切后的样品，柱前代表上样液，M代表蛋白marker。
将图1中A的1-6收集管的洗脱液、100％C液的洗脱液以及柱前样品进行还原SDS-PAGE分析，结果如图1中B所示，图1B中箭头所示为HA组分的条带。
图1表明，HA组分所在的样品为15％C液的洗脱液。
(三)收集含有HA组份的15％C液的洗脱液进行阴离子交换层析(色谱介质为Source 30Q(购自GE)，床层为
Figure PCTCN2015088748-appb-000002
检测波长为280nm，室温)。具体步骤如下：将15％C液的洗脱样品用A液(A液:1g/100mlTween20、5g/100ml的甘油，余量为20mM pH8.1的Tris-HCl)进行10倍稀释,以10ml/min的速度上样结束后，用B液平衡(B液:1g/100mlTween20、5g/100ml的甘油，余量为20mM pH7.5的Tris-HCl)，再分别用10％C液(该步骤的A液中添加终浓度为100mM的NaCl)、100％C液洗脱(该步骤的A液中添加终浓度为1M的NaCl)，最后用0.5M NaOH水溶液进行洗脱，得到各洗脱组分，结果如图2中A所示。
图2中，Q穿代表上样时未被Source 30Q结合的样品；10％C代表10％C液的洗脱液；100％C代表100％C液的洗脱液；NaOH代表0.5M NaOH水溶液的洗脱液。
将纯化前的样品(步骤(二)得到的15％C液的洗脱液)、没有被Q柱(Source30Q)吸附而穿出的样品、10％C液洗脱液(第一管收集液以及第二管收集液、第三管收集液)、利用100％C液进行洗脱的收集液，利用0.5M NaOH水溶液进行洗脱的收集液进行还原SDS-PAGE分析，结果如图2中B所示。图2B中，Q柱前代表纯化前的样品(步骤(二)得到的15％C液的洗脱液)，Q穿代表没有被Q柱(Source 30Q)吸附而穿出的样品，1代表10％C液洗脱第一管收集液，2代表10％C液洗脱第二管收集液，3代表10％C液洗脱第三管收集液，100％C代表100％C液进行洗脱的收集液，NaOH代表利用0.5M NaOH水溶液进行洗脱的收集液，M代表蛋白marker。箭头所示为HA条带。
图2表明，该步骤的纯化获得了纯度提高的HA样品，去除了部分杂蛋白，HA组分所在的样品为该步骤的10％C液的洗脱液。
(四)收集步骤(三)10％C液洗脱组分4ml，进行凝胶排阻色谱层析(色谱介质为Superdex200(购自GE),床层为
Figure PCTCN2015088748-appb-000003
检测波长为280nm，室温)，将步骤(三)10％C液洗脱组分4ml上样后，用洗脱液(洗脱液：含有0.1M NaCl的pH 7.0的20mM PB)进行洗脱，流速1ml/min，结果如图3A所示。
图3A中，箭头表示的第一个洗脱主峰为纯化的H7N9流感血凝素糖蛋白多聚物纳米颗粒，测定其蛋白浓度为0.33mg/ml。
将步骤(三)10％C液洗脱组分、EndoH酶切的凝胶排阻色谱层析纯化得到的 H7N9流感血凝素糖蛋白多聚物纳米颗粒以及凝胶排阻色谱层析纯化得到的H7N9流感血凝素糖蛋白多聚物纳米颗粒进行还原SDS-PAGE分析，结果如图3中B所示。
图3中，柱前代表步骤(三)10％C液洗脱组分，HA+endoh代表EndoH酶切的凝胶排阻色谱层析纯化得到的H7N9流感血凝素糖蛋白多聚物纳米颗粒，HA代表凝胶排阻色谱层析纯化得到的H7N9流感血凝素糖蛋白多聚物纳米颗粒，M代表蛋白marker。箭头所示为HA条带。
图3表明，该步纯化获得纯度较高的HA纯品，并利用EndoH糖苷酶进行了分析，表明其发生了正确的糖基化修饰。
五、流感病毒血凝素糖蛋白聚合物的结构分析
用N-糖苷酶F(PNGF)(购自NEB)处理，分析糖基切除前后的分子量变化，以分析步骤四制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒，具体步骤如下：取20ul步骤四纯化的H7N9流感血凝素糖蛋白多聚物纳米颗粒(浓度为330ug/ml)，按N-糖苷酶F(PNGF)的酶切方法对其进行处理，同时设不加酶对照组和不加样品对照组。将各样品进行还原SDS-PAGE检测，结果如图4所示。
图4中，PNGF代表N-糖苷酶F，HA+PNGF代表步骤四制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒中加入了N-糖苷酶F，HA代表步骤四制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒，M代表蛋白marker。箭头所示为HA条带。
图4表明，未用PNGF处理的H7N9流感血凝素糖蛋白多聚物纳米颗粒的HA0分子量约为64KD，PNGF处理后，分子量下降为约60KD，与未糖基化的HA0成熟蛋白的理论分子量(60172Da)一致。说明步骤四制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒是糖蛋白。
组成H7N9流感血凝素糖蛋白多聚物纳米颗粒的组分HA0的N端5个氨基酸序列分析结果为DKIXL，其中X为未测到，这与H7N9病毒HA成熟蛋白(HA0)N端理论序列一致(理论序列为DKICL，其中C在Edman法测序时会被破坏，不能测到)。说明HA的信号肽已被成功切除。
六、胰蛋白酶切割的HA制备
