CN109666067A - A kind of recombinant virus sample particle and its preparation method and application - Google Patents
A kind of recombinant virus sample particle and its preparation method and application Download PDFInfo
- Publication number
- CN109666067A CN109666067A CN201910013184.8A CN201910013184A CN109666067A CN 109666067 A CN109666067 A CN 109666067A CN 201910013184 A CN201910013184 A CN 201910013184A CN 109666067 A CN109666067 A CN 109666067A
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- virus
- hepatitis
- core antigen
- recombinant
- virus core
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
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- A61K2039/70—Multivalent vaccine
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- 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
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- 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
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- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10123—Virus like particles [VLP]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32311—Enterovirus
- C12N2770/32334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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Abstract
The present invention provides a kind of recombinant virus sample particles and its preparation method and application, described method includes following steps: collecting tunning after the cell for expressing recombinant virus capsid protein is fermented, depolymerization is dissolved using the urea solution of 2-6M, extracellular secondary assembling obtains the intact virus sample particle of recombinant virus capsid protein.The recombinant capsid protein of ammonium sulfate precipitation is dissolved simultaneously depolymerization, purifying, last extracellular secondary virus-like particle of the assembling formation with natural structure by the preparation method of recombinant hepatitis B virus core antigen particles of the invention under relatively mild conditions.This method is simple, efficiently, and prepared recombinant hepatitis B virus core antigen particles have the advantages that structural integrity, uniform and be free of host nucleic acids and foreign protein, is of great significance and wide application prospect.
Description
Technical field
The invention belongs to biomedicine technical field, it is related to a kind of recombinant virus sample particle and its preparation method and application,
Relate generally to a kind of recombinant virus sample particle and its preparation method and application of soluble form expression, and in particular to a kind of recombination second
Hepatovirus core antigen particles and its preparation method and application.
Background technique
Hepatitis B virus core antigen (HBc) is single-stranded capsid protein, can form the regular dodecahedron structure of free nucleic acid alone,
With the form similar with natural viral, size about 28nm.The surface that the dominant region of its principal immune (MIR) is located at particle is formed
Furcella, can effective present antigen, excite strong humoral immunity and cellular immunity.Furthermore the insertion of the area Qi MIR or replacement
For one section of other antigen, it will not influence it and form virus-like spheric granules structure.These advantages make HBc become virus-like
The vaccine carrier of most purposes prospect in grain (VLP).On the other hand the hollow structure of the HBc-VLP nano-scale without nucleic acid
Small-molecule drug or developer can be efficiently loaded, there is wide purposes prospect in terms of drug delivery and clinical diagnosis.
Currently, HBc-VLP successfully obtains expression in recombined eukaryotic cell and prokaryotic cell, the former is such as Africa xenopus
Xenopus oocytes, plant Ben Shi cigarette and Pichia pastoris etc., the latter such as Escherichia coli.Due to the complexity of virus-like particle structure
Property, tradition preparation mostly uses eukaryotic expression system to carry out culture expression, using the Protein processing ability of Higher cells itself, in born of the same parents
Interior completion particle assembling, is then separated in the form of assembly with other impurity of host.But it is this to prepare strategy there are following
Problem: first is that the particle of assembling intracellular there are structures inhomogenous, the incomplete phenomenon of particle, either thin by the Africa xenopus frog
Born of the same parents, plant or yeast cell to express, product is in addition to whole grain, and there is also a large amount of dimer, assembling intermediates even
It is the structure of mistake assembling, the vaccine quality after leading to direct purification is low, and activity is low, and it is low to be easy to aggregation stability.Second is that pure
Change process is easily destroyed particle assembly structure, since the active force between particle assembling body protein is weaker, with medium absorb-elute
Interaction the structure of assembly can be damaged, make partial particulate depolymerization, lead to purify yield and activity being greatly reduced.
Third is that partial impurities are not easy to remove, there are security risks for the vaccine product of direct purification, no matter to mammalian cell or elder brother
Worm cell is easy to host DNA or RNA and host protein being wrapped in inside in intracellular assembled, assembling intracellular
Protein body is also possible to be combined together with host protein.In order to remove such impurity, it is required to open host protein and particle
Combination and open original grain structure, this will necessarily bring change and destruction to the structure of completed assembled body so that
The complete depolymerization of grain.
Determine that protein steric conformation is identical with primary amino acid sequences, unique physicochemical property of viral capsid proteins determines
Capsid protein has in the extracellular ability for being self-assembled into virus-like particle.It is avoided that using assembling particle after first purifying subunit unit
Chromatography process can completely remove the impurity such as host nucleic acids to assembly structural damage, can also be significant by extracellular secondary assembling
Improvement granule-morphology improves homogeneity etc., therefore is expected to solve the upper of traditional vaccine preparation appearance using extracellular self-assembling method
State problem.It has been reported that the recombination HBc sucrose density gradient centrifuge results of Bacillus coli expression show and surpass from sample
Very wide distribution is presented in HBc sedimentation coefficient, there are the unassembled structure such as a large amount of dimer, show product structure it is serious not
It is uniform.Therefore the preparation of HBc-VLP takes extracellular self-assembling technique to be very important, complete to reach efficient, pure, particle
Purpose whole, structure is uniform.
“Wizemann,H.and Brunn,A.V.,Purification of E.coli-expressed HIS-
tagged hepatitis B core antigen by Ni2+-chelate affinity chromatography,
Journal of Virological Methods, 1999,189-197 " disclose a kind of side of assembled in vitro virus-like particle
Method, truncated HBc-VLP can the depolymerization under the conditions of 2M urea, pH 9.5, Ni FF assembles obtain VLP after purification, regrettably,
This method either purifies or assembles that equal yield is lower, and last product quality is not also high.
101848730 A of CN discloses the preparation that the above method is used to merge HBc-VLP, mainly urinates in denaturant
Element or guanidine hydrochloride existence condition under purify, then again re-assemble formed VLP, but existing method cannot obtain it is similar to natural structure
Viroid sample HBc particle.
