CN105233302B - A kind of preparation method of influenza hemagglutinin glycopolypeptide multimer object nano particle - Google Patents

A kind of preparation method of influenza hemagglutinin glycopolypeptide multimer object nano particle Download PDF

Info

Publication number
CN105233302B
CN105233302B CN201410328217.5A CN201410328217A CN105233302B CN 105233302 B CN105233302 B CN 105233302B CN 201410328217 A CN201410328217 A CN 201410328217A CN 105233302 B CN105233302 B CN 105233302B
Authority
CN
China
Prior art keywords
influenza virus
hemagglutinin
yeast
influenza
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201410328217.5A
Other languages
Chinese (zh)
Other versions
CN105233302A (en
Inventor
吴军
刘波
唱韶红
巩新
王莎
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Bioengineering Chinese Academy of Military Medical Sciences
Original Assignee
Institute of Bioengineering Chinese Academy of Military Medical Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Bioengineering Chinese Academy of Military Medical Sciences filed Critical Institute of Bioengineering Chinese Academy of Military Medical Sciences
Priority to CN201410328217.5A priority Critical patent/CN105233302B/en
Priority to PCT/CN2015/088748 priority patent/WO2016004904A2/en
Publication of CN105233302A publication Critical patent/CN105233302A/en
Application granted granted Critical
Publication of CN105233302B publication Critical patent/CN105233302B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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

Abstract

The invention discloses a kind of preparation methods of influenza virus hemagglutinin glycoprotein polymer nano granules, and the complete influenza virus hemagglutinin glycoprotein polymer of space structure can be prepared using this method.The present invention discloses a kind of method preparing influenza virus hemagglutinin glycoprotein polymer nano granules, includes the following steps：So that N-terminal upstream is contained signal peptide sequence and includes that the hemagglutinin gene of the influenza virus of the ends C transmembrane domains sequence is expressed in yeast；The yeast is subjected to clasmatosis, detergent is added, obtains the solution of the glycoprotein containing influenza virus hemagglutinin；The solution is purified, the influenza virus hemagglutinin glycoprotein polymer nano granules with hemagglutination activity are prepared.Preparing influenza virus hemagglutinin glycoprotein polymer nano granules with recombination yeast also has the characteristics that engineered strain construction schedule is short, growth is fast, is easy to large-scale production, safe.