为了制备被蛋白酶特异切割成HA1和HA2的HA，取浓度为330ug/ml的H7N9流感血凝素糖蛋白多聚物纳米颗粒1ml,加TPCK处理的胰蛋白酶16ug，冰浴处理1小时，作为实验组。同时另取同样的H7N9流感血凝素糖蛋白多聚物纳米颗粒1ml，100℃水浴处理5分钟进行变性，冰浴冷却，加TPCK处理的胰蛋白酶16ug，同样冰浴处理1小时，作为对照组。
蛋白酶特异切割不仅可用于制备切割H7N9流感血凝素糖蛋白多聚物纳米颗粒，而且也是分析HA0的高级结构是否正确的重要方法，具有流感病毒三聚体高级结构的HA0只有HA1与HA2间的碱性氨基酸位点裸露，才可以被胰蛋白酶特异性地切割为分子量约40KD的HA1和分子量约25KD的HA2。而不具有正确高级结构的HA0则可以被胰蛋白酶切成大小不等的各种片段。
将H7N9流感血凝素糖蛋白多聚物纳米颗粒、实验组和对照组进行还原SDS-PAGE， 检测结果如图5所示。
图5中，HA代表H7N9流感血凝素糖蛋白多聚物纳米颗粒，HA+胰酶为实验组，HA变性+胰酶为对照组。箭头代表HA0条带。
图5表明，HA0被胰蛋白酶切割后，非还原SDS-PAGE电泳分析其分子量为64KD与未切割HA0相似，而还原电泳分析发现，HA0已被特异性切割成了分子量为40kD和24kD的两个片段，与HA1和HA2的分子量一致。说明本发明获得的HA0被胰蛋白酶特异性切割成了由二硫键连接的HA1和HA2亚基构成的HA。而该HA0经加热破坏高级结构后，再用胰酶切割，非还原电泳和还原电泳分析均未发现特异性条带，说明HA0已被胰蛋白酶切成了大小不等的各种片段。因此本发明的HA0具有流感病毒血凝素相同的高级结构。
七、血凝实验
取上述制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒，先用PBS按照体积比1：10稀释，然后再用PBS按照体积比1：2系列稀释，以生理盐水为对照，用1％鸡红细胞进行血凝活性分析，具体方法见“郭元吉等《流行性感冒病毒及其实验技术》，北京，中国三峡出版社，1997”。
血凝活性检测结果如图6所示。
图6中，第一排从左至右为从1：20开始2倍系列稀释的H7N9流感血凝素糖蛋白多聚物纳米颗粒，第二排为生理盐水对照。
图6表明，H7N9流感血凝素糖蛋白多聚物纳米颗粒具有明显的血凝活性，鸡血血凝效价达到1：8000。因此本发明的流感血凝素(H7N9流感血凝素糖蛋白多聚物纳米颗粒)具有唾液酸受体结合活性。
八、为了分析制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒的分子大小，对其进行SEC(分子排阻凝胶色谱)分析，仪器为Agilent1290高效液相色谱仪(Agilent Technologies Co)，紫外检测器，检测波长214nm，TSKG4000SWXL
Figure PCTCN2015088748-appb-000004
色谱柱和TSK Guardcolumn SWXL保护柱(购自Tosoh Bioscience LLC)分析，以含有100mM NaCl的20mM PB为流动相，流速0.5ml/min。用SEC分子量标准(购自苏州赛分科技有限公司)分析标定，结果如图7所示。
图7中，A的上图为分子量标准蛋白的色谱图，各分子量的保留时间分别为：670KD,20.593min；150KD,22.478min；44KD,24.074min；17.6KD,25.363min；1.35KD,26.966min)。A的下图为H7N9流感血凝素糖蛋白多聚物纳米颗粒的色谱图。
图7B中的面积百分比报告如下：
Figure PCTCN2015088748-appb-000005
图7B表明H7N9流感血凝素糖蛋白多聚物纳米颗粒纯度达到了99.69％。图7表明，H7N9流感血凝素糖蛋白多聚物纳米颗粒在该条件分析下，保留时间为16.971min,说明其分子量明显大于670KD，由于HA0单体的分子量为64KD，其三聚体的分子量约为180KD，说明H7N9流感血凝素糖蛋白多聚物纳米颗粒为含9个以上HA0单体形成的多聚体，胰蛋白酶切割实验和血凝实验均表明H7N9流感血凝素糖蛋白多聚物纳米颗粒具有流感血凝素的三聚体高级结构，因此，该H7N9流感血凝素糖蛋白多聚物纳米颗粒为三个以上HA0三聚体形成的多聚体。
将制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒负染后用50,000倍电子显微镜照相，结果如图8所示。图8中的标尺为20纳米。图8表明，H7N9流感血凝素糖蛋白多聚物纳米颗粒为至少3个以上血凝素三聚体(此处的血凝素三聚体是指三个HA0单体聚合形成的三聚体)尾部在内聚合，头部向外突出，形成的多聚体颗粒，直径约20-50纳米。
实施例2、用重组毕赤酵母制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒制备流感疫苗
将实施例1制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒用pH7.4的PBS稀释至7.5μg/50μl，添加等体积的1.2mg/ml Al(OH)3佐剂(购自GE公司，商品名为Rehydragel@LV)，制成流感疫苗，为实验组注射液。同时，设其中不加H7N9流感血凝素糖蛋白多聚物纳米颗粒的溶液为对照组注射液。