The structure of virus-like particle is applied to important influence, therefore it provides a kind of easy to operate, high income,
Purification effect is good, and the uniform complete recombinant virus sample preparation method of granules of virus-like particle structure is of great significance.
Summary of the invention
In view of the deficiencies of the prior art and actual demand, the present invention provides a kind of recombinant virus sample particle and its preparation side
Method and purposes, recombinant virus sample particle of the invention are free of host nucleic acids, and with high purity and structural integrity is uniform, with natural viral shape
State is identical, and preparation method is simple, high income, and purification effect is good, is of great significance and wide application prospect.
To achieve this purpose, the present invention adopts the following technical scheme:
In a first aspect, the present invention provides a kind of preparation method of recombinant virus sample particle, described method includes following steps:
After the cell for recombinant virus capsid protein expressing is fermented collect tunning, it is molten using the urea solution of 2-6M
Depolymerization is solved, extracellular secondary assembling obtains the intact virus sample particle of recombinant virus capsid protein.
It include intact virus in tunning by gene recombination technology expression hepatitis B virus core antigen in the present invention
It also include a large amount of incomplete virus-like particles while sample particle, direct purification tunning yield is low, obtained virus-like
Seed activity is poor, and comprising nucleic acid, there are security risks, seriously affect subsequent applications, and the present invention uses the urea solution of 2-6M, dissolution
It is ill that depolymerization institute can be both resuspended in complete and incomplete virus-like particle in depolymerization tunning within the scope of the urea concentration
Malicious sample particle, while two three-level space conformations of antigen protein will not be caused to destroy, it prepares for extracellular secondary assembling.Urea concentration
It is too low cannot effective depolymerization virus-like particle, and urea concentration is excessively high, is easy to change second level, the tertiary structure of albumen, generates hydrophobic
Aggregation, cannot carry out extracellular assembling again, or the grain structure being assembled into is different from natural viral particle, lose its application meaning
Justice.
Preferably, the recombinant virus capsid protein includes recombinant hepatitis B virus core antigen.
Method of the invention is suitable for all recombinant virus sample particles expressed with soluble form, is particularly suitable for recombination second
Hepatovirus core antigen particles.
Preferably, the recombinant hepatitis B virus core antigen include recombinant hepatitis B virus core antigen truncate and/or
Recombinant hepatitis B virus core antigen overall length containing other antigens.
Preferably, the truncate and other Antigen Fusions are expressed.
Preferably, the recombinant hepatitis B virus core antigen overall length and other Antigen Fusions are expressed.
Method of the invention is particularly suitable for extracellular secondary assembling recombinant hepatitis B virus core antigen particles, wherein can be with
It is expressed after directly hepatitis B virus core antigen is truncated, it can also be by the overall length of hepatitis B virus core antigen or its truncate
It is expressed after carrying out Gene Fusion with other epitopes.
Preferably, the recombinant protein expression system used that recombinates includes eukaryotic expression system and/or prokaryotic expression system
System.
Preferably, the eukaryotic expression system includes in Africa xenopus Xenopus oocytes, plant Ben Shi cigarette or Pichia pastoris
It is any or at least two combination.
Preferably, the prokaryotic expression system includes Escherichia coli.
Preferably, the concentration of the urea solution is 3.5-4.5M, preferably 4M.
In the present invention, the final concentration of urea solution, can virus-like effectively in depolymerization tunning within the scope of 3.5-4.5M
Particle is conducive to the purifying yield and purity that improve following protein, and does not influence the efficiency of extracellular secondary assembling.
Preferably, the pH value of the urea solution is 7.0-9.0, for example, can be 7.0,7.2,7.5,7.8,8.0,8.2,
8.5,8.8 or 9.0.
In the present invention, the pH of urea solution is neutral or alkalescent, pH value it is too low cannot effective depolymerization tunning, pH value mistake
Height easily causes albuminous degeneration, only in the pH value range, cooperates preparation process of the invention, can just obtain structural integrity
Uniform virus-like particle.
Preferably, the collection tunning specifically comprises the following steps:
(1) supernatant is collected by centrifugation in smudge cells；
(2) ammonium sulfate is added in supernatant, stirs, precipitating is collected in centrifugation, obtains the tunning.
Preferably, the mode of step (1) described smudge cells includes ultrasonication and/or high-pressure homogenization.
Preferably, the final concentration of 0.05-4M of ammonium sulfate described in step (2), for example, can be 0.05M, 0.1M,
0.2M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.5M, 2M, 2.5M, 3M, 3.5M or 4M, preferably 0.1-1M.
Preferably, step (2) the ammonium sulfate pH value is 7.0-8.0, for example, can be 7.0,7.2,7.4,7.6,
7.8、8.0。
Preferably, the temperature of step (2) described stirring is 1-6 DEG C, such as can be 1 DEG C, 2 DEG C, 3 DEG C, 4 DEG C, 5 DEG C or 6
DEG C, preferably 4 DEG C.
Preferably, the extracellular secondary assembling includes the following steps:
(1 ') assembling unit after depolymerization is purified；
(2 ') dialysis reduces urea concentration, adjust the assembling pH value of buffer solution, ionic strength, protein concentration, temperature and
Time obtains the intact virus sample particle of recombinant virus capsid protein by extracellular assembling.
Preferably, step (the 1 ') purification process includes affinity chromatography, ion exchange or gel filtration, preferably affine
Chromatography.
Preferably, the affinity chromatography includes nickel ion metal chelate affinity chromatography (Ni Sepharose FF
) and/or heparin affinity chromatography (6 Fast Flow of Heparin Sepharose) cOmplete.
Preferably, step (2 ') the reduction urea concentration is by the way of gradient dialysis.