Description

A kind of preparation method of influenza hemagglutinin glycopolypeptide multimer object nano particle
Technical field
The present invention relates to a kind of preparation methods of influenza hemagglutinin glycopolypeptide multimer object nano particle, belong to biotechnology neck Domain.
Background technology
Influenza abbreviation influenza, be by first, second, the third three type influenza viruses respectively caused by acute respiration road transmission Disease.Influenza vaccinations are the important means of flu-prevention.Influenza hemagglutinin is one of major protein of influenza surface, with Trimeric form exists, and is the important component that influenza surface participates in host cell absorption and invasion, the neutrality induced Antibody therefore can be the main component of influenza vaccines with blocking virus in the absorption and invasion of host cell surface.Influenza blood clotting Element variation re-matches fast, and various new type influenzas take place frequently, the H5N1 highly pathogenic bird flus that such as break out in recent years, H1N1 Flu-As, H7N9 highly pathogenic bird flus etc. cause serious problem.After new Epidemic outbreak of disease, the quick research and development and production of vaccine are epidemic diseases The key of feelings control.
Existing influenza vaccines are mainly inactivated virus vaccine, split vaccine or the Asia list for purifying acquisition on this basis Position vaccine, these vaccines are all to obtain virus by the chick embryo culture of attenuated influenza virus strain or reassortant, through viral purification, are gone out It lives or further cracks, purify preparation.Main problem existing for chicken embryo influenza vaccines production technology is that virus production is limited to The supply of qualified chicken embryo, re-matching, attenuation and the chicken embryo of virus stain meet the needs of the plenty of time, and exist uncertain.It prepares Vaccine in addition to the influenza hemagglutinin as principle active component, also contain virulent other albumen and chicken embryo derived Protein.Epidemic disease The purity of protein of seedling is low, easily causes the side reactions such as allergy.And some hypotypes virus after the inactivation of inactivator formaldehyde etc., antigen Property may change, and cause vaccine fractions invalid.Mammalian cell substitutes the technology of chick embryo culture influenza virus in recent years Development is obtained, but the technology solves only the problem of chicken embryo supply is limited with GMP productions, above-mentioned other problems still remain.And And it is also limited to the ability of cell large-scale production.By technique for gene engineering, highly purified influenza is recombinantly expressed and prepared Hemagglutinin is expected to provide new way for the solution of these problems.
The name of influenza virus hemagglutinin HA albumen is from virion by HA albumen and specificity containing sialic acid Receptor combines, and is aggregated erythrocyte.It is that matter net synthesis in the cell contains 562~566 first after transcribing, translating that it, which is synthesized, The HA amyloid protein precursors (HA0) of amino acid, i.e. hemagglutinin precursor；Influenza Virus RNA coding hemagglutinin (HA) maturation protein containing about 550 amino acid residues, including heavy chain (HA1) and light chain (HA2) two parts, the basic amino acid site among the two is in maturation Virion to extracellular bud give birth to discharge when, by cells specific protein enzyme hydrolysis cut, become by disulfide bond be connected HA1 and HA2.And the influenza hemagglutinin before being cut without protease is otherwise known as influenza hemagglutinin precursor (HA0).This species specificity is cut Necessary but unrelated with the combination of influenza hemagglutinin and receptor, the differential protein digestion that is that influenza virus is merged with host cell membrane It cuts front and back influenza hemagglutinin antigenicity all having the same and receptor-binding activity, HA0 molecules is hydrolyzed to HA1 and HA2, be disease The infective prerequisite of poison.For convenience of narration, unless otherwise specified, influenza hemagglutinin or HA of the present invention include influenza The HA1 and HA2 that the disulfide bond formed after hemagglutinin precursor (HA0) and differential protein cleavage is connected.
The molecular weight of influenza hemagglutinin HA monomers is about 60KD, and the HA of influenza surface is with tripolymer (HA-trimer) Form forms furcella, and this Trimeric structures are necessary to it is combined with sialic acid receptor.Some animals such as chicken, cavy etc. it is red Cell surface has the sialylated glycosyl that can be combined with influenza virus HA tripolymers.Multiple HA furcellas of virus surface from it is different Multiple saliva acidizing sugar base junctions of erythrocyte surface are closed, multiple sialylated glycosyls of erythrocyte surface again with multiple virus surfaces HA furcellas combine, virus and the mutual crosslinking of red blood cell can be formed, form blood clotting phenomenon.Due to influenza infection, host is thin Process of sticking when born of the same parents is also to be implemented in combination with by the saliva acidizing sugar base of influenza virus HA tripolymers and host cell surface. Therefore hemagglutination activity is the important method for examining HA receptor-binding activities, and blood clotting Inhibition test is whether research antibody has blocking The important method for the neutralization activity that influenza virus HA tripolymers are combined with receptor.The weight that different expression systems and preparation method obtain There are significantly different in structure, glycosylation, the ability of induction neutralizing antibody etc. by group HA.(the Virology such as Athmaram, TN Journal2011,8:524；Virus Genes201245：440-451；J Ind Microbiol Biotechnol2013, 40:The HA0 for obtaining 2009 novel H1N1 influenzas 245-255) is expressed and purified with yeast successful secretion, but the HA0 prepared is used Mainly exist with monomer and a small amount of trimeric form when FPLC is purified, mouse is only immunized twice with the dosage of 50 μ g/ only with 10 μ g/ Blood clotting inhibitory activity is only 1 afterwards:32.Have no that yeast prepares the report of influenza hemagglutinin glycopolypeptide multimer object nano particle.
Invention content
The invention discloses a kind of preparation methods of influenza hemagglutinin glycopolypeptide multimer object nano particle.
A method of influenza virus hemagglutinin glycoprotein polymer nano granules are prepared, are included the following steps：Make on N-terminal It swims containing signal peptide sequence and includes that the hemagglutinin gene of influenza virus of the ends C- transmembrane domains sequence is expressed in yeast；It will The yeast carries out clasmatosis, and detergent is added, and obtains the solution of the glycoprotein containing influenza virus hemagglutinin；By the solution into Row purifying, is prepared the influenza virus hemagglutinin glycoprotein polymer nano granules with hemagglutination activity；
Contain signal peptide sequence and include the hemagglutinin HA bases of the influenza virus of the ends C- transmembrane domains sequence in the N-terminal upstream Because also containing Kozak sequences before signal peptide sequence, Kozak sequences are 5 '-aaacg-3 '；
It is specific between addition detergent and the clasmatosis also to contain the step of centrifuging to precipitate；
Specifically also contain between the solution that detergent is added and obtains the glycoprotein containing influenza virus hemagglutinin and centrifuges The step of supernatant；
The molecular weight of the influenza virus hemagglutinin glycoprotein polymer nano granules is more than 670KD；
Rose is presented under Electronic Speculum in the influenza virus hemagglutinin glycoprotein polymer nano granules with hemagglutination activity Kranz structure, it was demonstrated that it is at least formed by 3 or more tripolymers, what 3 influenza virus hemagglutinin amyloid protein precursor HA0 were formed HA0 trimeric molecules amounts are about 180KD, thus prove influenza virus hemagglutinin glycoprotein polymer nano granules at least by 9 The above influenza virus hemagglutinin amyloid protein precursor HA0 participates in forming polymer nano granules, wherein before influenza virus hemagglutinin albumen Body HA0 forms HA0 tripolymers.
In the above method, the N-terminal upstream contains signal peptide sequence and includes the influenza virus of the ends C- transmembrane domains sequence The method that hemagglutinin gene is expressed in yeast is that signal peptide sequence is contained in N-terminal upstream and includes the ends C- transmembrane domains sequence Influenza virus hemagglutinin gene recombinant expression carrier transformed yeast, cultivate the yeast that is converted, then induce and make base Because of expression；
The yeast is Pichia pastoris, Hansenula yeast or Kluyveromyces lactis；
The step of induction makes gene expression specially cultivates the yeast of the conversion, and the table of induced gene It reaches；
The culture specially shaking flask culture or fermentation tank culture, not according to the promoter of the control HA gene expressions Together, the yeast for first cultivating the conversion may be used to certain density, then the method for inducing HA gene expressions, can also uses The method for inducing HA gene expressions while the yeast of the conversion is cultivated, wherein former approach, which is more advantageous to, improves yeast work The stability of journey bacterium；
The signal peptide is the signal peptide of the HA genes itself or other other letters that can be worked in corresponding yeast Number peptide；
Other other signals peptides that can be worked in corresponding yeast are saccharomyces cerevisiae α mating factors signal peptide, the shallow lakes α The signal peptide of powder enzyme signal peptide or albumin；
The signal peptide of the albumin is specially sero-abluminous signal peptide.
In any of the above-described method, the hemagglutinin HA of the influenza virus is H1, H3, H5 or H7 serotype influenza disease The HA of poison.
In any of the above-described method, the HA of H1, H3, H5 or H7 serotype influenza virus be respectively H1N1, The HA of H3N2, H5N1 or H7N9 influenza virus.
In any of the above-described method, the recombinant expression carrier is that signal peptide sequence, simultaneously is contained in the N-terminal upstream It is obtained including the hemagglutinin gene of the influenza virus of the ends C- transmembrane domains sequence is inserted into the carrier containing AOX promoters, specifically The hemagglutinin gene of the influenza virus containing signal peptide sequence and the ends C- transmembrane domains sequence is inserted into the NotI of pPICZ α Between NspV restriction enzyme sites, and BglII linearizes to obtain；
The yeast is Pichia pastoris；
The influenza virus is specially H7N9 avian influenza virus, and the N-terminal upstream contains signal peptide sequence and includes the ends C- The hemagglutinin gene of the influenza virus of transmembrane domains sequence is specific as shown in SEQ ID No.4；
The H7N9 avian influenza virus is A/Hongzhou/1/2013 (H7N9)；
Or,
The influenza virus is specially H1N1virus, and the N-terminal upstream contains signal peptide sequence and includes C- Hold the hemagglutinin gene of the influenza virus of transmembrane domains sequence specific as shown in SEQ ID No.14；
The H1N1virus is specially A/FortMonmouth/1/47 (H1N1)；
Or,
The influenza virus is specially A type H3N2 influenza viruses, and the N-terminal upstream contains signal peptide sequence and includes C- Hold the hemagglutinin gene of the influenza virus of transmembrane domains sequence specific as shown in SEQ ID No.21；
The A type H3N2 influenza viruses are specially A/reassortant/NYMC X-223A (Texas/50/2012x PuertoRico/8/1934)(H3N2)。
In any of the above-described method, the recombinant expression carrier is that signal peptide sequence, simultaneously is contained in the N-terminal upstream It is obtained including the hemagglutinin gene of the influenza virus of the ends C- transmembrane domains sequence is inserted into the carrier containing MOX promoters, specifically Signal peptide sequence is contained into the N-terminal upstream and includes the hemagglutinin gene insertion of the influenza virus of the ends C- transmembrane domains sequence Between NotI the and BstB I restriction enzyme sites of pPICZ α, intermediate carrier 1 is obtained；The AOX promoters of intermediate carrier 1 are replaced with into the Chinese again The alcohol oxidase promoter MOX of inferior yeast, then linearize to obtain with BglII；
The yeast is Hansenula yeast, specially Hansenula polymorpha；
The MOX promoters are using the genomic DNA of Hansenula polymorpha as template, with SEQ ID No.9 and SEQ ID DNA molecular shown in No.10 is that primer progress PCR amplification obtains MOX promoters；
The method of the alcohol oxidase promoter MOX that the AOX promoters of intermediate carrier 1 are replaced with to Hansenula yeast is specific MOX promoters to obtain the PCR amplification carry out BglII digestions, and carry out phosphorylation, and it is BglII digestions to obtain 5 ' ends Cohesive end, the MOX promoters of 3 ' end phosphorylations；It will be large stretch of with Klenow fragment after the NspV single endonuclease digestions of intermediate carrier 1 Section enzyme and dNTP filling-in, obtains segment, after recycling segment, then uses BglII digestions, cuts off AOX promoters, obtains cutting off AOX and open The intermediate carrier 1 of mover；It is BglII digestion cohesive ends by 5 ' ends, MOX promoters and the excision AOX of 3 ' end phosphorylations start The intermediate carrier 1 of son connects；
The influenza virus is specially H5N1 avian influenza virus, and the N-terminal upstream contains signal peptide sequence and includes the ends C- The hemagglutinin gene of the influenza virus of transmembrane domains sequence is specific as shown in SEQ ID No.8；
The H5N1 avian influenza virus is specially A/duck/Guangxi/27/2003 (H5N1).
In any of the above-described method, the recombinant expression carrier is that signal peptide sequence, simultaneously is contained in the N-terminal upstream It is obtained including the hemagglutinin gene of the influenza virus of the ends C- transmembrane domains sequence is inserted into the carrier containing LAC4 promoters, specifically Signal peptide sequence is contained into the N-terminal upstream and includes the hemagglutinin gene insertion of the influenza virus of the ends C- transmembrane domains sequence Between HindIII the and NotI restriction enzyme sites of pKLAC1, then with SacII linearize to obtain；
The yeast is Kluyveromyces lactis；
The influenza virus is specially H7N9 avian influenza virus, and the N-terminal upstream contains signal peptide sequence and includes the ends C- The hemagglutinin gene of the influenza virus of transmembrane domains sequence is specific as shown in SEQ ID No.17.
The H7N9 avian influenza virus is specially A/Hongzhou/1/2013 (H7N9).
In any of the above-described method, the method for the clasmatosis is physical method, biological method or chemical method；
The physical method is specially bead succusion, high-pressure homogenization or ball-milling method；
The biological method is specially enzymatic lysis method；
The chemical method is specially alkaline lysis；
The detergent is non-ionic detergent or weak ionic detergent；
The non-ionic detergent is specially logical Qula, tween or Nonidet P40；
The weak ionic detergent is specially dexycholate or 3- [(3- courages amidopropyl) dimethylammonio] -1- Propane sulfonic acid salt；