实验组和对照组每组5只小鼠，每组每只小鼠后腿肌肉注射相应组的注射液100μl。第一次注射三个星期后加强免疫一次，加强免疫后一个星期采血得血清进行血凝抑制实验，血凝抑制实验的标准血凝素为WHO(世界卫生组织)推荐的英国国立生物标准与参考品研究所NIBSC(National Institute for Biological Standards and Control,a centre of the Medical and Healthcare products Regulatory Agency(MHRA),United Kingdom of Great Britain and Northern Ireland，)提供的H7N9流感重配疫苗株(NIBRG-268)的鸡胚培养病毒经1：2000甲醛灭活后制备。H7N9流感重配疫苗株(NIBRG-268)的鸡胚培养病毒经1：2000甲醛灭活后制备的病毒制备标准血凝素、稀释和血凝抑制实验方法见“郭元吉等《流行性感冒病毒及其实验技术》，北京，中国三峡出版社，1997”。
各组小鼠血清中中和抗体的血凝抑制活性结果如图9所示。
图9中，纵坐标为血凝抑制效价(HI)，横坐标为分组，0.0为对照组，7.5为实验组。图9表明，对照组小鼠的血清没有产生血凝抑制，实验组小鼠血清血凝抑制效价均大于1：40，平均血凝抑制效价为1：640。一般认为流感疫苗诱导的血凝抑制效价大于1：40即可为机体提供有效的免疫保护。因此，用重组酵母制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒可以用于制备流感疫苗。
实施例3、汉逊酵母制备的H5N1高致病性禽流感病毒血凝素糖蛋白多聚物纳米颗粒
一、重组表达载体的构建
(一)根据Genbank(EU263353.1)(A/duck/Guangxi/27/2003(H5N1))的流感HA全长氨基酸序列，根据酵母偏爱密码子和基因高表达原则优化编码基因，合成SEQ ID No.5所示的DNA分子。
(二)设计并合成如下引物：
HA5-5：5’-ATCTTCGAAACGatggagaaaatagtgcttc-3’(SEQ ID No.6)
下划线所示序列为BstB I酶切识别位点。
HA5-3：5’-ATCGCGGCCGCttaaatgcaaattctgcattg-3’。(SEQ ID No.7)
下划线所示序列为NotI酶切识别位点。
(三)以SEQ ID No.5所示的DNA分子为模板，以HA5-3和HA5-5为引物，进行PCR扩增，得到PCR扩增产物，该产物的序列如SEQ ID No.8所示，该序列从自5’末端起依次包含了Kozak序列、信号肽编码序列、HA基因(包含C端穿膜区序列)。
NotI和BstB I双酶切SEQ ID No.8所示的DNA分子，得到基因片段；NotI和BstB I双酶切pPICZα载体得到载体大片段；将基因片段与载体大片段连接，得到重组质粒，将其命名为pPICZ-(H5N1)HA。将pPICZ-(H5N1)HA测序，结果正确。
(四)为了适应在汉逊酵母中表达，将pPICZ-(H5N1)HA中的AOX启动子替换为汉逊酵母的醇氧化酶启动子MOX。
1、根据汉逊酵母启动子的核苷酸序列，合成引物MOX5：5’-ATCAGATCTTCGACGCGGAGAACGATCT-3’(SEQ ID No.9，下划线部分为BglII酶切识别位点)和MOX3：5’-TGTTTTTGTACTTTAGATTGATG-3’(SEQ ID No.10)。
2、以多型汉逊酵母ATCC 26012(可美国典型微生物菌种保藏中心获得)基因组为模板，以MOX5和MOX3为引物，利用Pyrobest DNA聚合酶进行PCR扩增，得到MOX启动子片段。
PCR体系：1μg基因组DNA，4μl dNTP(2.5mM)，5μl 10×Pyrobest Buffer，1μl 10μM MOX5引物，1μlμM MOX3引物,0.5μl Pyrobest DNA聚合酶，37.5μl水，总体积50μl。
PCR程序：94℃预变性5min；94℃变性30sec，55℃退火30sec，72℃延伸2min，30个循环；72℃延伸10min，4℃保存。
PCR扩增产物为约1.5kb的目的片段，将其用BglII酶切，并对酶切片段进行磷酸化，获得5’端为BglII酶切粘性末端，3’端磷酸化的1.5kb的MOX启动子。
3、将pPICZ-(H5N1)HA载体用NspV单酶切后用Klenow fragment大片段酶和dNTP补平，电泳回收后，再用BglII酶切，切除AOX启动子。
电泳回收切除AOX启动子片段的载体，将该载体与上述5’端为BglII酶切粘性末端，3’端为磷酸化的1.5kb的MOX启动子连接，获得用汉逊酵母醇氧化酶启动子MOX控制的H5N1流感血凝素糖蛋白多聚物纳米颗粒基因表达的载体，记作pMOXZ-HA5。
二、将pMOXZ-HA5载体用BglII线性化后，电转化多型汉逊酵母(ATCC 26012(可美国典型微生物菌种保藏中心获得))，并将转化的细胞涂布到含100μg/mL Zeocin的YPD平板(酵母提取物10g/L，胰蛋白胨20g/L，葡萄糖20g/L，琼脂15g/L)上。30℃温箱培养2-4天，至形成单克隆。随机挑取单克隆接种到2ml YPD(酵母提取物10g/L，胰蛋白胨20g/L，葡萄糖20g/L)液体培养基中，30℃培养24-48h，按5％的接种量接种到BMGY培养基(酵母提取物10g/L，胰蛋白胨20g/L，pH6.0，100mmol/L磷酸缓冲液，1.34g/100ml的YNB，4×10-5g/100ml Biotin，1g/100ml的甘油)中，24h后加入体积百分含量为0.5％甲醇诱导表达，每12h补加1次，诱导60h后离心收集菌体和上清。破菌液的制备以及血凝活性分析同实施例1中步骤二。筛选高血凝活性的阳性菌株命名为Hans(pMOX-HA5)。