Include de-assembly unit and the urea of 4M in the present invention, in the solution after depolymerization, needs to remove change before assembling
Property agent-urea, can directly thoroughly to assembling buffer in, a step removal urea while start to assemble；It can also be dialysed with gradient, first will
Solution is dialysed into Tris elution buffer, reduces the urea concentration in original solution, the low concentration urea in Tris elution buffer can
Preferably to maintain the stability of assembling unit, when urea concentration is dialysed to 2M in solution, then it is dialyzed to assembling buffer
In assembled, be conducive to efficiently assemble complete recombinant virus sample particle, packaging efficiency is up to 95%.
Preferably, the gradient dialysis specifically comprises the following steps: the sample dialysis after the depolymerization for obtaining step (1 ')
Into Tris elution buffer, then dialyse into assembling buffer.
Preferably, the Tris elution buffer includes Tris-HCl and urea.
Preferably, the final concentration of the urea in the Tris elution buffer be not more than 2M, such as can be 0.5M, 1M,
1.5M or 2M.
Preferably, the pH value of the Tris elution buffer be 8.0-9.0, such as can be 8.0,8.2,8.5,8.8 or
9.0。
Preferably, step (2 ') the assembling buffer includes Tris-HCl.
Preferably, the assembling buffer further includes NaCl.
Preferably, the final concentration of 0.2-1.5M of the NaCl, for example, can be 0.2M, 0.5M, 0.8M, 1M, 1.2M or
1.5M, preferably 0.5M.
In the present invention, the aggregation between the virus-like particle that secondary assembling is formed is can be effectively suppressed in the NaCl in the range,
The effect of surfactant is substituted, while accelerating the extracellular packaging efficiency and rate of assembling unit, NaCl concentration is too low, assembles
Rate is slack-off and is easy to produce intergranular aggregation, and excessive concentration then will lead to the precipitating of albumen, influence yield.
Preferably, the pH value of step (2 ') the assembling buffer is 7.0-8.0, for example, can be 7.0,7.2,7.4,
7.5,7.8 or 8.0, preferably 7.4.
In the present invention, constituent, ionic strength, the pH value, assembling unit in buffer are assembled by combination matching
Protein concentration, temperature and time obtain the condition for being most suitable for the extracellular secondary assembling of recombinant virus sample particle, of the present invention
The packaging efficiency that can effectively improve assembling unit in condition and range obtains complete recombinant virus sample particle.
Preferably, step (the 2 ') protein concentration be 0.8-500 μM, such as can be 0.8 μM, 1 μM, 2 μM, 3 μM,
5μM、8μM、10μMμM、20μM、30μM、40μM、50μM、60μM、70μM、80μM、90μM、100μM、120μM、150μM、180μ
M, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450 μM or 500 μM, preferably 0.8-100 μM.
In the present invention, the protein concentration of the assembling unit after depolymerization is greater than 0.8 μM and is just able to satisfy assembling requirement, realizes
Extracellular assembling obtains intact virus sample particle.
Preferably, step (the 2 ') temperature is 1-6 DEG C, such as can be 1 DEG C, 2 DEG C, 3 DEG C, 4 DEG C, 5 DEG C or 6 DEG C, excellent
It is selected as 4 DEG C.
Preferably, step (the 2 ') time no less than for 24 hours, such as can be for 24 hours, 26h, 28h, 32h, 40h, 45h,
48h, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days,
19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days or 30 days, preferably for 24 hours -3 days.
In the present invention, by the various conditions of combinatorial regulation, the NaCl that suitable concentration is added remarkably promotes the born of the same parents of assembling unit
Outer assembling can harvest a large amount of complete recombinant virus core antigen particles after 24h, and prepared by the method for the present invention
Assembled in vitro virus-like particle can be stable in the presence of in buffer at least 30 days.
Preferably, the condition of the extracellular assembling is the Tris-HCl buffer comprising NaCl, pH of buffer 7.4, NaCl
Final concentration 0.5M, final protein concentration are greater than 0.8 μM, dialyse 4 DEG C of temperature, and dialysis time is greater than for 24 hours.
In the present invention, by adjusting extracellular assembling condition appropriate, under the cooperation of various conditions, it is anti-that virus may be implemented
Former particle is assembled in extracellular high efficient secondary, forms complete virus-like particle.
Preferably, a kind of preparation method of recombinant hepatitis B virus core antigen particles, specifically comprises the following steps:
1) pass through gene recombination technology expression hepatitis B virus core antigen；
2) tunning is collected by centrifugation, the smudge cells by way of ultrasonication or high-pressure homogenization are collected by centrifugation
Clearly；
3) final concentration 0.1-1M is added in supernatant, pH value is the ammonium sulfate of 7.0-8.0, and 4 DEG C of stirrings, it is heavy that centrifugation is collected
It forms sediment；
4) urea solution of final concentration of 4M is added in precipitating, dissolves and depolymerization is complete and incomplete hepatitis B virus core
Antigen package assembly, while two three-level space conformations of antigen protein are not destroyed；
5) assembling unit after depolymerization is purified；
6) dialysis reduces urea concentration to 2M hereinafter, being dialysed again to assembling buffer, thoroughly removes urea, wherein assembling unit
Protein concentration is greater than 0.8 μM, and assembling buffer includes the Tris-HCl, the NaCl of final concentration 0.2-1.5M, pH value of 20mM
7.0-8.0,1-6 DEG C of temperature of dialysis, dialysis time are greater than for 24 hours, obtain recombinant hepatitis B virus core antigen by extracellular assembling
Intact virus sample particle.
In the present invention, using the ammonium sulfate of neutral low concentration, target protein can be effectively precipitated, is played with foreign protein
Separating effect, and the precipitating is easy to that depolymerization is resuspended.Using the alkalescent urea solution of suitable concentration, it is complete to can be only achieved resuspension depolymerization
With incomplete grain structure, and do not cause recombinant protein to be denaturalized, generates irreversible hydrophobic aggregation.Existed using high concentration albumen
Dialysis in the Tris-HCl buffer solution of NaCl concentration 0.2-1.5M, can be realized recombination hepatitis B to alkalescent under cryogenic conditions
The extracellular high efficient secondary of virus core antigen assembles.The method of present aspect is dense by combinatorial regulation ammonium sulfate, urea and NaCl's
Degree and pH condition, the comprehensive extracellular packaging efficiency for improving hepatitis B virus core antigen particle, while improving gained recombination hepatitis B disease
The structural integrity and homogeneity of malicious core antigen particles are conducive to the application of its later period.