The logical Qula is specially TritonX-100；
The tween is specially Tween20；
The Nonidet P40 is specially NP-40；
It is located on the cell membrane of the yeast after the influenza virus hemagglutinin glycoprotein polymer nano granules expression；
The effect of the detergent is that the influenza virus hemagglutinin glycoprotein polymerization is dissolved from the yeast cells film Object nano particle simultaneously preferably keeps its structure.
In any of the above-described method, the method that the solution is purified includes cation-exchange chromatography And/or anion-exchange chromatography and/or gel exclusion chromatography.
The filler of the cation-exchange chromatography is specially Sepharose FF SP；
The filler of the anion-exchange chromatography is specially Source30Q；
The filler of the gel exclusion chromatography is specially Superdex200；
In the purification process according to each step purified components whether have HA bands and hemagglutination activity judge its whether be containing The component for having the influenza virus hemagglutinin glycoprotein polymer nano granules, if the component has HA bands and hemagglutination activity For the component containing the influenza virus hemagglutinin glycoprotein polymer nano granules.
Non- influenza virus hemagglutinin in the influenza virus hemagglutinin glycoprotein polymer nano granules obtained after purification Protein content≤20%, specially≤10%；
The HA is connected comprising the disulfide bond formed after influenza virus hemagglutinin precursor HA0 and differential protein cleavage HA1 and HA2.
It is prepared by the influenza virus hemagglutinin glycoprotein polymer nano granules that any of the above-described method is prepared Influenza vaccines also belong to protection scope of the present invention；
The vaccine also includes specifically adjuvant；
The adjuvant is specially aluminium adjuvant, then specially aluminum hydroxide adjuvant.
The molecular weight of the influenza hemagglutinin glycopolypeptide multimer object nano particle prepared using the method for the present invention is more than 670KD, monomer (HA0) is the glycoprotein that molecular weight is 60KD or so, and vaccine prepared therefrom can induce generation efficient The neutralizing antibody of valence.Present invention firstly discloses the method for preparing influenza hemagglutinin glycopolypeptide multimer object nano particle with yeast, Not the problem of nano particle does not merge the carrier protein of external source, and there is no inoculation is caused to carrier protein immune response repeatedly, and Recombinant yeast provided by the invention has the characteristics that construction schedule is short, growth is fast, is easy to large-scale production, is safe, makes It is very suitable for happening suddenly under infectious disease and other emergency conditions in influenza etc., carries out vaccine efficiently research and development and large-scale production.
Description of the drawings
Fig. 1 is that the cation of H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by recombinant yeast pichia pastoris exchanges The testing result of chromatography.
Fig. 2 is the anion exchange of H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by recombinant yeast pichia pastoris The testing result of chromatography.
Fig. 3 is the gel exclusion color of H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by recombinant yeast pichia pastoris Compose the testing result of chromatography.
Fig. 4 is the glycosylation inspection of H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by recombinant yeast pichia pastoris It surveys.
Fig. 5 is the TPCK processing of H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by recombinant yeast pichia pastoris Tryptic digestion result.
Fig. 6 is the hemagglutination activity inspection of H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by recombinant yeast pichia pastoris It surveys.
Fig. 7 is the molecular exclusion color of H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by recombinant yeast pichia pastoris Spectrum analysis.
Fig. 8 is the electron microscope of H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by recombinant yeast pichia pastoris Photograph.
Fig. 9 is the influenza prepared with H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by recombinant yeast pichia pastoris Vaccine effect is evaluated.
Figure 10 is the H5N1 highly pathogenic bird flu hemagglutinin glycopolypeptide multimer object nano particles for recombinating Hansenula yeast and preparing Glycosylation detection.
Figure 11 is the H5N1 highly pathogenic bird flu hemagglutinin glycopolypeptide multimer object nano particles for recombinating Hansenula yeast and preparing Molecular-exclusion chromatography analysis.
Figure 12 is the H5N1 highly pathogenic bird flu hemagglutinin glycopolypeptide multimer object nano particles for recombinating Hansenula yeast and preparing Hemagglutination activity detection.
Figure 13 is the stream for recombinating H5N1 highly pathogenic avian influenza virus hemagglutinin glycoprotein polymer prepared by Hansenula yeast Influenza vaccine effect assessment.
Figure 14 is the glycosylation inspection of H1N1 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by recombinant yeast pichia pastoris It surveys.
Figure 15 is the molecular exclusion of H1N1 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by recombinant yeast pichia pastoris Chromatography.
Figure 16 is the hemagglutination activity of H1N1 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by recombinant yeast pichia pastoris Detection.
Figure 17 is the stream that the H1N1 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared with recombinant yeast pichia pastoris obtain Influenza vaccine effect assessment.
Figure 18 is the sugar for recombinating H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by Kluyveromyces lactis Baseization detects.
Figure 19 is point for recombinating H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by Kluyveromyces lactis Sub- exclusion chromatographic analysis.
Figure 20 is the blood for recombinating H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by Kluyveromyces lactis Solidifying Activity determination.
Figure 21 is that the H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared with recombination lactic acid kluyveromyces obtain The influenza vaccine efficacy evaluation arrived.
Figure 22 is the glycosylation inspection of H3N2 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by recombinant yeast pichia pastoris It surveys.
Figure 23 is the hemagglutination activity of H3N2 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by recombinant yeast pichia pastoris Detection.
Figure 24 is the stream that the H3N2 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared with recombinant yeast pichia pastoris obtain Influenza vaccine effect assessment.
Figure 25 is the hemagglutination activity detection in embodiment 7.
Figure 26 is the hemagglutination activity detection in embodiment 8.
Figure 27 is the hemagglutination activity detection in embodiment 9.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
For convenience of narration, unless otherwise specified, influenza hemagglutinin or HA of the present invention include influenza hemagglutinin precursor (HA0) HA1 and HA2 being connected with the disulfide bond formed after differential protein cleavage.
PPICZ α carriers are purchased from Invitrogen companies.
Pichia pastoris X-33 is purchased from Invitrogen companies.
TPCK handles trypsase (TPCK-Trypsin) and is purchased from Sigma companies.
H7N9 influenzas re-match vaccine strain (NIBRG-268), and in document, " Yang Juan, Zheng Yaming, Feng Luzhao, remaining macro outstanding people use It is disclosed in H7N9 avian influenza vaccine Recent Progresses In The Developments, Chinese Journal of Preventive Medicine, 2014,48 (2) ", the public can be from Chinese people Biological Engineering Inst., Academy of Military Medicine Scineces, CPLA obtains.
Pyrobest archaeal dna polymerases are purchased from precious bioengineering (Dalian) Co., Ltd.
PKLAC1 is purchased from NEB.
Kluyveromyces lactis is purchased from NEB.
H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by embodiment 1, recombinant yeast pichia pastoris
One, the structure of recombinant expression carrier
(1) according to the influenza HA full length amino acid of Genbank (KC853766) (A/Hongzhou/1/2013 (H7N9)) Sequence synthesizes DNA shown in SEQ ID No.1 according to yeast biased codons and gene high expression principle Optimized Coding Based gene Molecule.
(2) following primer is designed and synthesized：
HA7-3：5’-ATCGCGGCCGCTTAAATACAGATAGTACATCTCAT-3’(SEQ ID No.2)
Sequence shown in underscore is NotI digestion recognition sites.
HA7-5：5’-ATCTTCGAAACGATGAACACCCAAATACTGGTTTTC-3’(SEQ ID No.3)
Sequence shown in underscore is NspV digestion recognition sites.
(3) using DNA molecular is template shown in SEQ ID No.1, using HA7-3 and HA7-5 as primer, PCR expansions are carried out Increase, obtains pcr amplification product, the sequence of the product is as shown in SEQ ID No.4, in SEQ ID No.4 the 8th from 5 ' ends To the 12nd be Kozak sequences, the 13rd to the 66th be signal peptide sequence, the 67th to the 1695th be HA genes, the 1585th It is C-terminal transmembrane domains sequence to the 1668th.
(4) DNA molecular shown in NspV and NotI double digestions SEQ ID No.4, obtains genetic fragment；NspV and NotI Double digestion pPICZ α carriers obtain carrier large fragment；Genetic fragment is connect with carrier large fragment, recombinant plasmid is obtained, is ordered Entitled pPICZ α-HA7.PPICZ α-HA7 are sequenced, as a result correctly.
Two, the construction and screening of recombination yeast
It by about 10 μ g pPICZ α-HA7 plasmids, is linearized with BglII, with the 3M sodium acetate aqueous solutions of 1/10 volume and 3 times The plasmid of the absolute alcohol precipitation linearisation of volume.The ethanol aqueous wash for being 70% with volumn concentration is twice to remove it In salt, dry, be added about 30 μ L water be resuspended precipitation, obtain for conversion pPICZ α-HA7 linearization plasmids.
Reference books of the method with reference to Invitrogen companies of yeast Electroporation-competent cells are prepared in following steps " Molecular Cloning, A laboratory Manual (Fourth Edition) ", 2012Cold Spring Harbor Laboratory Press,Cold Spring Harbor,New YorK。
By pichia pastoris X-33 in YPD tablets (yeast extract 10g/L, tryptone 20g/L, glucose 20g/L, fine jade Fat 15g/L) on method of scoring detach monoclonal, 28 DEG C of incubator cultures 2 days.A monoclonal is inoculated with to one equipped with 10mL YPD In the 50mL triangular flasks of fluid nutrient medium (yeast extract 10g/L, tryptone 20g/L, glucose 20g/L), 28 DEG C overnight It cultivates to OD600About 2, obtain bacterium solution.0.1-0.5mL bacterium solutions are inoculated into the 3.5L containing 500mLYPD fluid nutrient mediums again In shaking flask, overnight incubation to OD600To between 1.3-1.5.Bacterium solution is transferred in sterile centrifugal bottle, 4 DEG C, 1500g centrifugations 10 Minute.Thalline is resuspended with the sterile water of 500mL precoolings, 4 DEG C, 1500g centrifuges 10 minutes harvest cells, the nothing being pre-chilled with 250mL Bacterium water is washed once again.Thalline is resuspended with the sterile 1M sorbierites of 20mL precoolings, 4 DEG C, 1500g centrifuges 10 minutes harvest cells, uses It is 1.5mL that thalline to final volume, which is resuspended, in the 1M sorbierites of precooling, obtains bacteria suspension.
Take 80 μ L bacteria suspensions and 10 μ L for the pPICZ α-HA7 linearization plasmids of conversion, the mixing in microcentrifugal tube, Mixture is obtained, 5min on ice is set, mixture is transferred in an ice-cold 0.2cm electricity revolving cup, electroporation of cells (Bio-Rad Gene Pulser, 2000V, 25 μ F, 200 Ω), then the ice-cold 1M sorbierites of 1mL are added into electric revolving cup immediately, And carefully mixture (transformed cells) is transferred in 15mL culture tubes.
Culture tube is placed on 28 DEG C of incubation 1h, not shaken.Then it is added after 1mL YPD fluid nutrient mediums 28 DEG C, it is incubated 3h in the shaking table of 250rpm.200 μ L transformed cells are taken to be applied on the YPD tablets containing 100 μ g/mL Zeocin.28 DEG C incubator culture 2-5 days, until forming monoclonal.
Random picking monoclonal is inoculated into 2ml YPD fluid nutrient mediums, 28 DEG C of culture 48h, 5% inoculation by volume Amount be inoculated into BMGY culture mediums (yeast extract 10g/L, tryptone 20g/L, pH6.0,100mmol/L phosphate buffer, 1.34g/100ml YNB, 4 × 10-5The glycerine of g/100ml Biotin, 1g/100ml) in, volumn concentration is added afterwards for 24 hours It expresses for 0.5% methanol induction, is added 1 time per 12h, thalline were collected by centrifugation after induction 60h.It is harvested by centrifugation per 1ml bacterium solutions Thalline is resuspended with 100 μ l PBS, is added the pickling glass pearl (425-600 μm of diameter or 0.5mm) of 1/4 volume, each sample with Maximum speed vortex concussion 1 minute, repeats six times, shakes intermediate ice bath two minutes per being vortexed twice to prevent protein degradation.With low 4 DEG C of warm microcentrifuge, 3500g are centrifuged 1 minute, sedimented glass pearl and unbroken cell, and it is broken bacterium solution to obtain supernatant.It takes Broken bacterium solution first uses PBS according to 1：After the dilution of 20 volume ratios, then with PBS according to 1：It is red thin with 1% chicken after 2 volume ratios are serially diluted Born of the same parents carry out hemagglutination activity analysis, and (method is shown in " Guo Yuanji etc.《Influenza virus and its experimental technique》, Beijing, China Three Gorges Publishing house, 1997).Picking has the corresponding clone's (recombinant yeast pichia pastoris monoclonal) of broken bacterium solution of hemagglutination activity, is used for influenza The preparation of hemagglutinin glycopolypeptide multimer object nano particle.
Three, Engineering Yeast ferments
Seed culture：The high recombinant yeast pichia pastoris monoclonal of the hemagglutination activity that step 2 obtains is inoculated into fresh MD tablets (1.34g/100mlYNB, 4 × 10-5G/100ml Biotin, 1g/100ml glucose, 1.5g/100ml agar powders) on, culture, And the monoclonal colonies of picking thereon are inoculated in YPD fluid nutrient mediums, 24 DEG C, 250rpm cultivates about 48h.It is transferred again in YPD In liquid submerged culture base 300mL, inoculum concentration 1%, 25 DEG C, 250rpm cultures are planted until strain density OD600 is more than 10 Sub- liquid.
Fermented and cultured：Prepare fermentation medium 2.1L (H3PO43.5mL/L, K2SO42.4g/L, KOH0.65g/L, CaSO4 (anhydrous) 0.14g/L, MgSO4·7H2O1.95g/L, the biotin of glycerine 40.0g/L, PTM11.2mL/L, 0.02g/100ml 0.5mL/L, surplus are water.The wherein group of PTM1 becomes：CuSO4·5H2O6.0g/L, MnSO4·H2O3.0g/L, FeSO4· 7H2O65g/L, ZnSO4·7H2O20g/L, CoCl2·6H2O0.5g/L, NaMoO4·2H2O0.2g/L, KI0.1g/L are dense H2SO45mL/L.), it is added to 5L fermentation tanks, 121 DEG C, 30min high pressure sterilizations.It waits for that fermentation tank is down to room temperature, is adjusted with ammonium hydroxide pH6.0。