三、H5N1高致病性禽流感血凝素糖蛋白多聚物纳米颗粒的制备
挑取Hans(pMOX-HA5)单菌落接种于3mlYPD液体培养基中，30℃,250rpm生长1-2天，至菌密度OD600大于10，以体积比2％转接至装有100mlYPD液体培养基的1L摇瓶中，30℃,250rpm培养24小时，以体积比5％转接至10个装有150mlBMGY液体培养基的1L摇瓶中，30℃,250rpm培养24小时，加入体积百分含量为0.5％的甲醇诱导表达，每12h补加1次甲醇，诱导60h后离心收集菌体。用实施例1中步骤二的方法破菌并测定其血凝活性，挑取具有血凝活性的破菌液对应的克隆用实施例1中步骤三的方法进行工程酵母发酵，采用实施例1中步骤四的方法纯化获得H5N1高致病性禽流感血凝素糖蛋白多聚物纳米颗粒，将其记为(H5N1)HA(Hans)。
四、还原SDS-PAGE分析(H5N1)HA(Hans)分子量约为66KD(此处的分子量约为66KD的条带是H5N1高致病性禽流感血凝素糖蛋白多聚物纳米颗粒的HA0组分)。
用N-糖苷酶F(PNGF)处理H5N1高致病性禽流感血凝素糖蛋白多聚物纳米颗粒，方法同实施例1中的步骤五，分析糖基切除前后的分子量变化，发现用PNGF切除糖基后(H5N1)HA(Hans)(该H5N1高致病性禽流感血凝素糖蛋白多聚物纳米颗粒的HA0组分)的分子量下降为60kD，结果如图10所示。
对(H5N1)HA(Hans)进行分子排阻色谱(SEC)分析，方法同实施例1中的步骤八，结果如图11所示。图11中，上图为分子量标准蛋白的色谱图，下图为(H5N1)HA(Hans)的色谱图，发现(H5N1)HA(Hans)的保留时间小于670kD的分子量标准的保留时间，该蛋白多聚物纳米颗粒是分子量大于670kD的多聚体。
对(H5N1)HA(Hans)进行鸡红细胞血凝活性分析，方法同实施例1中的步骤七，结果如图12所示，图12中，第一排从左至右为从1：20开始2倍系列稀释的(H5N1)HA(Hans)，第二排为生理盐水对照，发现(H5N1)HA(Hans)具有明显的血凝活性。
用实施例2相同的方法制备(H5N1)HA(Hans)流感疫苗、免疫小鼠，并在加强免疫后一个星期采血得血清进行血凝抑制实验(方法见“郭元吉等《流行性感冒病毒及其实验技术》，北京，中国三峡出版社，1997”)，结果如图13所示，图13中，纵坐标为血凝抑制效价(HI)，横坐标为分组，0.0为对照组，7.5为实验组，发现实验组小鼠血清的平均血凝抑制效价达到1:240。
因此，汉逊酵母制备的H5N1高致病性禽流感血凝素糖蛋白多聚物纳米颗粒可以用于制备流感疫苗。
实施例4、毕赤酵母制备的H1N1流感血凝素糖蛋白多聚物纳米颗粒
一、重组表达载体的构建
(一)合成来源于H1N1流感病毒(A/FortMonmouth/1/47(H1N1))的SEQ ID No.11。
(二)设计并合成如下引物：
HA1-5：5’-ATCTTCGAAACGatgaaagcaaaactactgatc-3’(SEQ ID No.12)
下划线所示序列为NspV酶切识别位点。
HA1-3：5’-gatGCGGCCGCtcagatgcatattctgcattg-3’(SEQ ID No.13)
下划线所示序列为NotI酶切识别位点。
(三)以SEQ ID No.11为模板，以HA1-5和HA1-3为引物，利用Pyrobest DNA聚合酶进行PCR扩增，得到PCR扩增产物，如SEQ ID No.14所示，SEQ ID No.14中自5’末端起第8至第12位为Kozak序列，第13至第63位为信号肽序列，第64位至第1713位为HA基因，第1594至第1677位为C端穿膜区序列。
NspV和NotI双酶切SEQ ID No.14所示的DNA分子，得到基因片段；NspV和NotI双酶切pPICZα载体得到载体大片段；将基因片段与载体大片段连接，得到重组质粒，将其命名为pPICZα-HA1。将pPICZα-HA1测序，结果正确。
二、重组酵母的构建和筛选
将约10μg pPICZα-HA1质粒，用BglII线性化，并转化毕赤酵母菌X-33，最终获得具有血凝活性的破菌液对应的克隆(重组毕赤酵母单克隆)，用于H1N1流感血凝素糖蛋白多聚物纳米颗粒的制备，具体方法同实施例1中的步骤二。
三、工程酵母发酵同实施例1中的步骤三。
四、H1N1流感血凝素糖蛋白多聚物纳米颗粒的纯化同实施例1中的步骤四,将其记为(H1N1)HA。
五、还原SDS-PAGE分析((H1N1)HA分子量约为66KD(此处的分子量约为66KD的条带是H1N1流感血凝素糖蛋白多聚物纳米颗粒的HA0组分)。
用N-糖苷酶F(PNGF)处理H1N1流感病毒血凝素糖蛋白聚合物(H1N1)HA，方法同实施例1中的步骤五，分析糖基切除前后的分子量变化，发现用PNGF切除糖基后血 凝素糖蛋白(H1N1流感血凝素糖蛋白多聚物纳米颗粒的HA0组分)的分子量下降为61kD，结果如图14所示。
对H1N1流感血凝素糖蛋白多聚物纳米颗粒(H1N1)HA进行分子排阻色谱(SEC)分析，方法同实施例1中的步骤八，结果如图15所示。图15中，上图为分子量标准蛋白的色谱图，下图为(H1N1)HA的色谱图，发现(H1N1)HA的保留时间小于670kD的分子量标准的保留时间，该流感血凝素糖蛋白多聚物纳米颗粒是分子量大于670kD的多聚体。