The present invention is by constantly groping, adjustment ammonium sulfate, urea solution, elution buffer and the pH for assembling buffer
Weight is better achieved within the scope of ammonium sulfate of the present invention, the concentration of urea solution and buffer ionic strength in value
Precipitating, depolymerization and the assembling of group virus-like particle, keep whole process milder, utmostly keep the structure of assembling unit steady
It is qualitative, extracellular secondary assembling is efficiently carried out, a large amount of complete recombinant virus sample particles are obtained.
Second aspect, the present invention provide a kind of recombinant hepatitis B virus core antigen particles, the method as described in first aspect
It is prepared.
Preferably, the recombinant hepatitis B virus core antigen particles include the truncate of hepatitis B virus core antigen.
Preferably, the truncate is at least 140 amino acid that hepatitis B virus core antigen N-terminal rises, such as be can be
140,141,142,143,144,145,146,147,148,149,150,151,152
It is a, 153,154,155,156,157,158,159,160,161,162,163,164,
165,166,167,168,169,170,171,172,173,174,175,176,177
A, 178,179,180,181,182 or 183, preferably 144-149.
Preferably, the amino acid sequence of the truncate includes the amino acid sequence as shown in SEQ ID NO.1:
MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMNL
ATWVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPIL.
183 amino acid of amino acid sequence overall length (SEQ ID NO.2) of hepatitis B virus core antigen, are verified by experiments,
Truncate includes at least 140 amino acid, so that it may is prepared by means of the present invention identical as natural viral capsid protein
The recombinant hepatitis B virus core antigen particles of form, the preferred hepatitis B virus core antigen N-terminal of the present invention play 144-149 amino acid
Truncate preparation and reorganization virus-like particle, avoid the combination rich in arginic sequence and host nucleic acids, 144 amino
The sequence of acid is as shown in SEQ ID NO.3, and the sequence of 149 amino acid is as shown in SEQ ID NO.4.But hepatitis B virus core is anti-
Former full-length proteins and not applicable this method need stronger depolymerisation conditions such as 6M guanidine hydrochloride that could open because of its stable structure,
It is not easy to carry out secondary assembling.
Hepatitis B virus core antigen full-length proteins HBc183(SEQ ID NO.2): MDIDPYKEFGASVELLSFLPSDFFP
SIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMNLATWVGSNLEDPASRELVVSYVNVNMGLKIRQLL
WFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQ
SRESQC.
The truncate HBc of hepatitis B virus core antigen144(SEQ ID NO.3): MDIDPYKEFGASVELLSFLPSDFFP
SIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMNLATWVGSNLEDPASRELVVSYVNVNMGLKIRQLL
WFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLP.
The truncate HBc of hepatitis B virus core antigen149(SEQ ID NO.4): MDIDPYKEFGASVELLSFLPSDFFP
SIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMNLATWVGSNLEDPASRELVVSYVNVNMGLKIRQLL
WFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVV.
Preferably, the recombinant hepatitis B virus core antigen particles include the hepatitis B containing other at least one epitopes
Virus core antigen full-length proteins or truncate.
Preferably, the hepatitis B virus core antigen is to merge with the connection type of other epitopes.
Preferably, the recombinant hepatitis B virus core antigen particles and natural viral capsid protein have same modality.
The third aspect, the present invention provide a kind of use of recombinant hepatitis B virus core antigen particles as described in second aspect
On the way, the purposes is including loading drug and/or preparing vaccine.
Preferably, the drug includes small molecule anticancer drug, nucleic acid drug or developer.
Preferably, the vaccine further includes other antigens.
Preferably, other described antigens are merged or are coupled with hepatitis B virus core antigen.
In the present invention, the recombinant hepatitis B virus core antigen particles being prepared by first aspect the method can be merged
Or other antigens are coupled as preventative or therapeutic vaccine.
Preferably, other described antigens include influenza virus memebrane protein M2e, hand-foot-and-mouth disease virus VP 1, hepatitis B
Any one of PreS1, hepatitis B virus Pres 2, hepatitis B HBsAg or human papilloma virus E7 or at least two combination.
In the present invention, recombinant hepatitis B virus core antigen particles belong to nano-scale particle, can target and continue in vivo
Efficiently stimulation lymph node, can be used as vaccine carrier, by the way that other epitopes are inserted into mainly exempting from for hepatitis B virus core antigen
The dominant region of epidemic disease, improves the immunogenicity of other antigens, and good biocompatibility can stimulate body to generate immune answer without adjuvant
It answers, can be used for preventative or therapeutic vaccine preparation.The typical but non-localized antigen that can be merged or be coupled includes influenza disease
The Structural protein VP1 of malicious memebrane protein M2e, hand-foot-and-mouth disease viral (FMDV), hepatitis B (HBV) envelope protein PreS1, PreS2,
The structural proteins E7 of hepatitis B virus surface antigen HBsAg or human papilloma virus (HPV).
Preferably, the method for loading drug includes the following steps:
(A) urea solution depolymerization recombinant hepatitis B virus core antigen particles as described in second aspect are used；
(B) in the hepatitis B virus core antigen solution after adding drug to step (A) depolymerization；
(C) step (B) acquired solution is dialysed in buffer, isolates and purifies to obtain the recombinant hepatitis B virus for loading drug
Core antigen particles simultaneously test and analyze.
Preferably, the final concentration of 2-4M of step (A) described urea solution, such as can be 2M, 2.5M, 3M, 3.5M or 4M.
Preferably, step (C) the assembling buffer includes Tris-HCl.