Seed liquor is accessed into fermentation tank with 10% inoculum concentration, ammonium hydroxide controls pH6.0, and temperature is 28 DEG C, adjusts stirring and turns Speed and ventilatory capacity maintain 10% or more dissolved oxygen.When glycerol depletion, dissolved oxygen gos up, and starts flow feeding growth medium ((PTM1 containing 12mL/L, 2mL/L500 × biotin are (glad through section's biotechnology purchased from Beijing for the glycerine water solution of 50g/100ml Co., Ltd)), 40ml/h, stream adds 6-8h, stops feed supplement.Start methanol induction, temperature maintains 24 DEG C, will with ammonia spirit PH is adjusted to 6.4.Initial period, absolute methanol start stream with 2.4mL/h and add, increase 2.4mL100% methanol per hour, increase after 5h To 12mL/h, it is at this time induction 0 hour, is sampled later per 12h.Terminate fermentation after inducing 48h, zymotic fluid is in 4 DEG C, 7000rpm/ Min centrifuges 20min.The suspension for being 40g/100ml is resuspended with water, high-pressure homogenization instrument breaks bacterium (1200bar breaks bacterium 3 times), must be homogenized Liquid, the homogenate are used for the purifying of influenza hemagglutinin glycopolypeptide multimer object nano particle.
Four, the purifying of influenza hemagglutinin glycopolypeptide multimer object nano particle
(1) add 50g PEG2000, stirring and dissolving 0.5h, 7000rpm centrifugation in the homogenate that 500ml step 3 obtains 20min collects precipitation, abandons supernatant；Solution (Tris-HCl containing 10mM, the volume basis in the solution of precipitation plus 500ml volumes Content 2%TritonX-100 (Qula is logical, non-ionic detergent), the glycerine of 5g/100ml, surplus is water) it is resuspended, stirring is molten Solve 2h.8000rpm centrifuges 20min, collects supernatant, and it is 6.0 to adjust pH with phosphate, is diluted with water to conductance and is less than 2.5ms/ Cm obtains sample solution.
(2) (chromatographic media is Sepharose FF SP (being purchased from GE) to cation chromatographic column, and bed is Detection wavelength is 280nm, room temperature) first A liquid is used to balance (A liquid：Tween20,5g/100ml glycerine containing 1g/100ml, surplus For the PB of 20mM pH6.0) after, by the sample solution of step (1) with 50ml/min flow velocity loadings, with the same flow velocity of A liquid rinse to A280 is less than 0.2, uses B liquid (B liquid instead：Tween20,5g/100ml glycerine containing 1g/100ml, surplus are 20mM pH6.86 PB) balance, with 15%C liquid (NaCl that final concentration of 150mM is added in A liquid), 100%C liquid elution (in A liquid add eventually it is dense Degree is the NaCl of 1M), the eluting peak of 15%C liquid, 100%C liquid is obtained, as a result as shown in A in Fig. 1.
In Fig. 1,1-6 represents the eluent for the 1-6 collecting pipes that 15%C liquid affords, and 100%C represents 100%C liquid Eluent, 3+endoh represent sample of the eluent after EndoH digestions of 3 collecting pipes, represent sample solution before column, M represents albumen marker。
Sample before the eluent of the 1-6 collecting pipes of A, the eluent and column of 100%C liquid in Fig. 1 is subjected to reduction SDS- PAGE is analyzed, and as a result as shown in B in Fig. 1, arrow show the band of HA components in Figure 1B.
Fig. 1 shows that the sample where HA components is the eluent of 15%C liquid.
(3) eluent for collecting the 15%C liquid containing HA components carries out anion-exchange chromatography (chromatographic media is Source30Q (is purchased from GE), and bed isDetection wavelength is 280nm, room temperature).It is as follows：By 15% The elution samples of C liquid A liquid (A liquid:The glycerine of 1g/100ml Tween20,5g/100ml, surplus are 20mM pH8.1's Tris-HCl 10 times of dilutions) are carried out, after the speed end of the sample of 10ml/min, (B liquid is balanced with B liquid:1g/100ml The glycerine of Tween20,5g/100ml, surplus are the Tris-HCl of 20mM pH7.5), then the 10%C liquid (A of the step is used respectively The NaCl of final concentration of 100mM is added in liquid), 100%C liquid elution (NaCl that final concentration of 1M is added in the A liquid of the step), It is finally eluted with 0.5M NaOH aqueous solutions, obtains each elution fraction, as a result as shown in A in Fig. 2.
In Fig. 2, Q wears the sample not combined by Source30Q when representing loading；10%C represents the eluent of 10%C liquid； 100%C represents the eluent of 100%C liquid；NaOH represents the eluent of 0.5M NaOH aqueous solutions.
By before purification sample (eluent for the 15%C liquid that step (2) obtains), not by Q columns (Source30Q) inhale It is attached and be pierced by sample, 10%C liquid eluent (the first pipe collection liquid and the second pipe collection liquid, third pipe collection liquid), utilize The collection liquid that 100%C liquid is eluted, the collection liquid eluted using 0.5M NaOH aqueous solutions carry out reduction SDS-PAGE Analysis, as a result as shown in B in Fig. 2.
In Fig. 2 B, sample (eluent for the 15%C liquid that step (2) obtains) before purification is represented before Q columns, Q, which wears representative, not to be had There is the sample for being adsorbed and be pierced by by Q columns (Source30Q), 1, which represents 10%C liquid, elutes the first pipe collection liquid, and 2 represent 10%C liquid The second pipe collection liquid is eluted, 3 represent 10%C liquid elution third pipe collection liquid, and 100%C represents the receipts that 100%C liquid is eluted Liquid collecting, NaOH represent the collection liquid eluted using 0.5M NaOH aqueous solutions, and M represents albumen marker.Arrow show HA Band.
Fig. 2 shows that the purifying of the step obtains the HA samples of purity raising, eliminates part foreign protein, HA components institute Sample be the step 10%C liquid eluent.
(4) collection step (three) 10%C liquid elution fraction 4ml, carrying out gel exclusion chromatography chromatography, (chromatographic media is Superdex200 (is purchased from GE), and bed isDetection wavelength is 280nm, room temperature), by step (3) 10%C liquid After elution fraction 4ml loadings, with eluent (eluent：The 20mM PB of pH7.0 containing 0.1M NaCl) it is eluted, flow velocity 1ml/min, as a result as shown in Figure 3A.
In Fig. 3 A, first main elution peak that arrow indicates is the H7N9 influenza hemagglutinin glycopolypeptide multimer object nanometers of purifying Particle measures a concentration of 0.33mg/ml of its albumen.
The H7N9 streams that the gel exclusion chromatography chromatographic purifying of step (3) 10%C liquid elution fraction, EndoH digestions is obtained The H7N9 influenza hemagglutinin sugar eggs that sense hemagglutinin glycopolypeptide multimer object nano particle and gel exclusion chromatography chromatographic purifying obtain White polymer nano particle carries out reduction SDS-PAGE analyses, as a result as shown in B in Fig. 3.
Step (3) 10%C liquid elution fractions are represented in Fig. 3, before column, HA+endoh represents the gel exclusion of EndoH digestions The H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles that chromatographic purification obtains, it is pure that HA represents gel exclusion chromatography chromatography Change obtained H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles, M represents albumen marker.Arrow show HA bands.
Fig. 3 shows that step purifying obtains the higher HA sterlings of purity, and is analyzed using EndoH glycosidases, shows It has occurred correctly glycosylation modified.
Five, the structural analysis of influenza virus hemagglutinin glycoprotein polymer
With N- glycosidases F (PNGF) (being purchased from NEB) processing, the front and back the change of molecular weight of analysis glycosyl excision, with analysis step The rapid four H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared, are as follows：20ul step 4 is taken to purify H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles (a concentration of 330ug/ml), by the digestion side of N- glycosidases F (PNGF) Method handles it, while setting not enzyme control group and being not added with sample controls group.Each sample is subjected to reduction SDS-PAGE inspections It surveys, the results are shown in Figure 4.
In Fig. 4, PNGF represents N- glycosidases F, and the H7N9 influenza hemagglutinin glycoprotein that HA+PNGF represents step 4 preparation is more N- glycosidases F is added in polymers nano particle, the H7N9 influenza hemagglutinin glycopolypeptide multimer objects that HA represents step 4 preparation are received Rice grain, M represent albumen marker.Arrow show HA bands.
Fig. 4 shows the HA0 molecular weight of the H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles of unused PNGF processing about For 64KD, after PNGF processing, molecular weight and molecular weight is about 60KD, the theoretical molecular weight with the HA0 maturation proteins being not glycosylated (60172Da) is consistent.Illustrate that H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by step 4 are glycoprotein.
Form 5 amino acid sequence analysis of N-terminal of the component HA0 of H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles As a result it is DKIXL, wherein X is not measure, this (theoretical sequence consistent with H7N9 virus HA maturation protein (HA0) N-terminal theoretical sequences It can be destroyed, cannot measure when Edman methods are sequenced for DKICL, wherein C).Illustrate that the signal peptide of HA is successfully cut off.
Six, prepared by the HA of trypsase cutting
In order to prepare the HA for specifically being cut into HA1 and HA2 by protease, the H7N9 influenza blood clottings of a concentration of 330ug/ml are taken Plain glycopolypeptide multimer object nano particle 1ml, the trypsase 16ug for adding TPCK to handle, ice bath is handled 1 hour, as experimental group. Same H7N9 influenza hemagglutinins glycopolypeptide multimer object nano particle 1ml, 100 DEG C of water bath processings is separately taken to be become for 5 minutes simultaneously Property, ice bath cooling, the trypsase 16ug for adding TPCK to handle, same ice bath processing 1 hour, as a control group.
Specifically cutting cannot be only used for preparing cutting H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles protease, and And be also the whether correct important method of higher structure of analysis HA0, the HA0 with influenza virus tripolymer higher structure only has Basic amino acid site between HA1 and HA2 is exposed, just can be specifically cut into molecular weight about 40KD's by trypsase The HA2 of HA1 and molecular weight about 25KD.HA0 without correct higher structure can be then cut by trypsase and differ in size Various segments.
H7N9 influenza hemagglutinin glycopolypeptide multimer objects nano particle, experimental group and control group are subjected to reduction SDS-PAGE, Testing result is as shown in Figure 5.
In Fig. 5, HA represents H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles, and HA+ pancreatin is experimental group, HA denaturation + pancreatin is control group.Arrow represents HA0 bands.
After Fig. 5 shows that HA0 is cut by trypsase, non-reduced its molecular weight of SDS-PAGE electrophoretic analysis is for 64KD and not It is similar to cut HA0, and restores electrophoretic analysis discovery, it is two of 40kD and 24kD that HA0 has cut into molecular weight by specificity Segment, it is consistent with the molecular weight of HA1 and HA2.Illustrate that the HA0 that the present invention obtains has been cut by trypsase specificity by two sulphur The HA that the HA1 and HA2 subunits of key connection are constituted.And after the heated destruction higher structures of the HA0, then cut with pancreatin, it is non-reduced Electrophoresis and reduction electrophoretic analysis do not find specific band, illustrate HA0 be cut by trypsase differ in size it is various Segment.Therefore the HA0 of the present invention has the identical higher structure of influenza virus hemagglutinin.
Seven, blood coagulation tests
The H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles of above-mentioned preparation are taken, first use PBS according to volume ratio 1：10 Then dilution uses PBS according to volume ratio 1 again：2 are serially diluted, and are control with physiological saline, blood clotting is carried out with 1% chicken red blood cell Activity analysis, specific method are shown in " Guo Yuanji etc.《Influenza virus and its experimental technique》, Beijing, Chinese Sanxia Publishing House, 1997”。
Hemagglutination activity testing result is as shown in Figure 6.
In Fig. 6, first row is from 1 from left to right：20 start 2 times of H7N9 influenza hemagglutinin glycopolypeptide multimers being serially diluted Object nano particle, second row are saline control.
Fig. 6 shows that H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles have apparent hemagglutination activity, chicken blood blood clotting Potency reaches 1：8000.Therefore the influenza hemagglutinin (H7N9 influenza hemagglutinin glycopolypeptide multimer objects nano particle) of the present invention has Sialic acid receptor combines activity.
Eight, in order to analyze preparation H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles molecular size, to its into Row SEC (exclusion gel chromatography) is analyzed, and instrument is Agilent1290 high performance liquid chromatographs (Agilent Technologies Co), UV detector, Detection wavelength 214nm, TSKG4000SWXL Chromatographic column and TSK Guardcolumn SWXLGuard column (being purchased from Tosoh Bioscience LLC) analysis, with the 20mM PB containing 100mM NaCl For mobile phase, flow velocity 0.5ml/min.It is demarcated with SEC molecular weight standards (being purchased from Suzhou Sepax Technologies, Inc.) analysis, as a result As shown in Figure 7.
In Fig. 7, the upper figure of A is the chromatogram of molecular weight marker proteins, and the retention time of each molecular weight is respectively：670KD, 20.593min；150KD,22.478min；44KD,24.074min；17.6KD,25.363min；1.35KD,26.966min).A Figure below be H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles chromatogram.
Fig. 7 B show that H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particle purity has reached 99.69%.
Fig. 7 shows H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles under the condition analysis, and retention time is 16.971min illustrates that its molecular weight is significantly greater than 670KD, since the molecular weight of HA0 monomers is 64KD, the molecule of tripolymer Amount is about 180KD, illustrates that H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles are more to be formed containing 9 or more HA0 monomers Aggressiveness, and trypsase cutting experiment and blood coagulation tests show that H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles have The tripolymer higher structure of influenza hemagglutinin, therefore, the H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles be three with The polymer that upper HA0 tripolymers are formed.
By after the H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particle negative staining of preparation with 50,000 times of electron microscopes Photograph, the results are shown in Figure 8.
Scale in Fig. 8 is 20 nanometers.
Fig. 8 shows that H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles are the above hemagglutinin tripolymer of at least three (hemagglutinin tripolymer herein refers to the tripolymer that three HA0 monomer polymerizations are formed) tail portion polymerize inside, and head protrudes outward, The polymer particle of formation, about 20-50 nanometers of diameter.
Embodiment 2 prepares stream with H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by recombinant yeast pichia pastoris Influenza vaccine
H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by embodiment 1 are diluted to the PBS of pH7.4 7.5 μ g/50 μ l, add isometric 1.2mg/ml Al (OH)3Adjuvant (is purchased from GE companies, trade name Rehydragel@ LV), influenza vaccines are made, are experimental group injection.Meanwhile if being wherein not added with H7N9 influenza hemagglutinin glycopolypeptide multimer object nanometers The solution of particle is control group injection.