对H1N1流感血凝素糖蛋白多聚物纳米颗粒(H1N1)HA进行鸡红细胞血凝活性分析，方法同实施例1中的步骤七，结果如图16所示，图16中，第一排从左至右为从1：20开始2倍系列稀释的(H1N1)HA，第二排为生理盐水对照，发现(H1N1)HA具有良好的血凝活性。
电镜照片显示(H1N1)HA形成20-50nm的多聚体颗粒。
用实施例2相同的方法制备H1N1流感血凝素糖蛋白多聚物纳米颗粒(H1N1)HA流感疫苗、免疫小鼠，并在加强免疫后一个星期采血得血清进行血凝抑制实验(方法见“郭元吉等《流行性感冒病毒及其实验技术》，北京，中国三峡出版社，1997”)，结果如图17所示，图17中，纵坐标为血凝抑制效价(HI)，横坐标为分组，0.0为对照组，7.5为实验组，发现实验组小鼠血清的平均血凝抑制效价达到1:320。
因此，毕赤酵母制备的H1N1流感血凝素糖蛋白多聚物纳米颗粒(H1N1)HA可以用于制备流感疫苗。
实施例5、乳酸克鲁维酵母制备H7N9流感血凝素糖蛋白多聚物纳米颗粒
一、重组表达载体的构建
(一)设计并合成如下引物：
HA7-HindIII-5：5’-ATCAAGCTTACGATGAACACCCAAATACTGGTTTTC-3’(SEQ ID No.15)
下划线所示序列为HindIII酶切识别位点。
HA7-3：5’-ATCGCGGCCGCTTAAATACAGATAGTACATCT-3’(SEQ ID No.16)。
下划线所示序列为NotI酶切识别位点。
(二)以SEQ ID No.1所示的DNA分子为模板，以HA7-3和HA7-HindIII-5为引物，进行PCR扩增，得到PCR扩增产物，该产物的序列如SEQ ID No.17所示，该分子自5’末端起依次包含Kozak序列，信号肽编码序列，HA基因(含有C端穿膜区序列)。
(三)HindIII和NotI双酶切SEQ ID No.17所示的DNA分子，得到基因片段；HindIII和NotI双酶切乳酸克鲁维酵母表达载体pKLAC1得到载体大片段；将基因片段与载体大片段连接，得到重组质粒，将其命名为pKLAC1-(H7N9)HA7。
二、重组酵母的构建和筛选
将约10μg pKLAC1-(H7N9)HA7质粒，用SacII线性化，用1/10体积的3M醋酸钠 和3倍体积的无水酒精沉淀线性化的质粒。用体积百分含量为70％的乙醇水溶液洗两次以除去其中的盐，晾干，约30μL水重悬沉淀，获得用于转化的pKLAC1-(H7N9)HA7线性化质粒。
制备乳酸克鲁维酵母电转化感受态细胞的方法同实施例1中的步骤二。
取80μL感受态细胞与10μL用于转化的pKLAC1-(H7N9)HA7线性化质粒，在微量离心管中混匀，得到混合物。将其置冰上5min，将混合物转移到一个冰冷的0.2cm电转杯中。电穿孔细胞(Bio-Rad Gene Pulser，1500V，25μF，200Ω)，再立即向电转杯中加入1mL冰冷的1M山梨醇，并小心地将混合物(转化细胞)转移至15mL培养管中。
将离心管放在28℃温箱孵育2h，不要摇动。取200μL转化细胞涂布于含有5mM乙酰胺的YCB平板(制备方法参见NEB公司提供的K.lactis Protein Expression Kit)上28℃培养3-4天直至单克隆出现。
随机挑取单克隆接种到2ml YPD液体培养基中，28℃培养48h，按体积比5％的接种量接种到YPGal液体培养基(酵母提取物10g/L，胰蛋白胨20g/L，半乳糖20g/L，余量为水)中，60h后离心收集菌体。每1ml菌液离心收获的菌体用100ulPBS重悬，制备破菌液的方法同实施例1中的步骤二，并用1％鸡红细胞进行血凝活性分析(方法见“郭元吉等《流行性感冒病毒及其实验技术》，北京，中国三峡出版社，1997)。挑取具有血凝活性的破菌液对应的克隆(重组毕赤酵母单克隆)用于H7N9流感病毒血凝素糖蛋白聚合物的制备。
三、工程酵母发酵
种子培养方法和发酵培养方法同实施例1中的步骤三，仅将诱导剂由半乳糖替代甲醇。
四、H7N9流感血凝素糖蛋白多聚物纳米颗粒的纯化方法同实施例1中的步骤四，将其记为(H7N9)HA(K.lactis)。
五、流感血凝素糖蛋白多聚物纳米颗粒的结构分析方法同实施例1中的步骤五。
还原SDS-PAGE分析(H7N9)HA(K.lactis)(HA0单体)分子量约为66KD，用PNGF切除糖基后分子量下降为61KD，结果如图18所示。
六、对H7N9流感血凝素糖蛋白多聚物纳米颗粒(H7N9)HA(K.lactis)进行分子排阻色谱(SEC)分析，方法同实施例1中的步骤八，结果如图19所示。图19中，上图为分子量标准蛋白的色谱图，下图为(H7N9)HA(K.lactis)的色谱图，发现(H7N9)HA(K.lactis)的保留时间小于670kD的分子量标准的保留时间，该流感血凝素糖蛋白多聚物纳米颗粒是分子量大于670kD的多聚体，由于HA0单体的分子量为64KD，三聚体的分子量约为180KD，说明(H7N9)HA(K.lactis)为含9个以上HA0单体形成的多聚体。
七、将制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒(H7N9)HA(K.lactis)负染后用50,000倍电子显微镜照相发现该流感血凝素为至少3个以上HA0三聚体形成的多聚体颗粒。