Preferably, step (C) the assembling buffer further includes NaCl.
Preferably, the final concentration of 0.2-1.5M of the NaCl, for example, can be 0.2M, 0.5M, 0.8M, 1M, 1.2M or
1.5M, preferably 0.5M.
Preferably, the temperature of step (C) described dialysis is 1-6 DEG C, such as can be 1 DEG C, 2 DEG C, 3 DEG C, 4 DEG C, 5 DEG C or 6
DEG C, preferably 4 DEG C.
Preferably, the time of step (C) described dialysis be greater than for 24 hours, such as can be for 24 hours, 26h, 28h, 32h, 40h,
45h, 48h, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days,
18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days or 30 days, preferably for 24 hours -3
It.
Preferably, the pH value of step (C) the assembling buffer is 7.0-8.0, for example, can be 7.0,7.2,7.4,
7.6,7.8 or 8.0, preferably 7.4.
Preferably, step (C) it is described test and analyze include SDS-PAGE, CD, FL, DLS, TEM, gel permeation chromatography,
Any one of Native-agarose gel electrophoresis or MALDI-TOF-MS or at least two combination.
In the present invention, SDS-PAGE is lauryl sodium sulfate-polyacrylamide gel electrophoresis, CD be circular dichroism,
FL is fluorescence spectrum, DLS is dynamic light scattering, TEM is transmission electron microscope, Native-agarose gel electrophoresis
It is Matrix-assisted laser desorption ionization for agarose gel electrophoresis, MALDI-TOF-MS.
Preferably, gel permeation chromatography medium includes 200 10/300GL and/or TSK G4000 SWxl of Superdex.
Preferably, the resolving gel concentration of the SDS-PAGE is 8-15%, for example, can be 8%, 9%, 10%, 11%,
12%, 13%, 14% or 15%.
Preferably, step (C) mode isolated and purified is gel permeation chromatography.
Preferably, the method for merging other antigens includes the following steps:
(a) other epitopes and hepatitis B virus core antigen are subjected to Gene Fusion；
(b) using method preparation and reorganization hepatitis B virus core antigen particle as described in relation to the first aspect；
(c) recombination fusion hepatitis B virus core antigen particle is tested and analyzed.
Preferably, step (a) hepatitis B virus core antigen includes hepatitis B virus core antigen truncate and/or hepatitis B
Virus core antigen full-length proteins.
Preferably, step (c) it is described test and analyze include SDS-PAGE, CD, FL, DLS, TEM, gel permeation chromatography,
Any one of Native-agarose gel electrophoresis or MALDI-TOF-MS or at least two combination.
Preferably, the resolving gel concentration of the SDS-PAGE is 8-15%.
Preferably, the method for being coupled other antigens includes the following steps:
(I) hepatitis B virus core antigen mutation is no less than 1 specific site；
(II) using method preparation and reorganization hepatitis B virus core antigen particle as described in relation to the first aspect；
(III) in hepatitis B virus core antigen particle surface or internal other antigens of connection or developer；
(IV) the recombinant hepatitis B virus core antigen particles for being coupled other antigens obtained by step (III) are isolated and purified and is examined
Survey analysis.
Preferably, step (III) it is described test and analyze include SDS-PAGE, CD, FL, DLS, TEM, gel permeation chromatography,
Any one of Native-agarose gel electrophoresis or MALDI-TOF-MS or at least two combination.
Preferably, the resolving gel concentration of the SDS-PAGE is 8-15%.
Compared with prior art, the invention has the following beneficial effects:
(1) method of the invention specifies depolymerisation conditions for the yield of protein, structure, purifying and secondary group for the first time
The influence of dress provides suitable urea concentration, efficiently completes depolymerization, purifying and the secondary assembling of HBc-VLP；
(2) present invention carries out albumen precipitation using suitable concentration and the ammonium sulfate of pH value, purifies under depolymerisation conditions,
Purification process is simple, purity is high；
(3) operation of the present invention is simple, and preparation efficiency is high, and adopt can be completed with the conventional methods in the field；
(4) present invention is suitable for all recombinant virus sample particles expressed with soluble form；
(5) the recombinant hepatitis B virus core antigen particles prepared by the present invention have similar structure with natural viral,
Grain is complete, and structure is uniform, is free of host nucleic acids or foreign protein.