Every group of 5 mouse of experimental group and control group, 100 μ of injection of every group of every mouse hind leg muscle injection respective sets l.Booster immunization is primary after three week of injection for the first time, take a blood sample in booster immunization the latter week serum carries out blood clotting and inhibits real It tests, the standard hemagglutinin of blood clotting Inhibition test is the state-run biological standard of Britain and reference material that WHO (World Health Organization) recommends Research institute 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 H7N9 influenzas that provide re-match vaccine strain (NIBRG-268) Chick embryo culture virus is through 1：It is prepared after 2000 formalin-inactivateds.H7N9 influenzas re-match the chick embryo culture disease of vaccine strain (NIBRG-268) Poisons 1：Viral preparation standard hemagglutinin, dilution and the blood clotting Inhibition test method prepared after 2000 formalin-inactivateds is shown in " Guo Yuanji Deng《Influenza virus and its experimental technique》, Beijing, Chinese Sanxia Publishing House, 1997 ".
The results are shown in Figure 9 for the blood clotting inhibitory activity of neutralizing antibody in each group mice serum.
In Fig. 9, ordinate is that blood clotting inhibits potency (HI), and abscissa is is grouped, and 0.0 is control group, and 7.5 be experimental group.
Fig. 9 shows that the serum of control group mice does not generate blood clotting inhibition, and experimental mice serum blood clotting inhibits potency equal More than 1：40, it is 1 that average blood clotting, which inhibits potency,：640.It is generally acknowledged that the blood clotting of influenza vaccines induction inhibits potency to be more than 1：40 are Effective immunoprotection can be provided for body.Therefore, the H7N9 influenza hemagglutinin glycopolypeptide multimer objects prepared with recombination yeast are received Rice grain can be used for preparing influenza vaccines.
H5N1 highly pathogenic avian influenza virus hemagglutinin glycopolypeptide multimer object nanometers prepared by embodiment 3, Hansenula yeast Grain
One, the structure of recombinant expression carrier
(1) according to the influenza HA overall length of Genbank (EU263353.1) (A/duck/Guangxi/27/2003 (H5N1)) Amino acid sequence, according to yeast biased codons and gene high expression principle Optimized Coding Based gene, shown in synthesis SEQ ID No.5 DNA molecular.
(2) following primer is designed and synthesized：
HA5-5：5’-ATCTTCGAAACGatggagaaaatagtgcttc-3’(SEQ ID No.6)
Sequence shown in underscore is BstB I digestion recognition sites.
HA5-3：5’-ATCGCGGCCGCttaaatgcaaattctgcattg-3’。(SEQ ID No.7)
Sequence shown in underscore is NotI digestion recognition sites.
(3) using DNA molecular is template shown in SEQ ID No.5, using HA5-3 and HA5-5 as primer, PCR expansions are carried out Increase, obtains pcr amplification product, as shown in SEQ ID No.8, which contains the sequence of the product successively from N-terminal to C-terminal Kozak sequences, signal peptide sequence, HA genes (including C-terminal transmembrane domains sequence).
DNA molecular shown in NotI and BstB I double digestion SEQ ID No.8, obtains genetic fragment；NotI and BstB I Double digestion pPICZ α carriers obtain carrier large fragment；Genetic fragment is connect with carrier large fragment, recombinant plasmid is obtained, is ordered Entitled pPICZ-(H5N1)HA.By pPICZ-(H5N1)HA is sequenced, as a result correctly.
(4) in order to adapt to express in Hansenula yeast, by pPICZ-(H5N1)AOX promoters in HA replace with the inferior ferment of the Chinese Female alcohol oxidase promoter MOX.
1, according to the nucleotide sequence of Hansenula yeast promoter, synthetic primer MOX5：5’- ATCAGATCTTCGACGCGGAGAACGATCT-3 ' (SEQ ID No.9, underscore part be BglII digestions recognition site) and MOX3：5’-TGTTTTTGTACTTTAGATTGATG-3’(SEQ ID No.10).
2, using Hansenula polymorpha ATCC26012 (can American Type Culture collection obtain) genome as mould Plate carries out PCR amplification using Pyrobest archaeal dna polymerases, obtains MOX promoter fragments using MOX5 and MOX3 as primer.
PCR system：1 μ g genomic DNAs, 4 μ l dNTP (2.5mM), 5 μ l10 × Pyrobest Buffer, 1 μ l10 μM MOX5 primers, 1 μ l μM MOX3 primers, 0.5 μ l Pyrobest archaeal dna polymerases, 37.5 μ l water, 50 μ l of total volume.
PCR programs：94 DEG C of pre-degeneration 5min；94 DEG C denaturation 30sec, 55 DEG C annealing 30sec, 72 DEG C extension 2min, 30 Cycle；72 DEG C of extension 10min, 4 DEG C of preservations.
Pcr amplification product is the target fragment of about 1.5kb, it is used to BglII digestions, and carry out phosphoric acid to endonuclease bamhi Change, it is BglII digestion cohesive ends, the MOX promoters of the 1.5kb of 3 ' end phosphorylations to obtain 5 ' ends.
3, by pPICZ-(H5N1)It is mended with Klenow fragment klenow fragments and dNTP after HA carriers NspV single endonuclease digestions It is flat, after electrophoresis recycling, then BglII digestions are used, excision AOX promoters.
The carrier and above-mentioned 5 ' end are BglII digestion viscosity end by the carrier of electrophoresis recycling excision AOX promoter fragments End, the MOX promoters connection that 3 ' ends are the 1.5kb of phosphorylation are obtained with the MOX controls of Hansenula yeast alcohol oxidase promoter The carrier of H5N1 influenza hemagglutinin glycopolypeptide multimer object nano particle gene expressions, is denoted as pMOXZ-HA5.
Two, after linearizing pMOXZ-HA5 carriers with BglII, (ATCC26012 (can the U.S. for electrotransformation Hansenula polymorpha Type Culture Collection obtains)), and the cell of conversion is applied to the YPD tablets containing 100 μ g/mL Zeocin On (yeast extract 10g/L, tryptone 20g/L, glucose 20g/L, agar 15g/L).30 DEG C of incubator cultures 2-4 days, until Form monoclonal.Random picking monoclonal is inoculated into 2ml YPD (yeast extract 10g/L, tryptone 20g/L, glucose 20g/L) in fluid nutrient medium, 30 DEG C of culture 24-48h are inoculated into BMGY culture medium (yeast extracts by 5% inoculum concentration 10g/L, tryptone 20g/L, pH6.0,100mmol/L phosphate buffer, the YNB of 1.34g/100ml, 4 × 10-5g/100ml The glycerine of Biotin, 1g/100ml) in, volumn concentration is added afterwards for 24 hours and is expressed for 0.5% methanol induction, 1 is added per 12h Secondary, thalline were collected by centrifugation and supernatant after induction 60h.The preparation of broken bacterium solution and hemagglutination activity are analyzed with step 2 in embodiment 1. The positive strain for screening high hemagglutination activity is named as Hans (pMOX-HA5).
Three, the preparation of H5N1 highly pathogenic bird flus hemagglutinin glycopolypeptide multimer object nano particle
Picking Hans (pMOX-HA5) single bacterium colony is inoculated in 3mlYPD fluid nutrient mediums, 30 DEG C, and 250rpm grows 1-2 It is forwarded to volume ratio 2% in the 1L shaking flasks equipped with 100mlYPD fluid nutrient mediums, 30 until strain density OD600 is more than 10 DEG C, 250rpm is cultivated 24 hours, is forwarded in 10 1L shaking flasks equipped with 150mlBMGY fluid nutrient mediums with volume ratio 5%, 30 DEG C, 250rpm is cultivated 24 hours, and the methanol induction that volumn concentration is 0.5% is added and expresses, adds 1 methanol per 12h, lures Lead after 60h that thalline were collected by centrifugation.Bacterium is broken with the method for step 2 in embodiment 1 and measures its hemagglutination activity, and picking has blood clotting The corresponding clone of active broken bacterium solution carries out Engineering Yeast fermentation with the method for step 3 in embodiment 1, using being walked in embodiment 1 Rapid four method purifying obtains H5N1 highly pathogenic bird flu hemagglutinin glycopolypeptide multimer object nano particles, is denoted as(H5N1) HA(Hans)。
Four, reduction SDS-PAGE analyses(H5N1)HA(Hans)Molecular weight is about the 66KD (items that molecular weight herein is about 66KD Band is the HA0 components of H5N1 highly pathogenic bird flu hemagglutinin glycopolypeptide multimer object nano particles).
H5N1 highly pathogenic bird flu hemagglutinin glycopolypeptide multimer object nano particles, side are handled with N- glycosidases F (PNGF) Method is found with the step five in embodiment 1, the front and back the change of molecular weight of analysis glycosyl excision after cutting off glycosyl with PNGF(H5N1) HA(Hans)The molecular weight and molecular weight of (the HA0 components of the H5N1 highly pathogenic bird flu hemagglutinin glycopolypeptide multimer object nano particles) is 60kD, the results are shown in Figure 10.
It is right(H5N1)HA(Hans)It carries out molecular-exclusion chromatography (SEC) to analyze, method is with the step eight in embodiment 1, as a result such as Shown in Figure 11.In Figure 11, upper figure is the chromatogram of molecular weight marker proteins, and figure below is(H5N1)HA(Hans)Chromatogram, hair It is existing(H5N1)HA(Hans)Molecular weight standard of the retention time less than 670kD retention time, which is Molecular weight is more than the polymer of 670kD.
It is right(H5N1)HA(Hans)Chicken red blood cell hemagglutination activity analysis is carried out, method is with the step seven in embodiment 1, as a result as schemed Shown in 12, Tu12Zhong, first row is from 1 from left to right：What 20 2 times of beginnings were serially diluted(H5N1)HA(Hans), second row is physiology Saline control is found(H5N1)HA(Hans)With apparent hemagglutination activity.
It is prepared with 2 identical method of embodiment(H5N1)HA(Hans)Influenza vaccines, immune mouse, and in booster immunization the latter Take a blood sample in week serum carries out blood clotting Inhibition test (method is shown in " Guo Yuanji etc.《Influenza virus and its experimental technique》, north Capital, Chinese Sanxia Publishing House, 1997 "), as a result as shown in figure 13, Tu13Zhong, ordinate is that blood clotting inhibits potency (HI), horizontal seat It is designated as being grouped, 0.0 is control group, and 7.5 be experimental group, it is found that the average blood clotting of experimental mice serum inhibits potency to reach 1: 240。
Therefore, the H5N1 highly pathogenic bird flu hemagglutinin glycopolypeptide multimer object nano particles that prepared by Hansenula yeast can be used In preparation influenza vaccines.
H1N1 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by embodiment 4, Pichia pastoris
One, the structure of recombinant expression carrier
(1) synthesis source is in the SEQ ID No.11 of H1N1 influenza viruses (A/FortMonmouth/1/47 (H1N1)).
(2) following primer is designed and synthesized：
HA1-5：5’-ATCTTCGAAACGatgaaagcaaaactactgatc-3’(SEQ ID No.12)
Sequence shown in underscore is NspV digestion recognition sites.
HA1-3：5’-gatGCGGCCGCtcagatgcatattctgcattg-3’(SEQ ID No.13)
Sequence shown in underscore is NotI digestion recognition sites.
(3) using SEQ ID No.11 as template, using HA1-5 and HA1-3 as primer, Pyrobest archaeal dna polymerases are utilized Carry out PCR amplification, obtain pcr amplification product, as shown in SEQ ID No.14, in SEQ ID No.14 from 5 ' ends the 8th to 12nd be Kozak sequences, the 13rd to the 63rd be signal peptide sequence, the 64th to the 1713rd be HA genes, the 1594th to 1677th is C-terminal transmembrane domains sequence.
DNA molecular shown in NspV and NotI double digestion SEQ ID No.14, obtains genetic fragment；The bis- enzymes of NspV and NotI It cuts pPICZ α carriers and obtains carrier large fragment；Genetic fragment is connect with carrier large fragment, recombinant plasmid is obtained, is named as pPICZα-HA1.PPICZ α-HA1 are sequenced, as a result correctly.
Two, the construction and screening of recombination yeast
It by about 10 μ g pPICZ α-HA1 plasmids, is linearized with BglII, and converts Pichia yeast X-33, finally had There is the corresponding clone's (recombinant yeast pichia pastoris monoclonal) of the broken bacterium solution of hemagglutination activity, is used for H1N1 influenza hemagglutinin glycopolypeptide multimers The preparation of object nano particle, specific method is the same as the step two in embodiment 1.
Three, Engineering Yeast fermentation is the same as the step three in embodiment 1.
Four, the purifying of H1N1 influenza hemagglutinins glycopolypeptide multimer object nano particle is remembered with the step four in embodiment 1 For(H1N1)HA。
Five, reduction SDS-PAGE analyses((H1N1)HA molecular weight is about that (band that molecular weight herein is about 66KD is 66KD The HA0 components of H1N1 influenza hemagglutinin glycopolypeptide multimer object nano particles).
H1N1 influenza virus hemagglutinin glycoprotein polymer is handled with N- glycosidases F (PNGF)(H1N1)HA, method is the same as implementation Step five in example 1, the front and back the change of molecular weight of analysis glycosyl excision are found with hemagglutinin glycoprotein after PNGF excision glycosyls The molecular weight and molecular weight of (the HA0 components of H1N1 influenza hemagglutinin glycopolypeptide multimer object nano particles) is 61kD, as a result such as Figure 14 institutes Show.
To H1N1 influenza hemagglutinin glycopolypeptide multimer object nano particles(H1N1)HA carries out molecular-exclusion chromatography (SEC) and analyzes, Method is with the step eight in embodiment 1, as a result as shown in figure 15.In Figure 15, upper figure is the chromatogram of molecular weight marker proteins, under Figure is(H1N1)The chromatogram of HA is found(H1N1)The retention time of molecular weight standard of the retention time of HA less than 670kD, the influenza Hemagglutinin glycopolypeptide multimer object nano particle is the polymer that molecular weight is more than 670kD.
To H1N1 influenza hemagglutinin glycopolypeptide multimer object nano particles(H1N1)HA carries out chicken red blood cell hemagglutination activity analysis, side Method is with the step seven in embodiment 1, and as a result as shown in figure 16, Tu16Zhong, first row is from 1 from left to right：20 start 2 times of series It is diluted(H1N1)HA, second row are saline control, are found(H1N1)HA has good hemagglutination activity.
Electromicroscopic photograph is shown(H1N1)HA forms the polymer particle of 20-50nm.
H1N1 influenza hemagglutinin glycopolypeptide multimer object nano particles are prepared with 2 identical method of embodiment(H1N1)HA influenza epidemic diseases Seedling, immune mouse, and take a blood sample in booster immunization the latter week serum carries out blood clotting Inhibition test (method is shown in " Guo Yuanji etc. 《Influenza virus and its experimental technique》, Beijing, Chinese Sanxia Publishing House, 1997 "), as a result as shown in figure 17, Figure 17 In, ordinate is that blood clotting inhibits potency (HI), and abscissa is is grouped, and 0.0 is control group, and 7.5 be experimental group, it is found that experimental group is small The average blood clotting of mouse serum inhibits potency to reach 1:320.
Therefore, the H1N1 influenza hemagglutinin glycopolypeptide multimer object nano particles that prepared by Pichia pastoris(H1N1)HA can be used for making Standby influenza vaccines.
Embodiment 5, Kluyveromyces lactis prepare H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles
One, the structure of recombinant expression carrier
(1) following primer is designed and synthesized：
HA7-HindIII-5：5’-ATCAAGCTTACGATGAACACCCAAATACTGGTTTTC-3’(SEQ ID No.15)
Sequence shown in underscore is HindIII digestion recognition sites.
HA7-3：5’-ATCGCGGCCGCTTAAATACAGATAGTACATCT-3’(SEQ ID No.16)。
Sequence shown in underscore is NotI digestion recognition sites.
(2) using DNA molecular is template shown in SEQ ID No.1, using HA7-3 and HA7-HindIII-5 as primer, into Row PCR amplification, obtains pcr amplification product, the sequence of the product as shown in SEQ ID No.17, the molecule from N-terminal to C-terminal successively Including Kozak sequences, signal peptide sequence, HA genes (contain C-terminal transmembrane domains sequence).
(3) DNA molecular shown in HindIII and NotI double digestions SEQ ID No.17, obtains genetic fragment；HindIII Carrier large fragment is obtained with NotI double digestion Kluyveromyces lactis expression vectors pKLAC1；By genetic fragment and carrier large fragment Connection, obtains recombinant plasmid, is named as pKLAC1-(H7N9)HA7。