八、对H7N9流感血凝素糖蛋白多聚物纳米颗粒(H7N9)HA(K.lactis)进行鸡红细胞血凝活性分析，方法同实施例1中的步骤七，结果如图20所示，图20中，第一排从左至右为从1：20开始2倍系列稀释的(H7N9)HA(K.lactis)，第二排为生理盐水对照，发现(H7N9)HA(K.lactis)具有良好的血凝活性。
九、用重组乳酸克鲁维酵母制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒(H7N9)HA(K.lactis)制备流感疫苗
将(H7N9)HA(K.lactis)用pH7.4的PBS分别稀释到0μg、6μg、12μg/50μl三个不同的剂量组，同时，每组均添加等体积的1.2mg/mlAl(OH)3佐剂，制成流感疫苗，为实验组注射液。同时，设上述溶液中不加(H7N9)HA(K.lactis)的溶液为对照组注射液。
免疫方案、方式、采血时间和血凝抑制的标准血凝素的步骤同实施例2。
血清中中和抗体的血凝抑制活性结果如图21所示。
图21表明，对加强免疫后7天的血清进行血凝抑制活性分析，实验组小鼠的平均血凝抑制效价达到1:320。
因此，用重组乳酸克鲁维酵母制备的H7N9流感血凝素糖蛋白多聚物纳米颗粒(H7N9)HA(K.lactis)可以用于制备流感疫苗。
实施例6、毕赤酵母制备的H3N2流感血凝素糖蛋白多聚物纳米颗粒
一、重组表达载体的构建
(一)根据Genbank(A/reassortant/NYMC X-223A(Texas/50/2012 x PuertoRico/8/1934)(H3N2))(请确认该病毒的名称是准确的)上获得流感HA全长序列，合成SEQ ID No.18所示的DNA分子。
(二)设计并合成如下引物：
HA3-5：5’-ATCTTCGAAACGatgaagactatcattgcttt-3’(SEQ ID No.19)
下划线所示序列为NspV酶切识别位点。
HA3-3：5’-gatgcggccgctcaaatgcaaatgttgcacctaatgttgccctt-3’(SEQ ID No.20)
下划线所示序列为NotI酶切识别位点。
(三)以SEQ ID No.18所示的DNA分子为模板，以HA3-5和HA3-3为引物，利用Pyrobest DNA聚合酶进行PCR扩增，得到PCR扩增产物，如SEQ ID No.21所示，。
SEQ ID No.21所示的DNA分子从N端到C端依次含有Kozak序列、信号肽、HA基因序列(含有C端穿膜区序列)
SEQ ID No.21中自5’末端起第8位至第12位为Kozak序列、第13至第60位为信号肽序列，第61至第1713位为HA基因，第1600至第1674位为C端穿膜区序列。
NspV和NotI双酶切SEQ ID No.21所示的DNA分子，得到基因片段；NspV和NotI双酶切pPICZα载体得到载体大片段；将基因片段与载体大片段连接，得到重 组质粒，将其命名为pPICZα-HA3,将pPICZα-HA3测序，结果正确。
二、重组酵母的构建和筛选
将约10μg pPICZα-HA3质粒，用BglII线性化，并转化毕赤酵母菌X-33，最终获得具有血凝活性的破菌液对应的克隆(重组毕赤酵母单克隆)，用于H3N2流感血凝素糖蛋白多聚物纳米颗粒的制备，具体方法同实施例1中的步骤二。
三、工程酵母发酵同实施例1中的步骤三。
四、H3N2流感血凝素糖蛋白多聚物纳米颗粒的纯化同实施例1中的步骤四,将其记为(H3N2)HA。
五、还原SDS-PAGE分析((H3N2)HA分子量约为66KD，该条带是H3N2流感血凝素糖蛋白多聚物纳米颗粒的HA0组分。
用N-糖苷酶F(PNGF)处理(H3N2)HA，方法同实施例1中的步骤五，分析糖基切除前后的分子量变化，发现用PNGF切除糖基后血凝素糖蛋白HA0组分的分子量下降为61kD，结果如图22所示。
六、对H3N2流感血凝素糖蛋白多聚物纳米颗粒(H3N2)HA进行分子排阻色谱(SEC)分析，方法同实施例1中的步骤八，结果显示H3N2流感血凝素糖蛋白多聚物纳米颗粒(H3N2)HA是分子量大于670kD的多聚体。
七、对H3N2流感血凝素糖蛋白多聚物纳米颗粒(H3N2)HA进行鸡红细胞血凝活性分析，方法同实施例1中的步骤七，结果如图23所示，图23中，第一排从左至右为从1：20开始2倍系列稀释的((H3N2)HA，第二排为生理盐水对照，发现(H3N2)HA具有良好的血凝活性。
八、用实施例2相同的方法制备H3N2流感血凝素糖蛋白多聚物纳米颗粒(H3N2)HA流感疫苗、免疫小鼠，并在加强免疫后一个星期采血得血清进行血凝抑制(方法见“郭元吉等《流行性感冒病毒及其实验技术》，北京，中国三峡出版社，1997”)，结果如图24所示。
图24中，纵坐标为血凝抑制效价(HI)，横坐标为分组，0.0为对照组，7.5为实验组。图24表明，实验组小鼠血清的平均血凝抑制效价达到1:320。
因此，毕赤酵母制备的H3N2流感血凝素糖蛋白多聚物纳米颗粒(H3N2)HA可以用于制备流感疫苗。
实施例7、流感血凝素糖蛋白多聚物纳米颗粒阳离子交换层析
将实施例1中步骤四的(一)得到的上样液只进行步骤(二)的阳离子色谱柱纯化，得到15％C液洗脱峰，将该组分进行血凝活性检测，方法同实施例1中步骤七，结果如图25所示。
图25中，第一排从左至右为从1：20开始2倍系列稀释的15％C液洗脱峰，第二排为生理盐水对照。图25表明，该组分中含有流感血凝素糖蛋白多聚物纳米颗粒，且具有血凝活性。
实施例8、流感病毒血凝素蛋白聚合物纳米颗粒阴离子交换层析
将实施例1中步骤四的(一)得到的上样液直接进行步骤(三)阴离子交换层析，得到10％C液洗脱峰，将其进行血凝活性检测，方法同实施例1中步骤七，结果如图26所示。