Detailed description of the invention
Figure 1A is HBc in the embodiment of the present invention 1149Recombinate growth curve of the E.coli in 20L fermentor；Figure 1B is
The result figure of SDS-PAGE after IPTG induction, wherein M is marker, and swimming lane 1 is full bacterium before inducing, and swimming lane 2 is full bacterium after induction,
Swimming lane 3 is bacteria break supernatant, and swimming lane 4 is broken bacterium precipitating；Fig. 1 C is the TEM result figure of product after induction；
Fig. 2 is the SDS-PAGE figure of different depolymerisation conditions protein dissolutions in the embodiment of the present invention 2, and wherein S is different depolymerization
The supernatant of condition dissolution, P are the precipitating that different depolymerisation conditions fail dissolution；
Fig. 3 A is difference depolymerisation conditions albumen Ni FF purifying chromatography chromatogram in the embodiment of the present invention 2；Fig. 3 B is corresponding
SDS-PAGE figure, wherein S is the corresponding Ni FF upper prop sample of different depolymerisation conditions, P1-P5For the corresponding Ni of different depolymerisation conditions
FF eluting peak；
Fig. 4 A is that albumen affinity purification chromatography amplifies chromatogram under the conditions of 4M urea in the embodiment of the present invention 2；Fig. 4 B is corresponding
SDS-PAGE figure, wherein S be Ni FF upper prop sample, FT be flow through, P1For 0.15M imidazoles step eluting peak, P2For 0.5M miaow
Azoles step eluting peak, M marker；
Fig. 5 A is 200 characterization result of Superdex of HBc VLP in the embodiment of the present invention 2；Fig. 5 B is the TEM of HBc VLP
Characterization result；Fig. 5 C is fluorescence spectra under different depolymerisation conditions；Fig. 5 D is the knot of Superdex 200 under different depolymerisation conditions
Fruit figure；Fig. 5 E is the TEM result figure of HBc VLP；Fig. 5 F is the TEM result figure under the conditions of 2M urea concentration；Fig. 5 G is 4M urea concentration
Under the conditions of TEM result figure；Fig. 5 H is the TEM result figure under the conditions of 6M urea concentration；Fig. 5 I is the TEM knot under the conditions of 8M urea concentration
Fruit figure；Fig. 5 J is the TEM result figure under the conditions of 6M guanidine hydrochloride；
Fig. 6 A is the gel chromatography figure of the secondary assembling of albumen after different depolymerisation conditions in the embodiment of the present invention 2；Fig. 6 B is 2M
The TEM of the secondary assembling of albumen under the conditions of urea concentration schemes；Fig. 6 C is the TEM figure of the secondary assembling of albumen under the conditions of 4M urea concentration；Fig. 6 D
It is the TEM figure of the secondary assembling of albumen under the conditions of 6M urea concentration；Fig. 6 E is the TEM figure of the secondary assembling of albumen under the conditions of 8M urea concentration；
Fig. 6 F is the TEM figure of the secondary assembling of albumen under the conditions of 6M guanidine hydrochloride；
Fig. 7 A is the influence that the temperature of solution microenvironment in the embodiment of the present invention 3 assembles HBc VLP；Fig. 7 B is pH pairs
The influence of HBc VLP assembling；Fig. 7 C is the influence that surfactant assembles HBc VLP, and wherein PBST is tween experimental group,
PBS is control group；Fig. 7 D is the influence assembled to HBc VLP the time；Fig. 7 E is the influence that ionic strength assembles HBc VLP；
Fig. 7 F is that TEM detects assembling effect figure under optimal assembling policy condition；
Fig. 8 A is the inducing expression SDS-PAGE qualification figure of pET28a-HBc-MHC I in the embodiment of the present invention 4, and wherein M is
Maker, swimming lane 1 are not induce full bacterium, and swimming lane 2 is full bacterium after induction, and swimming lane 3 is bacteria break supernatant, and swimming lane 4 is broken bacterium precipitating；Figure
8B is TEM result figure；
Fig. 9 A is the SDS-PAGE figure that pET28a-HBc-MHC I is crushed supernatant ammonium sulfate precipitation in the embodiment of the present invention 4,
Middle M is Maker, and swimming lane 1 is bacteria break supernatant, and swimming lane 2 is broken bacterium precipitating, and swimming lane 3 is the supernatant being added after ammonium sulfate, and swimming lane 4 is
The undissolved part of 4M urea, swimming lane 5 are 4M urea depolymerized sample；Fig. 9 B is that 6 FF of Heparin purifies thin layer chromatography under depolymerisation conditions
Figure；Fig. 9 C is the SDS-PAGE result that 6 FF of Heparin is purified under depolymerisation conditions；
Figure 10 A is the TSK G4000 SWxl result of the secondary assembling of pET28a-HBc-MHC I in the embodiment of the present invention 4
Figure；Figure 10 B is the TEM characterization result figure of the secondary assembling of pET28a-HBc-MHC I；Figure 10 C is Capto core purifying chromatography
Chromatogram；Figure 10 D is the TEM characterization result figure of Capto core after purification.
Specific embodiment
Further to illustrate technological means and its effect adopted by the present invention, below by way of specific embodiment come into
One step illustrates technical solution of the present invention, but the present invention is not limited in scope of embodiments.
The expression and identification of 1 recombinant hepatitis B virus core antigen of embodiment
The HBc of full genome synthesis149Segment is inserted into carrier pET28a by BamH I and III site Hind, is named as
pET28a-HBc149, subclone to competent cell BL21 (DE3), HBc149Fragment amino acid sequence such as SEQ ID NO.3 institute
Show.It is seeded to 20L ferment tank, to OD600When to 5-8,1mM IPTG induction 4h (thalli growth curve is shown in Figure 1A) is added,
Thallus is collected in centrifugation (inducing expression qualification result is shown in Figure 1B).According to the ratio of 1:10 be added broken bacterium solution (20mM Tris-HCl,
3mM EDTA, 0.5%Triton X-100,1mM PMSF, pH 8.0), under the pressure of 800MPa, pass through the side of high-pressure homogenization
Formula circulation is broken three times, and supernatant, i.e. recombinant hepatitis B virus core antigen, transmission electron microscope (TEM) identification knot are collected in centrifugation
Fruit sees Fig. 1 C.
SEQ ID NO.4:
MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMNL
ATWVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLP
ETTVV.
By Figure 1A it is found that working as cell density OD600Reach 6.2, thallus has entered logarithmic growth phase at this time, and 1mM is added
IPTG induces HBc149The expression of albumen terminates fermentation after inducing 4h.After carrying out centrifugal treating to fermentation liquid, thallus is collected about
200g.Supernatant and HBc in precipitating after the SDS-PAGE full bacterium in detection induction front and back and broken bacterium149Expression.HBc149Theory
Molecular weight is 19kDa, by Figure 1B it is found that it can be seen that the position protein content obviously increases after induction.Target protein after broken bacterium
(black frame region) is all located in supernatant, illustrates HBc149Albumen is expressed with soluble form.TEM is further passed through to broken supernatant
Observation by Fig. 1 C it is found that there are the virus-like particle of structural integrity (white arrow are signified) in supernatant, but exists big at the same time
The incomplete particle or intermediate structure of amount (Dark grey arrow is signified).
Although expression has the HBc of whole grain in Escherichia coli supernatant, the overwhelming majority or with incomplete particle or
The form of person's intermediate structure exists, and furthermore virus-like particle physicochemical property is different from general proteins, surface charge, hydrophobic property
It is related with its spherical structure, it is difficult to effectively be purified by Conventional chromatography, is first unified knot by all particle depolymerization therefore
Structure is purified again, and then extracellular secondary assembling, prepares virus-like particle.
2 depolymerisation conditions of embodiment are to protein yield, purifying, structure and the extracellular influence assembled again of VLP
1, influence of the depolymerisation conditions to protein yield
By broken supernatant 1M ammonium sulfate precipitation, using the denaturant of different type and concentration to albumen depolymerization, Zhi Houyong
Bradford method surveys protein concentration (protein yield is shown in Table 1), and the solution after depolymerization carries out SDS-PAGE identification (result is shown in Fig. 2).
Influence of the denaturant of 1 different type of table and concentration to protein yield
| Depolymerisation conditions | Protein concentration mg/ml | Protein yield |
| 2M urea | 3.64 | 72.8% |
| 4M urea | 4.59 | 91.8% |
| 6M urea | 4.83 | 96.6% |
| 8M urea | 4.94 | 98.8% |
| 6M GdnHCl | 4.99 | 99.8% |
By table 1 and Fig. 2 it is found that when denaturant is greater than 4M urea concentrations above, almost without target protein in precipitating.It therefore can be with
Think that target protein can be completely dissolved depolymerization by the urea of 4M and concentrations above.
2, influence of the depolymerisation conditions to protein purification
The albumen of depolymerization is resuspended through Ni FF chromatographic purifying in the denaturant of different type and concentration, and type of elution is taken
The elution of 0.15M imidazoles step, correspondence chromatogram after purification are shown in Fig. 3 A, and correspondence SDS-PAGE figure after purification is shown in Fig. 3 B.
As shown in figs 3 a and 3b, when urea concentration is lower than 6M, albumen can be preferably in conjunction with medium, wherein being in urea concentration
Protein purification yield highest under the depolymerisation conditions of 4M, purity of protein is higher under the conditions of SDS-PAGE result also shows these, greatly
In 95%.On the basis of determining 4M urea as depolymerisation conditions, affinity chromatography amplification test is carried out to HBc.Existing for 4M urea
In the case of, 200mg protein sample is purified through Ni FF, obtains 26.35mg albumen, yield 13.2%, and purity is greater than 95%, knot
Fruit is as shown in figs. 4 a-4b.
3, influence of the depolymerisation conditions to protein structure
It is verified using reverse method, i.e., first obtains assembly pure, that structure is uniform, be depolymerized to different journeys later
Degree recycles gel permeation chromatography, fluorescence spectrum and TEM to analyze it.Specifically: assembled VLP is verified, is verified
As a result see Fig. 5 A-5B, respectively take the assembled VLP of 2mg, 1M ammonium sulfate is added, all albumen are precipitated, are added into precipitating
Various concentration and the denaturant of type are to final concentration of 1mg/mL.Bradford method surveys protein concentration, and albumen is complete as the result is shown
By ammonium sulfate precipitation, and all it is resuspended and is dissolved.The solution that dissolution is resuspended is subjected to fluorescence detection, fluorescence results (Fig. 5 C) display
With the increase of denaturation, 343nm of the albumen maximum absorption band from the 325.8nm red shift of assembly to 6M guanidine hydrochloride sample,
Illustrate that the hydrophobic region degree of exposure of albumen increases.In conjunction with Superdex 200 (Fig. 5 D) as a result, assembly is in outer water appearance；2M urea
Cannot be completely by its depolymerization, there are part VLP structures, occur moiety intermediate at the same time；4M, 6M and 8M urea chromatograph behavior base
This is similar, and albumen exists with intermediate forms, and urea concentration increases, and protein structure is further opened, and intermolecular hydrophobic is assembled gradually
Increase；6M guanidine hydrochloride depolymerized sample hydrophobic region sufficiently exposes, and exists completely in the form of hydrophobic aggregation.TEM result (Fig. 5 E-5J)
It is similar.
4, influence of the depolymerisation conditions to the secondary assembling of albumen
Above-mentioned each denaturation depolymerized sample is dialysed first to 20mM Tris-HCl, 2M urea, and dialysis extremely assembles pH 8.0 respectively again
Buffer (20mM Tris-HCl, 0.5M NaCl, pH 7.4), detects its assembling effect using Superdex 200 and TEM.
200 result of Superdex is shown in Fig. 6 A, and corresponding TEM result is shown in Fig. 6 B-6F.By Fig. 6 A-6F it is found that the albumen of Urea denaturation can
Be assembled into VLP again, and the albumen of guanidine hydrochloride denaturation cannot, only exist with aggregated forms；When urea concentration is greater than 4M, with denaturation
Agent concentration increases, and packaging efficiency reduces, and imperfect particle increases.
Therefore, depolymerisation conditions have apparent influence to the yield of HBc albumen, purifying, structure and secondary assembling.In 2M
Under this low concentration denaturant conditions of urea, HBc VLP can not be fully opened, and there is also affect its yield to part VLP structure
With and medium combination；Albumen is completely dissolved when more than 4M urea, but with the increase of denaturant concentration, especially 8M urea and 6M salt
Under the conditions of sour guanidine, albumen hydrophobic region degree of exposure increases, intermolecular easily to generate irreversible aggregation, influences the knot with medium
It closes, the assembling that can not be effectively carried out when denaturant removes.Comprehensive analysis, HBc albumen can either under conditions of 4M urea
Adequately dissolution, and its natural low structure is not destroyed, it is combined with medium, removing denaturant can also obtain group again completely
Dress, is optimum depolymerisation conditions.
Influence of the extracellular assembling condition of embodiment 3 to the viruslike particle assembling of HBc
The extracellular assembling of virus-like particle is mainly strong by temperature, solution ph, surfactant, reaction time and ion
The influence of degree, this experiment investigate it one by one.After purification through Ni FF by the sample of 4M urea depolymerization, it is dialysed first to 2M urea
In, it is dialysed later to each assembling buffer.
Characterization method mainly takes TSK G4000SWxl, flow velocity 0.6ml/min, by corresponding with TEM result, determines
Peak time 9.5min is aggregation, and 10.6min is assembly and 19.4min is unassembled structure.
1, influence of the reaction temperature to assembling: reaction temperature mainly influences reaction rate, as shown in Figure 7 A, in room temperature condition
Lower albumen folding rate is too fast, overwhelming majority precipitating, and albumen precipitation-free under the conditions of 4 DEG C, and when according to gel appearance
Between calculate mainly in the form of VLP exist.
2, influence of the pH to assembling: the net charge and distribution of charges of protein surface can be changed by theoretically changing pH value, thus
The electrostatic interaction between protein protomer is influenced, to influence assembly structure.As shown in Figure 7 B, albumen is in pH 6.4, absolutely
Most of albumen precipitation；And under the conditions of 8.4 pH, albumen does not assemble；Only under conditions of neutral slightly meta-alkali, albumen
Largely assemble.
3, influence of the surfactant to assembling: since the assembling of virus-like particle is mainly by electrostatic interaction and hydrophobic effect
Etc. weak interactions the phenomenon that maintaining, being easy to produce aggregation between VLP, therefore a certain amount of surfactant, such as Tween is added
80 facilitate the dispersion of VLP.Such as anticipation, 0.1%Tween80 is added, assembles between VLP and significantly reduces, and assembles effect
Rate increased (Fig. 7 C).
4, influence of the time to assembling: the assembling of virus-like particle is a relatively slow process, when needing certain
Between complete, in 6h, about 80% albumen assembles, and occurs to assemble (Fig. 7 D) to the albumen for being for 24 hours more than 95%.
5, influence of the ionic strength to assembling: in the assembling process of virus-like particle, electrostatic repulsion is main
Interaction, then protein surface charge density and density are different for ionic strength difference, and the increase of ionic strength facilitates HBc
Assembling.As seen in figure 7e, with the increase of NaCl concentration, packaging efficiency increases, but when NaCl is greater than 1M, albumen can sink
It forms sediment, precipitating will not occur for albumen under the conditions of 0.5M NaCl and packaging efficiency is greater than 95%, simultaneously the NaCl of higher concentration
The effect that can substitute surfactant is introduced, i.e. aggregation and raising packaging efficiency between reduction VLP, this may be strong with ion
When spending high, the mutual electrostatic repulsion of albumen increases related.
On the basis of integrated survey temperature, solution ph, surfactant, reaction time and ionic strength, really
Fixed optimal packaging strategy: 20mM Tris-HCl, 0.5M NaCl, pH 7.4 is assembling buffer, is dialysed more than for 24 hours at 4 DEG C.?
It is greater than 95% to packaging efficiency, particle is complete, the uniform VLP of structure, as shown in Figure 7 F.
The purifying and assembling of recombinant hepatitis B virus core antigen (HBc) particle of 4 fused antigen of embodiment
MHC I class antigen a kind of in Humanmachine tumour related antigen MAGE A3 gene is inserted into the area HBc MIR, is connected
Enter in pET28a, is named as pET28a-HBc-MHC I.Conversion to competent cell BL21 (DE 3) post-fermentation is expressed, and centrifugation is received
Collect thallus.Tunning is characterized after ultrasonication, SDS-PAGE and TEM result respectively as shown in Fig. 8 A, 8B,
PET28a-HBc-MHC I is expressed in supernatant, is existed on a small quantity in the form of virus-like particle.
With HBc149Preparation method is similar, and bacteria break supernatant first uses 1M ammonium sulfate precipitation (the SDS-PAGE knot of precipitating of neutral pH
Fruit sees Fig. 9 A), the 4M urea depolymerization of alkalescent pH is added, 6 FF of Heparin purifying is carried out under depolymerisation conditions, collects and flows through
Peak obtains purer assembling unit, 6 FF of Heparin purifying chromatography chromatogram and SDS-PAGE result difference under depolymerisation conditions
As shown in figures 9 b and 9 c.
Assembling unit after purification is dialysed to 20mM Tris-HCl, is placed in 4 DEG C of refrigerators 3 days in pH 7.4, gel result
The a large amount of recombination VLP of display generate (Figure 10 A-10B).Exquisite purifying, assembling are carried out to the sample after dialysis using Capto core
Body flows through, and the structure of unassembled or wrong assembling achievees the purpose that exquisite purifying in conjunction with medium.Collection penetrates peak, utilizes TEM
Characterization, the chromatogram and TEM characterization result difference of Capto core purifying are as illustrated in figs. loc and 10d, the results showed that, the present invention
The uniform assembly of the available structure of method.
In conclusion recombinant virus sample particle of the invention is free of host nucleic acids, with high purity and structural integrity is uniform, with day
Right morphology of virus is identical, and preparation method is simple, high income, and purification effect is good, is of great significance and wide application prospect.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office
Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
Claims (10)
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| WO2014116730A2 (en) * | 2013-01-23 | 2014-07-31 | The Board Of Trustees Of The Leland Stanford Junior University | Stabilized hepatitis b core polypeptide |
| CN106906230A (en) * | 2017-03-22 | 2017-06-30 | 厦门大学 | Reconstituted drug carrier peptide gene and preparation method and application |
| CN109134621A (en) * | 2018-09-21 | 2019-01-04 | 中国科学院过程工程研究所 | A kind of purification process of HBcAg-VLP or HBcAg-VLP derivative |
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| WO2014116730A2 (en) * | 2013-01-23 | 2014-07-31 | The Board Of Trustees Of The Leland Stanford Junior University | Stabilized hepatitis b core polypeptide |
| CN106906230A (en) * | 2017-03-22 | 2017-06-30 | 厦门大学 | Reconstituted drug carrier peptide gene and preparation method and application |
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| CN110092841A (en) * | 2019-05-15 | 2019-08-06 | 中国科学院过程工程研究所 | A kind of recombinant virus sample particle and its preparation method and application based on inclusion bodies expression |
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