Two, the construction and screening of recombination yeast
By about 10 μ g pKLAC1-(H7N9)HA7 plasmids, are linearized with SacII, with the 3M sodium acetates of 1/10 volume and 3 times of bodies The plasmid of long-pending absolute alcohol precipitation linearisation.The ethanol aqueous wash for being 70% with volumn concentration is twice to remove wherein Salt, dry, about 30 μ L water be resuspended precipitation, obtain for conversion pKLAC1-(H7N9)HA7 linearization plasmids.
The method of Kluyveromyces lactis Electroporation-competent cells is prepared with the step two in embodiment 1.
Take 80 μ L competent cells and 10 μ L for the pKLAC1- of conversion(H7N9)HA7 linearization plasmids, in microcentrifugal tube Middle mixing, obtains mixture.5min on ice is set, mixture is transferred in an ice-cold 0.2cm electricity revolving cup.Electroporation Cell (Bio-Rad Gene Pulser, 1500V, 25 μ F, 200 Ω), then the ice-cold mountains 1M 1mL are added into electric revolving cup immediately Pears alcohol, and carefully mixture (transformed cells) is transferred in 15mL culture tubes.
Centrifuge tube is placed on 28 DEG C of incubation 2h, not shaken.200 μ L transformed cells are taken to be coated on containing 5mM acetyl The upper 28 DEG C of trainings of YCB tablets (the K.lactis Protein Expression Kit that preparation method is provided referring to NEB companies) of amine It supports 3-4 days until monoclonal occurs.
Random picking monoclonal is inoculated into 2ml YPD fluid nutrient mediums, 28 DEG C of culture 48h, 5% inoculation by volume Amount is inoculated into YPGal fluid nutrient mediums (yeast extract 10g/L, tryptone 20g/L, galactolipin 20g/L, surplus are water) In, thalline were collected by centrifugation after 60h.The thalline being harvested by centrifugation per 1ml bacterium solutions is resuspended with 100ulPBS, and the method for preparing broken bacterium solution is same Step two in embodiment 1, being used in combination 1% chicken red blood cell to carry out hemagglutination activity analysis, (method is shown in " Guo Yuanji etc.《Influenza Virus and its experimental technique》, Beijing, Chinese Sanxia Publishing House, 1997).Picking has corresponding gram of the broken bacterium solution of hemagglutination activity Grand (recombinant yeast pichia pastoris monoclonal) is used for the preparation of H7N9 influenza virus hemagglutinin glycoprotein polymer.
Three, Engineering Yeast ferments
Seed culture method and fermentation culture method are only substituted derivant by galactolipin with the step three in embodiment 1 Methanol.
Four, the purification process of H7N9 influenza hemagglutinins glycopolypeptide multimer object nano particle, will with the step four in embodiment 1 It is denoted as(H7N9)HA(K.lactis)。
Five, the structure analysis method of influenza hemagglutinin glycopolypeptide multimer object nano particle is the same as the step five in embodiment 1.
Restore SDS-PAGE analyses(H7N9)HA(K.lactis)(HA0 monomers) molecular weight is about 66KD, after cutting off glycosyl with PNGF Molecular weight and molecular weight is 61KD, as a result as shown in figure 18.
Six, to H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles(H7N9)HA(K.lactis)Carry out molecular-exclusion chromatography (SEC) it analyzes, method is with the step eight in embodiment 1, as a result as shown in figure 19.In Figure 19, upper figure is molecular weight marker proteins Chromatogram, figure below is(H7N9)HA(K.lactis)Chromatogram, find(H7N9)HA(K.lactis)Retention time less than 670kD point The retention time of sub- amount standard, the influenza hemagglutinin glycopolypeptide multimer object nano particle are the polymers that molecular weight is more than 670kD, Since the molecular weight of HA0 monomers is 64KD, the molecular weight of tripolymer is about 180KD, explanation(H7N9)HA(K.lactis)For contain 9 with The polymer that upper HA0 monomers are formed.
Seven, by the H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles of preparation(H7N9)HA(K.lactis)With 50 after negative staining, 000 times of electron micrograph finds that the influenza hemagglutinin is the polymer particle that the above HA0 tripolymers of at least three are formed.
Eight, to H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles(H7N9)HA(K.lactis)Carry out chicken red blood cell blood clotting Activity analysis, method is with the step seven in embodiment 1, and as a result as shown in figure 20, Tu20Zhong, first row is from 1 from left to right：20 What 2 times of beginning was serially diluted(H7N9)HA(K.lactis), second row is saline control, is found(H7N9)HA(K.lactis)With good Hemagglutination activity.
Nine, the H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared with recombination lactic acid kluyveromyces(H7N9) HA(K.lactis)Prepare influenza vaccines
It will(H7N9)HA(K.lactis)It is diluted to 0 μ g, 6 μ g, 12 μ g/50 μ tri- different dosage of l respectively with the PBS of pH7.4 Group, meanwhile, every group is added isometric 1.2mg/mlAl (OH)3Influenza vaccines are made in adjuvant, are experimental group injection.Together When, if being not added in above-mentioned solution(H7N9)HA(K.lactis)Solution be control group injection.
The step of standard hemagglutinin that immunization protocol, mode, blood sampling time and blood clotting inhibit, is the same as embodiment 2.
The blood clotting inhibitory activity result of neutralizing antibody is as shown in figure 21 in serum.
Figure 21 shows to carry out blood clotting inhibitory activity analysis, the average blood of experimental mice to 7 days after booster immunization serum It is solidifying that potency is inhibited to reach 1:320.
Therefore, the H7N9 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared with recombination lactic acid kluyveromyces(H7N9) HA(K.lactis)It can be used for preparing influenza vaccines.
H3N2 influenza hemagglutinin glycopolypeptide multimer object nano particles prepared by embodiment 6, Pichia pastoris
One, the structure of recombinant expression carrier
(1) according to Genbank (A/reassortant/NYMC X-223A (Texas/50/2012x PuertoRico/ 8/1934) (H3N2)) influenza HA full length sequence, synthesis SEQ ID are obtained on (title of the PLSCONFM virus is accurate) DNA molecular shown in No.18.
(2) following primer is designed and synthesized：
HA3-5：5’-ATCTTCGAAACGatgaagactatcattgcttt-3’(SEQ ID No.19)
Sequence shown in underscore is NspV digestion recognition sites.
HA3-3：5’-gatgcggccgctcaaatgcaaatgttgcacctaatgttgccctt-3’(SEQ ID No.20)
Sequence shown in underscore is NotI digestion recognition sites.
(3) it using DNA molecular is template shown in SEQ ID No.18, using HA3-5 and HA3-3 as primer, utilizes Pyrobest archaeal dna polymerases carry out PCR amplification, obtain pcr amplification product, as shown in SEQ ID No.21,.
DNA molecular shown in SEQ ID No.21 contains Kozak sequences, signal peptide, HA gene sequences successively from N-terminal to C-terminal It arranges (containing C-terminal transmembrane domains sequence)
In SEQ ID No.21 from 5 ' ends the 8th to the 12nd be Kozak sequences, the 13rd to the 60th be signal peptide Sequence, the 61st to the 1713rd is HA genes, and the 1600th to the 1674th is C-terminal transmembrane domains sequence.
DNA molecular shown in NspV and NotI double digestion SEQ ID No.21, obtains genetic fragment；The bis- enzymes of NspV and NotI It cuts pPICZ α carriers and obtains carrier large fragment；Genetic fragment is connect with carrier large fragment, recombinant plasmid is obtained, is named as PPICZ α-HA3 pPICZ α-HA3 are sequenced, as a result correctly.
Two, the construction and screening of recombination yeast
It by about 10 μ g pPICZ α-HA3 plasmids, is linearized with BglII, and converts Pichia yeast X-33, finally had There is the corresponding clone's (recombinant yeast pichia pastoris monoclonal) of the broken bacterium solution of hemagglutination activity, is used for H3N2 influenza hemagglutinin glycopolypeptide multimers The preparation of object nano particle, specific method is the same as the step two in embodiment 1.
Three, Engineering Yeast fermentation is the same as the step three in embodiment 1.
Four, the purifying of H3N2 influenza hemagglutinins glycopolypeptide multimer object nano particle is remembered with the step four in embodiment 1 For(H3N2)HA。
Five, reduction SDS-PAGE analyses((H3N2)HA molecular weight is about 66KD, which is H3N2 influenza hemagglutinin glycoprotein The HA0 components of polymer nano particle.
It is handled with N- glycosidases F (PNGF)(H3N2)HA, method is with the step five in embodiment 1, before and after analysis glycosyl excision The change of molecular weight, it is found that the molecular weight and molecular weights of hemagglutinin glycoprotein h A0 components is 61kD after cutting off glycosyl with PNGF, as a result such as Shown in Figure 22.
Six, to H3N2 influenza hemagglutinin glycopolypeptide multimer object nano particles(H3N2)HA carries out molecular-exclusion chromatography (SEC) point As a result analysis, method show H3N2 influenza hemagglutinin glycopolypeptide multimer object nano particles with the step eight in embodiment 1(H3N2)HA is Molecular weight is more than the polymer of 670kD.
Seven, to H3N2 influenza hemagglutinin glycopolypeptide multimer object nano particles(H3N2)HA carries out chicken red blood cell hemagglutination activity point Analysis, method is with the step seven in embodiment 1, and as a result as shown in figure 23, Tu23Zhong, first row is from 1 from left to right：20 start 2 It is serially diluted again((H3N2)HA, second row are saline control, are found(H3N2)HA has good hemagglutination activity.
Eight, H3N2 influenza hemagglutinin glycopolypeptide multimer object nano particles are prepared with 2 identical method of embodiment(H3N2)HA flows Influenza vaccine, immune mouse, and take a blood sample in booster immunization the latter week serum carries out blood clotting inhibition (method is shown in " Guo Yuanji etc. 《Influenza virus and its experimental technique》, Beijing, Chinese Sanxia Publishing House, 1997 "), as a result as shown in figure 24.
In Figure 24, ordinate is that blood clotting inhibits potency (HI), and abscissa is is grouped, and 0.0 is control group, and 7.5 be experimental group.
Figure 24 shows that the average blood clotting of experimental mice serum inhibits potency to reach 1:320.
Therefore, the H3N2 influenza hemagglutinin glycopolypeptide multimer object nano particles that prepared by Pichia pastoris(H3N2)HA can be used for making Standby influenza vaccines.
Embodiment 7, influenza hemagglutinin glycopolypeptide multimer object nano particle cation-exchange chromatography
The sample solution that (one) of step 4 in embodiment 1 obtains only is carried out to the cation chromatography column purification of step (2), 15%C liquid eluting peaks are obtained, which are subjected to hemagglutination activity detection, method is with step 7 in embodiment 1, as a result such as Figure 25 institutes Show.
In Figure 25, first row is from 1 from left to right：20 start 2 times of 15%C liquid eluting peaks being serially diluted, and second row is Saline control.
Figure 25 shows to contain influenza hemagglutinin glycopolypeptide multimer object nano particle in the component, and has hemagglutination activity.
Embodiment 8, influenza virus hemagglutinin protein polymer nano particle anion-exchange chromatography
The sample solution that (one) of step 4 in embodiment 1 is obtained directly carries out step (3) anion-exchange chromatography, obtains To 10%C liquid eluting peaks, hemagglutination activity detection is carried out, method is with step 7 in embodiment 1, as a result as shown in figure 26.
In Figure 26, first row is from 1 from left to right：20 start 2 times of 10%C liquid eluting peaks being serially diluted, and second row is Saline control.
Figure 26 shows to contain influenza hemagglutinin glycopolypeptide multimer object nano particle in the component, and has hemagglutination activity.
Embodiment 9, influenza virus hemagglutinin protein polymer nano particle gel exclusion chromatography
The sample solution that (one) of step 4 in embodiment 1 obtains only is carried out to the gel exclusion chromatography chromatography of step (4), First main elution peak is collected, hemagglutinin activity detection is carried out to it, method is with step 7 in embodiment 1, as a result such as Figure 27 institutes Show.
In Figure 27, first row is from 1 from left to right：20 start 2 times of main elution peaks being serially diluted, and second row is physiology salt Water compares.
Figure 27 shows to contain influenza hemagglutinin glycopolypeptide multimer object nano particle in the component, and has hemagglutination activity.
Embodiment 10, ELISA method measure the influenza virus hemagglutinin albumen collected after different detergents dissolve in supernatant Polymer nano granules content
After step 3 obtains zymotic fluid in embodiment 1, by zymotic fluid in 4 DEG C, 7000rpm centrifuges 20min.It is resuspended with water For the suspension of 40g/100ml, high-pressure homogenization instrument breaks bacterium (1200bar breaks bacterium 3 times), homogenate is obtained, by yeast 500ml homogenates In plus 50g PEG2000, stirring and dissolving 0.5h, 7000rpm centrifuge 20min, collect precipitation, abandon supernatant；Precipitation plus 500ml volumes Solution (solution Tris-HCl containing 10mM, (Qula is logical, tween, Nonidet P40 or weaker ion for detergent Type detergent dexycholate, CHAPS (3- [(3- courages amidopropyl) dimethylammonio] -1- propane sulfonic acid salt)), 5g/100ml Glycerine, surplus are water) it is resuspended, stirring and dissolving 2h.Ultracentrifugation 2h collects supernatant, and it is 6.0 to adjust pH with phosphate, is taken each Sample does elisa assay.
Elisa assay method measures hemagglutinin content (method reference Wang Jiazheng, Fan Ming chief editor collected in supernatant《Albumen Matter technical manual》(Science Press, 2000))：Sample antigen coat liquid (0.15g/100ml Na2CO3, 0.293g/ 100ml NaHCO3, pH9.6, surplus is water) and 100 times of dilution, then respectively take 100 μ L samples to be added in elisa plate, 4 DEG C were coated with Night, primary antibody are rabbit-anti HA7 (1:500) (antibody is purchased from Beijing Bo Feikang Bioisystech Co., Ltd), secondary antibody (are diluted) with PBS For goat anti-rabbit igg-HRP (1:1000) (antibody is purchased from magnificent bioengineering Co., Ltd) (is diluted) with PBS, is added per hole 100 μ L developing solutions (1.84g/100ml Na2HPO4·12H2O, 0.5g/100ml citric acid add 0.04g/100ml again before use The H of OPD and volumn concentration 0.15%2O2), 37 DEG C of Incubation in dark 15min are last to add 20 μ L terminate liquids (2M per hole H2SO4) with color development stopping, certain color change is presented wherein in positive colony hole, reflects the content of HA, blank is (with no blood The hole of solidifying element sample is blank) and negative hole in without apparent color change.Data are read on enzyme-linked instrument at the 492nm, are tied Fruit is as shown in table 1.
Table 1 shows that various detergents are effective, can detect that the influenza virus hemagglutinin protein polymer in supernatant is received Rice grain, wherein 2g/100ml TritonX-100 (Qula logical) effect is best, secondly 5g/100ml Tween20 (tween) and 2g/100ml dexycholates are 2g/100ml NP-40 (Nonidet P40) and 2g/100ml CHAPS again, but Without detergent-treatment, it is nearly no detectable influenza virus hemagglutinin protein polymer nano particle, illustrates that detergent-treatment is One of important step in the present invention.
It is poly- that 1 elisa assay method of table measures the influenza virus hemagglutinin albumen collected in supernatant after different detergent dissolvings Close object nano-particle content