图26中，第一排从左至右为从1：20开始2倍系列稀释的10％C液洗脱峰，第二排为生理盐水对照。图26表明，该组分中含有流感血凝素糖蛋白多聚物纳米颗粒，且具有血凝活性。
实施例9、流感病毒血凝素蛋白聚合物纳米颗粒凝胶排阻层析
将实施例1中步骤四的(一)得到的上样液只进行步骤(四)的凝胶排阻色谱层析，收集第一个洗脱主峰，对其进行血凝素活性检测，方法同实施例1中步骤七，结果如图27所示。
图27中，第一排从左至右为从1：20开始2倍系列稀释的洗脱主峰，第二排为生理盐水对照。图27表明，该组分中含有流感血凝素糖蛋白多聚物纳米颗粒，且具有血凝活性。
实施例10、ELISA方法测定不同去污剂溶解后收集上清中的流感病毒血凝素蛋白聚合物纳米颗粒含量
实施例1中步骤三得到发酵液之后，将发酵液于4℃，7000rpm离心20min。用水重悬为40g/100ml的悬液，高压匀浆仪破菌(1200bar，破菌3次),得匀浆液，将酵母500ml匀浆液中加50g PEG2000，搅拌溶解0.5h,7000rpm离心20min，收集沉淀，弃上清；沉淀加500ml体积的溶液(该溶液含10mM Tris-HCl，去污剂(曲拉通、吐温、乙基苯基聚乙二醇或较弱的离子型去污剂脱氧胆酸盐、CHAPS(3-[(3-胆酰胺基丙基)二甲基铵基]-1-丙磺酸盐)),5g/100ml甘油，余量为水)重悬，搅拌溶解2h。超速离心2h，收集上清，用磷酸盐调节pH为6.0，取各个样品做ELISA分析。
ELISA分析方法测定收集上清中的血凝素含量(方法参照汪家政、范明主编《蛋白质技术手册》(科学出版社，2000年))：样品用抗原包被液(0.15g/100ml Na2CO3，0.293g/100ml NaHCO3，pH9.6，余量为水)稀释100倍，然后各取100μL样品加入酶联板中，4℃包被过夜，一抗为兔抗HA7(1:500)(用PBS稀释)(该抗体购自北京博菲康生物技术有限公司)，二抗为羊抗兔IgG-HRP(1:1000)(用PBS稀释)(该抗体购自华美生物工程有限公司)，每孔各加100μL显色液(1.84g/100ml Na2HPO4·12H2O，0.5g/100ml柠檬酸，临用前再加0.04g/100ml OPD和体积百分含量0.15％的H2O2)，37℃避光温育15min，最后每孔加20μL终止液(2M H2SO4)以终止显色，其中阳性克隆孔内呈现一定的颜色变化，反映出HA的含量，空白(以无血凝素样品的孔为空白)及阴性孔内没有明显的颜色变化。于492nm处酶联仪上读取数据，结果如表1所示。
表1表明，各种去污剂均有效果，能检测到上清中的流感病毒血凝素蛋白聚合物纳米颗粒，其中2g/100ml TritonX-100(曲拉通)效果最好，其次5g/100mlTween20(吐温)和2g/100ml脱氧胆酸盐，再次是2g/100ml NP-40(乙基苯基聚乙二醇)和2g/100ml CHAPS，但不用去污剂处理，几乎检测不到流感病毒血凝素蛋白聚合物纳米颗粒，说明去污剂处理是本发明中重要步骤之一。
表1ELISA分析方法测定不同去污剂溶解后收集上清中的流感病毒血凝素蛋白聚合物纳米颗粒含量
Figure PCTCN2015088748-appb-000006
工业应用
利用本发明的方法制备的流感血凝素糖蛋白多聚物纳米颗粒的分子量大于670KD，单体(HA0)为分子量为60KD左右的糖蛋白，并且由其制备的疫苗可以诱导产生高效价的中和抗体。本发明首次公开了用酵母制备流感血凝素糖蛋白多聚物纳米颗粒的方法，该纳米颗粒未融合外源的载体蛋白，不存在反复接种引起对载体蛋白免疫反应的问题，且本发明提供的重组酵母菌具有构建周期短、生长快、易于大规模生产、安全性高等特点，使其非常适合于在流感等突发传染病和其它应急条件下，进行疫苗高效研发和大规模生产。

Claims (27)

  1. 一种制备流感病毒血凝素糖蛋白聚合物纳米颗粒的方法，包括如下步骤：使N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因在酵母中表达；将所述酵母进行细胞破碎，加入去污剂，获得含流感病毒血凝素糖蛋白的溶液；将所述溶液进行纯化，制备得到具有血凝活性的流感病毒血凝素糖蛋白聚合物纳米颗粒。
  2. 根据权利要求1所述的方法，其特征在于：所述使N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因在酵母中表达的方法为：将含有N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因的重组表达载体转化酵母，培养得到转化的酵母，再诱导使基因表达。
  3. 根据权利要求1所述的方法，其特征在于：所述酵母为毕赤酵母、汉逊酵母或乳酸克鲁维酵母。
  4. 根据权利要求1所述的方法，其特征在于：所述流感病毒的血凝素HA为H1、H3、H5或H7血清型流感病毒的HA。
  5. 根据权利要求4所述的方法，其特征在于：所述H1、H3、H5或H7血清型流感病毒的HA分别为H1N1、H3N2、H5N1或H7N9流感病毒的HA。
  6. 根据权利要求2所述的方法，其特征在于：所述重组表达载体是将含有所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因插入含有AOX启动子的载体得到的。
  7. 根据权利要求6所述的方法，其特征在于：所述重组表达载体是将含有所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因插入pPICZα的NotI和NspV酶切位点间，并用BglII线性化得到的。
  8. 根据权利要求7所述的方法，其特征在于：所述流感病毒为H7N9禽流感病毒，所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因如SEQ ID No.4所示。
  9. 根据权利要求7所述的方法，其特征在于：所述流感病毒为甲型H1N1流感病毒，所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因如SEQ ID No.14所示。
  10. 根据权利要求7所述的方法，其特征在于：所述流感病毒为甲型H3N2流感病毒，所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因如SEQ ID No.21所示。
  11. 根据权利要求2所述的方法，其特征在于：所述重组表达载体是将含有所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因插入含有MOX启动子的载体得到的。
  12. 根据权利要求11所述的方法，其特征在于：所述重组表达载体是将含有 所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因插入pPICZα的NotI和BstB I酶切位点间，得到中间载体1；再将中间载体1的AOX启动子替换为汉逊酵母的醇氧化酶启动子MOX，再用BglII线性化得到的。
  13. 根据权利要求12所述的方法，其特征在于：所述汉逊酵母为多型汉逊酵母。
  14. 根据权利要求13所述的方法，其特征在于：所述MOX启动子是以多型汉逊酵母的基因组DNA为模板，以SEQ ID No.9和SEQ ID No.10所示的DNA分子为引物进行PCR扩增得到MOX启动子。
  15. 根据权利要求12所述的方法，其特征在于：所述将中间载体1的AOX启动子替换为汉逊酵母的醇氧化酶启动子MOX的方法为将所述PCR扩增得到的MOX启动子进行BglII酶切，并进行磷酸化，获得5’端为BglII酶切粘性末端，3’端磷酸化的MOX启动子；将中间载体1用NspV单酶切后用Klenow fragment大片段酶和dNTP补平，得到片段，回收片段后，再用BglII酶切，切除AOX启动子，得到切除AOX启动子的中间载体1；将5’端为BglII酶切粘性末端，3’端磷酸化的MOX启动子与切除AOX启动子的中间载体1连接。
  16. 根据权利要求12所述的方法，其特征在于：所述流感病毒为H5N1禽流感病毒，所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因具体如SEQ ID No.8所示。
  17. 根据权利要求2所述的方法，其特征在于：所述重组表达载体是将所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因插入含有LAC4启动子的载体得到的。
  18. 根据权利要求17所述的方法，其特征在于：所述重组表达载体是将所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因插入pKLAC1的HindIII和NotI酶切位点间，再用SacII线性化得到的。
  19. 根据权利要求18所述的方法，其特征在于：所述流感病毒为H7N9禽流感病毒，所述N端上游含有信号肽序列、并包含C-端穿膜区序列的流感病毒的血凝素HA的编码基因如SEQ ID No.17所示。
  20. 根据权利要求1所述的方法，其特征在于：所述细胞破碎的方法为物理方法、生物方法或化学方法。
  21. 根据权利要求20所述的方法，其特征在于：
    所述物理方法为玻璃珠振荡法、高压匀浆法或球磨法；
    所述生物方法为酶裂解法；
    所述化学方法为碱裂解法。
  22. 根据权利要求1所述的方法，其特征在于：所述去污剂为非离子型去污剂或弱离子型去污剂。
  23. 根据权利要求22所述的方法，其特征在于：
    所述非离子型去污剂为曲拉通、吐温或乙基苯基聚乙二醇；
    所述弱离子型去污剂为脱氧胆酸盐或3-[(3-胆酰胺基丙基)二甲基铵基]-1-丙磺酸盐。
  24. 根据权利要求1所述的方法，其特征在于：所述将所述溶液进行纯化的方法包括阳离子交换层析和/或阴离子交换层析和/或凝胶排阻层析。
  25. 根据权利要求24所述的方法，其特征在于：
    所述阳离子交换层析的填料为Sepharose FF SP；
    所述阴离子交换层析的填料为Source 30Q；
    所述凝胶排阻层析的填料为Superdex200；
    所述纯化过程中根据各步骤纯化组分是否有HA条带和血凝活性判断其是否为含有所述流感病毒血凝素糖蛋白聚合物纳米颗粒的组分，如果该组分有HA条带和血凝活性就为含有所述流感病毒血凝素糖蛋白聚合物纳米颗粒的组分。
  26. 由权利要求1所述方法制备得到的流感病毒血凝素糖蛋白聚合物纳米颗粒制备的流感疫苗。
  27. 根据权利要求26所述的流感疫苗，其特征在于：所述疫苗还包含佐剂；所述佐剂具体为铝佐剂，再具体为氢氧化铝佐剂。
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