Claims (10)

1. a kind of method preparing influenza virus hemagglutinin glycoprotein polymer nano granules, includes the following steps：Make N-terminal upstream Containing signal peptide sequence and include that the hemagglutinin gene of influenza virus of the ends C- transmembrane domains sequence is expressed in yeast；By institute It states yeast and carries out clasmatosis, detergent is added, obtain the solution of the glycoprotein containing influenza virus hemagglutinin；The solution is carried out Purifying, is prepared the influenza virus hemagglutinin glycoprotein polymer nano granules with hemagglutination activity.
2. according to the method described in claim 1, it is characterized in that：It is described so that N-terminal upstream is contained signal peptide sequence and include C- It is that signal peptide sequence is contained in N-terminal upstream to hold the method that the hemagglutinin gene of the influenza virus of transmembrane domains sequence is expressed in yeast The recombinant expression carrier transformed yeast for arranging and include the hemagglutinin gene of the influenza virus of the ends C- transmembrane domains sequence, is cultivated To the yeast of conversion, then induces and make gene expression；
The yeast is Pichia pastoris, Hansenula yeast or Kluyveromyces lactis.
3. method according to claim 1 or 2, it is characterised in that：The hemagglutinin HA of the influenza virus is H1, H3, H5 Or the HA of H7 serotype influenza viruses.
4. according to the method described in claim 3, it is characterized in that：The HA of H1, H3, H5 or H7 serotype influenza virus points Not Wei H1N1, H3N2, H5N1 or H7N9 influenza virus HA.
5. according to the method described in claim 4, it is characterized in that：The recombinant expression carrier is to contain the N-terminal upstream Signal peptide sequence and include the ends C- transmembrane domains sequence influenza virus hemagglutinin gene be inserted into the load containing AOX promoters Body obtains, and signal peptide sequence is specifically contained in the N-terminal upstream and includes the blood clotting of the influenza virus of the ends C- transmembrane domains sequence Plain HA genes are inserted between NotI the and NspV restriction enzyme sites of pPICZ α, and BglII is used in combination to linearize to obtain；
The yeast is Pichia pastoris；
The influenza virus is H7N9 avian influenza virus, and the N-terminal upstream contains signal peptide sequence and includes the ends C- transmembrane domains sequence The hemagglutinin gene of the influenza virus of row is as shown in SEQ ID No.4；
Or,
The influenza virus is H1N1virus, and the N-terminal upstream contains signal peptide sequence and includes the ends C- transmembrane domains The hemagglutinin gene of the influenza virus of sequence is as shown in SEQ ID No.14；
Or,
The influenza virus is A type H3N2 influenza viruses, and the N-terminal upstream contains signal peptide sequence and includes the ends C- transmembrane domains The hemagglutinin gene of the influenza virus of sequence is as shown in SEQ ID No.21.
6. according to the method described in claim 4, it is characterized in that：The recombinant expression carrier is to contain the N-terminal upstream Signal peptide sequence and include the ends C- transmembrane domains sequence influenza virus hemagglutinin gene be inserted into the load containing MOX promoters Body obtains, and signal peptide sequence is specifically contained in the N-terminal upstream and includes the blood clotting of the influenza virus of the ends C- transmembrane domains sequence Plain HA genes are inserted between NotI the and BstB I restriction enzyme sites of pPICZ α, obtain intermediate carrier 1；The AOX of intermediate carrier 1 is opened again Mover replaces with the alcohol oxidase promoter MOX of Hansenula yeast, then linearizes to obtain with BglII；
The yeast is Hansenula yeast；The Hansenula yeast is Hansenula polymorpha；
The MOX promoters are using the genomic DNA of Hansenula polymorpha as template, with SEQ ID No.9 and SEQ ID DNA molecular shown in No.10 is that primer progress PCR amplification obtains MOX promoters；
The method of the alcohol oxidase promoter MOX that the AOX promoters of intermediate carrier 1 are replaced with to Hansenula yeast is will be described The MOX promoters that PCR amplification obtains carry out BglII digestions, and carry out phosphorylation, and it is BglII digestion cohesive ends to obtain 5 ' ends, The MOX promoters of 3 ' end phosphorylations；Will intermediate carrier 1 with after NspV single endonuclease digestions with Klenow fragment klenow fragments and DNTP filling-in, obtains segment, after recycling segment, then uses BglII digestions, cuts off AOX promoters, obtains cutting off AOX promoters Intermediate carrier 1；It is BglII digestion cohesive ends by 5 ' ends, 3 ' hold in the MOX promoters of phosphorylations and excision AOX promoters Between carrier 1 connect；
The influenza virus is H5N1 avian influenza virus, and the N-terminal upstream contains signal peptide sequence and includes the ends C- transmembrane domains sequence The hemagglutinin gene of the influenza virus of row is specific as shown in SEQ ID No.8.
7. according to the method described in claim 4, it is characterized in that：The recombinant expression carrier is to contain the N-terminal upstream Signal peptide sequence and include the ends C- transmembrane domains sequence influenza virus hemagglutinin gene be inserted into the load containing LAC4 promoters Body obtains, and signal peptide sequence is specifically contained in the N-terminal upstream and includes the blood clotting of the influenza virus of the ends C- transmembrane domains sequence Plain HA genes are inserted between HindIII the and NotI restriction enzyme sites of pKLAC1, then linearize to obtain with SacII；
The yeast is Kluyveromyces lactis；
The influenza virus is H7N9 avian influenza virus, and the N-terminal upstream contains signal peptide sequence and includes the ends C- transmembrane domains sequence The hemagglutinin gene of the influenza virus of row is as shown in SEQ ID No.17.
8. according to the method described in claim or 2, it is characterised in that：The method of the clasmatosis is physical method, biology Method or chemical method；
The detergent is non-ionic detergent or weak ionic detergent.
9. according to the method described in claim 8, it is characterized in that：The physical method is bead succusion, high-pressure homogenization Method or ball-milling method；
The biological method is enzymatic lysis method；
The chemical method is alkaline lysis；
The non-ionic detergent is logical Qula, tween or Nonidet P40；
The weak ionic detergent is dexycholate or 3- [(3- courages amidopropyl) dimethylammonio] -1- propane sulfonic acid Salt.
10. method according to claim 1 or 2, it is characterised in that：The method that the solution is purified include sun from Sub- displacement chromatography and/or anion-exchange chromatography and/or gel exclusion chromatography；
The filler of the cation-exchange chromatography is Sepharose FF SP；
The filler of the anion-exchange chromatography is Source 30Q；
The filler of the gel exclusion chromatography is Superdex200；
Whether there are HA bands and hemagglutination activity to judge whether it is containing according to each step purified components in the purification process The component for stating influenza virus hemagglutinin glycoprotein polymer nano granules, if the component has HA bands and hemagglutination activity for containing There is the component of the influenza virus hemagglutinin glycoprotein polymer nano granules.
CN201410328217.5A 2014-07-10 2014-07-10 A kind of preparation method of influenza hemagglutinin glycopolypeptide multimer object nano particle Active CN105233302B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201410328217.5A CN105233302B (en) 2014-07-10 2014-07-10 A kind of preparation method of influenza hemagglutinin glycopolypeptide multimer object nano particle
PCT/CN2015/088748 WO2016004904A2 (en) 2014-07-10 2015-09-01 Method for preparing influenza hemagglutinin glycoprotein polymer nanoparticles

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201410328217.5A CN105233302B (en) 2014-07-10 2014-07-10 A kind of preparation method of influenza hemagglutinin glycopolypeptide multimer object nano particle

Publications (2)

Publication Number Publication Date
CN105233302A CN105233302A (en) 2016-01-13
CN105233302B true CN105233302B (en) 2018-10-19

Family

ID=55031286

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201410328217.5A Active CN105233302B (en) 2014-07-10 2014-07-10 A kind of preparation method of influenza hemagglutinin glycopolypeptide multimer object nano particle

Country Status (2)

Country Link
CN (1) CN105233302B (en)
WO (1) WO2016004904A2 (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106868025B (en) * 2017-03-13 2020-01-21 中国人民解放军军事医学科学院生物工程研究所 Method for preparing tripolymer Ebola virus glycoprotein mutant by using yeast
CN107034225B (en) * 2017-05-04 2020-01-17 中国人民解放军军事医学科学院生物工程研究所 Method for preparing fusion mutant of ebola virus glycoprotein and matrix protein
IT202100024620A1 (en) 2021-09-27 2023-03-27 Graficonsul S R L MODULAR TILE FOR DISPLAY OF ADVERTISING PRODUCTS

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101405026A (en) * 2006-02-02 2009-04-08 全球免疫股份有限公司 Yeast-based vaccine for inducing an immune response
CN103209990A (en) * 2010-10-30 2013-07-17 刘大才 Recombinant hemagglutinin protein of influenza virus and vaccine containing the same

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK2445928T3 (en) * 2009-06-24 2018-05-28 Medicago Inc CHEMICAL INFLUENZA VIRUS-LIKE PARTICLES INCLUDING HEMAGGLUTIN
CN104711278A (en) * 2013-12-16 2015-06-17 特菲(天津)生物医药科技有限公司 Recombinant sequence containing H7N9 virus HA gene, recombinant baculovirus and application of virus in vaccine preparation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101405026A (en) * 2006-02-02 2009-04-08 全球免疫股份有限公司 Yeast-based vaccine for inducing an immune response
CN103209990A (en) * 2010-10-30 2013-07-17 刘大才 Recombinant hemagglutinin protein of influenza virus and vaccine containing the same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Yeast expressed recombinant Hemagglutinin protein of Novel H1N1 elicits neutralizing antibodies in rabbits and mice;TN Athmaram et.al.;《Virology Journal》;20111231;第524卷(第8期);摘要、第1页右栏最后1段、第3页右栏第1段、第9-10页、Figure 1 *
甲型H1N1流感病毒血凝素HAl在糖基工程毕赤酵母中的表达;张晓红等;《生物技术通讯》;20120131;第23卷(第1期);第1-5页 *

Also Published As

Publication number Publication date
CN105233302A (en) 2016-01-13
WO2016004904A2 (en) 2016-01-14
WO2016004904A3 (en) 2016-03-17

Similar Documents

Publication Publication Date Title
CN108159409A (en) A kind of 3 type Cap protein vaccine of pig circular ring virus and its preparation method and application
CN110760006A (en) African swine fever immune system targeted genetic engineering vaccine
CN113512096B (en) Weever rhabdovirus recombinant G2 protein and application thereof
CN103992988B (en) A kind of monoclonal antibody of the anti-canine distemper disease viral N proteins of hybridoma cell strain and generation thereof
CN104524564B (en) A kind of crucian Simplex Virus combination vaccine preparation and preparation method and application
CN111393531A (en) Subunit fusion protein CD2V-Fc and preparation method and application thereof
CN105233302B (en) A kind of preparation method of influenza hemagglutinin glycopolypeptide multimer object nano particle
CN107973841B (en) Preparation method and application of recombinant bovine viral diarrhea virus E2 protein expressed by CHO (Chinese hamster ovary) cell and subunit vaccine
Yang et al. Lactobacillus plantarum displaying conserved M2e and HA2 fusion antigens induces protection against influenza virus challenge
CN104593388B (en) Crucian herpesvirus disease JDORF25 vaccine as well as preparation method and application thereof
CN110272473A (en) General virus-like particle of Flu-A and its preparation method and application
CN101015691B (en) Recombinant phages influenza vaccine
CN101879312B (en) Broad spectrum type influenza vaccine and preparation method thereof
CN102816246B (en) Human cytomegalo virus immunogen fusion protein as well as preparation method and usage thereof
CN102397559B (en) Broad spectrum type influenza vaccine and preparation method thereof
CN108348596A (en) With the recombinant virus sample particle of bovine immunodeficiency virus Gag albumen
CN105671109B (en) There is the method for the glycosylation modified influenza hemagglutinin glycoprotein of zooblast with the preparation of glycosyl Engineering Yeast
CN108939061A (en) A kind of multicomponent B group meningitis cocci vaccine and preparation method thereof
CN112142827B (en) gB subunit recombinant protein of porcine pseudorabies virus, and preparation method and application thereof
CN113827714B (en) H7N9 subtype avian influenza virus-like particle vaccine preparation, preparation and application
CN103554233B (en) Duck plague virus (DPV) UL13 intercepted recombinant protein and polyclonal antibody, preparation method and application thereof
CN107460173A (en) A kind of method for purifying Porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus or avian influenza virus
CN104152477A (en) Schistosoma japonicum recombinant antigen SjPDI (schistosoma japonicum protein disulfide isomerase) and preparation method and application thereof
CN113861277A (en) Bovine rotavirus recombinant VP8 protein and application thereof
CN102190701B (en) Method for separating and purifying influenza virus hemagglutinin on large scale

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant