CN102327611A - Vaccine taking HBeAg egg white and/or HBeAg epitope polypeptide as immunogen - Google Patents
Vaccine taking HBeAg egg white and/or HBeAg epitope polypeptide as immunogen Download PDFInfo
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Abstract
The invention discloses a vaccine taking HBeAg egg white and/or HBeAg epitope polypeptide as immunogen. The vaccine is prepared by mixing HBeAg egg white and/or HBeAg epitope polypeptide serving as immunogen with an auxiliary material. The invention provides preparation of a hepatitis B therapeutic HBeAg nano-vaccine by directly taking HBeAg as immunogen with a nanotechnology. According to the invention, the immunogenicity and immune effect of the HBeAg are improved, and an optimum strategy and a direct way for converting HBeAg blood serum are provided.
Description
Technical field
The invention belongs to field of biomedicine technology, relate to therapeutic hepatitis B vaccine, particularly with HBeAg albumen and/or HBeAg antigen epitope polypeptide as immunogenic vaccine.
Background technology
Chronic viral hepatitis B is the healthy global problem of harm humans, and human health and World Economics are caused very big influence.Therapeutic hepatitis B vaccine has become the focus of treating chronic hepatitis B research in this century, and the domestic market of following therapeutic hepatitis B vaccine is expected to reach 3,000,000,000 RMB, and with the speed sustainable growth in year 15%.Therefore, the basic and applied research of development therapeutic hepatitis B vaccine has great scientific meaning and huge social and economic benefit.
At present, in the world, the therapeutic hepatitis B vaccine of developing mainly contains heavy dose of recombinant hepatitis b vaccine, synthetic peptide vaccine, dna vaccination, immune complex vaccine, antibody antigen vaccine etc.At home; The therapeutic hepatitis B vaccine that Bears SIDONG, Wu Yu chapter research group are developed has respectively all been accomplished or has been carried out clinical trial, and shows remarkable therapeutic effect, still; Existing therapeutic hepatitis B vaccine still is in conceptual phase mostly, still faces many problems:
(1) existing therapeutic vaccine clinical research focuses mostly in utilizing pro-S antigen and S antigen, and through being that carrier activates T, B cell response with the extremely strong HBcAg of immunogenicity, has certain limitation, and is dissatisfied to part patient's therapeutic effect;
(2) hepatitis B patient host body immune tolerance state makes the vaccine design difficulty significantly increase, and existing therapeutic vaccine can not directly be realized the conversion of HBeAg serum, obtains satisfied therapeutic effect;
(3) safety of therapeutic vaccine and standardization issue wait further investigation.
Therefore; The research of following therapeutic hepatitis B vaccine answers analysis-by-synthesis chronic viral hepatitis B patient disease to lapse to rule and immunologic tolerance mechanism, screen the immunogenicity that new immunogen improves therapeutic hepatitis B vaccine; Optimize therapeutic hepatitis B vaccine layout strategy and immune programme for children, the suitable adjuvant of selection; Rationally auxiliary cytokine or the antiviral agents of using; The person's that is beneficial to recover the chronic HBV infection immune reactivity, break body immune tolerance state, realize the conversion of HBsAg serum, obtain protectiveness anti--HBs antibody, become the matter of utmost importance of current therapeutic hepatitis B vaccine research.
Complete hepatitis B virus is the Dane granule, is made up of adventitia, nucleocapsid.The DNA that nucleocapsid contains a Circular DNA molecular structure, DNA polymerase, have a protein kinase activity is conjugated protein.Adventitia carries hepatitis B surface antigen (HBsAg), and nucleocapsid carries HBcAg (HBcAg) and e antigen (HBeAg).The HBV genome has S, C, X, P, preceding-preceding-S and preceding-6 genes of X.
HBeAg and HBcAg are by the ORF-C coding of HBV.HBcAg has extremely strong immunogenicity and antigenicity (is that the B cell relies on antigen; Also be the non-dependence antigen of T cell); Its antigenicity is strong 100 times than HBeAg; Be unique HBV antigen that can cause significant immune response, can induce to produce CTL, and auxiliary protection property antigen (like S, M, L antigen) produces humoral immunization and strengthens cellullar immunologic response.Therefore, resisting-HBc antibody all can appear in nearly all HBV the infected.
Serum HBeAg is the non-particulate shape viral capsid proteins of secreted; Identical with about 75% aminoacid sequence of HBcAg; 34 aminoacid have been lacked than HBcAg at c-terminus; With Va1～149 is carboxyl terminal, and the disulfide bond of the cysteine formation in 61 in the cysteine in C district-7 and C district is the secondary structure that HBeAg is different from the uniqueness of HBcAg before being arranged in, and also is that HBeAg is had and diverse epitope of HBcAg and immunoreactive reason.
HBeAg is the distinctive immunologic tolerance factor of hepatitis B virus, expresses through downward modulation TLR2 to cause host immune tolerance (Stephen Locarnini).In addition, HBeAg can also block CTL, suppresses the cytotoxic activity of host T cell, immune attack is removed from the hepatocyte that infects, thereby formed the immunologic tolerance that HBV is infected.And the immunologic tolerance of hepatitis B patient is considered to the chronic viral hepatitis B protracted course of disease and one of major reason of badness come-off occurs, also is the most scabrous problem in the treatment.Perinatal stage wild type C genotype HBV infected patient positive with HBeAg, that HBVDNA titre height is characteristic is still the main patient colony of China's chronic viral hepatitis B.
In metainfective natural history of HBV and the therapeutic process; Its course of disease can be divided into incubation period, symptom phase, removing phase, duration of immunity four-stage; Whether it lapses to viral genotype, is prone to mutability, integrates; Host's sex, nutrition, immunity, infection age, whether merge other and have a liking for that hepatovirus infects or excessive drinking, it is relevant whether to accept multiple factors such as antiviral therapy, immune modulating treatment.In case the conversion of HBeAg serum occurs, body produces anti--HBe antibody, just mean that host immune response strengthens, the course of disease gets into the removing phase, and virus replication stops.On this basis, if HBsAg serum conversion (serum HBsAg is turned out cloudy) explains that then the course of disease gets into duration of immunity, produce protectiveness and resist-HBs antibody, the body inner virus is thoroughly removed.
It is thus clear that; HBeAg serum conversion the having become milestone event that hepatitis B patient well lapses to; The conversion of HBeAg serum; And produce the important indicator that anti--HBe antibody is judgement PD trend and curative effect of medication, and be final basis of thoroughly removing HBV, radical cure hepatitis B, also be the medium-term goal of treating chronic hepatitis B.Therefore, block the immunologic tolerance effect of HBeAg, break the immune tolerance state of body, might become the key and the novel targets of treating chronic hepatitis B.So, realize that the conversion of HBeAg serum should be as the primary goal of therapeutic hepatitis B vaccine design, and should be the direct way of realizing above-mentioned target as the immunogen preparing therapeutic hepatitis B vaccine with HBeAg.
Meanwhile, follow the fast development and the popularization of antigen group, epitope group, immune group, crystal X-ray diffraction, cryotronics microscopy, for furtheing investigate the HBeAg space structure, analyze its epitope, strong technical support being provided.
Nanotechnology is the science and technology of rising late 1980s.Particularly closely during the last ten years, fast-developing nanotechnology is permeated to every field with its power of influence beyond imagination, has demonstrated beat all application potential and effect in the medical research field.Nano vaccine also receives publicity in research and development prevention and treatment vaccine field as a kind of new generation vaccine technology of preparing, is demonstrating great application prospect aspect the lifting vaccine immunogenicity.In recent years multiple multi-form nano vaccine occurs.For example, oral vaccine adopts the Biodegradable nano granule to come the specific antigen of the special dendritic cell receptor of targeting.Nanomed company adopts the treatment vaccine of its patent nano-form engineering development targeting dendritic cell recently, to produce the immunne response to HIV-1 (HIV-1).Preliminary data prompting, with new nano-particle conveying protein obviously enhance immunity reply.As if in addition, some nano-particle can play adjuvant effect, improve the curative effect of vaccine.Biosante company reports that in July, 2007 the nanoparticle based adjuvant Biovant of its calcium phosphate (CaP) helps to develop new anti-H5N1 influenza strain vaccine.The rodent model experiment shows that its immunne response is bigger 4 times with H5N1 antigen than separately.A kind of novel nano Hepatitis B virus vaccine also comes out in the U.S..Obviously, the development of nanotechnology also provides new technical support for the research of novel therapeutic hepatitis B vaccine.
Summary of the invention
The problem that the present invention solves is the target of HBeAg serum conversion as the therapeutic hepatitis B vaccine design; Provide with HBeAg albumen and/or HBeAg antigen epitope polypeptide as immunogenic vaccine, this vaccine can bring out the antibody to HBeAg albumen or its epitope.
The present invention realizes through following technical scheme:
A kind of with HBeAg albumen and/or HBeAg antigen epitope polypeptide as immunogenic vaccine, be by as immunogenic HBeAg albumen and/or HBeAg antigen epitope polypeptide, mix with adjuvant and process;
The proteic aminoacid sequence of described HBeAg is shown in SEQ ID NO:1;
Described HBeAg antigen epitope polypeptide is one or more shown in SEQ ID NO:2～SEQ ID NO:11.
Described with HBeAg albumen and/or HBeAg antigen epitope polypeptide as immunogenic vaccine, be AL (OH) with 0.1～10 times of HBeAg albumen and/or HBeAg antigen epitope polypeptide and its quality
3, mixing in the phosphate buffer of pH6.8 adds AL (OH) again
3The mannitol that quality is 0.1～2 times, sucrose, albumin, gelatin hydrolysate, lactose or sorbitol mixing.
Described with HBeAg albumen and/or HBeAg antigen epitope polypeptide as immunogenic vaccine; Be that HBeAg albumen and/or HBeAg antigen epitope polypeptide are adsorbed in polylactic acid-polyglycol copolymer (PLA-PEG) through two emulsion systems; Forming particle diameter is the Nano microsphere of 0.5～10 μ m, polylactic acid-polyglycol copolymer drug loading greater than 15%, envelop rate is greater than 80%.
Described with HBeAg albumen and/or HBeAg antigen epitope polypeptide as immunogenic vaccine, be that HBeAg albumen and/or HBeAg antigen epitope polypeptide are adsorbed in the nanometer Al that particle diameter is 50～100nm (OH)
3In the middle of the colloidal sol, HBeAg albumen and/or HBeAg antigen epitope polypeptide: nanometer Al (OH)
3The mass ratio of colloidal sol=10～20 μ g: 1mg, nanometer Al (OH)
3The adsorption rate of colloidal sol is greater than 10 μ g/1mg.
Described with HBeAg albumen and/or HBeAg antigen epitope polypeptide as immunogenic vaccine; Be that HBeAg albumen and/or HBeAg antigen epitope polypeptide are scattered in the oil phase; Processing particle diameter then is the water-in-oil type nano-particle of 10～30nm, and the volume ratio of oil phase and water is 1: 3～5; Oil phase is Span-20: poloxamer 188: the mass ratio of soybean oil=80: 180: 100 mixes, and water is a distilled water; The mass ratio of HBeAg albumen and oil phase is 1: 200～300.
Described with HBeAg albumen and/or HBeAg antigen epitope polypeptide as immunogenic vaccine; Be HBeAg albumen and/or HBeAg antigen epitope polypeptide and polylactic acid-glycolic guanidine-acetic acid copolymer (PLGA) according to mass ratio stirring and emulsifying in organic solvent of 1: 20～50; Form W/O/W type emulsion; Remove organic solvent under the room temperature then, the resulting particle diameter of lyophilization is the microsphere of 100～400nm after the reuse water washing, and envelop rate is greater than 40.0%.
Described with HBeAg albumen and/or HBeAg antigen epitope polypeptide as immunogenic vaccine; Be that HBeAg albumen and/or HBeAg antigen epitope polypeptide are adsorbed in the chitosan particle; Forming particle diameter is the nano-particle of 100～200nm, HBeAg albumen and/or HBeAg antigen epitope polypeptide: the mass ratio of chitosan 1: 3～5.
Described with HBeAg albumen and/or HBeAg antigen epitope polypeptide as immunogenic vaccine, be that the HBeAg antigen epitope polypeptide is coupled to key hole limpet hemocyanin, the HBeAg antigen epitope polypeptide: the mass ratio of key hole limpet hemocyanin 1: 3000～2: 30; The HBeAg antigen epitope polypeptide is one or more in SEQ ID NO:2～11.
Compared with prior art, the present invention has following beneficial technical effects:
Through systematic analysis chronic hepatitis patient immunity of organism tolerance mechanism and treatment present situation; It directly is immunogen with HBeAg that the present invention proposes; The preparation of applying nano technology preparation hepatitis B therapeutic HBeAg nano vaccine; To improve immunogenicity and the immune effect of HBeAg, this is optimal strategy and a direct way of realizing the conversion of HBeAg serum.
The present invention adopts eukaryotic expression system or non-eukaryotic expression system to prepare HBeAg albumen, adopts the synthetic method; Preferred Fmoc solid-phase synthesis synthetic HBeAg antigen epitope polypeptide, directly with HBeAg albumen with (or) the HBeAg antigen epitope polypeptide separately with (or) mix the preparation that is used for therapeutic hepatitis B vaccine as main immunogens.This therapeutic hepatitis B vaccine is protein vaccine, polypeptide vaccine both, also novel nano vaccine.
Provided by the inventionly can bring out BALB/c mouse with HBeAg albumen and/or HBeAg antigen epitope polypeptide as immunogenic vaccine and produce the height anti--HBe antibody of tiring.Antibody titer promptly began to raise after immunity in 3 weeks, and after this institute's immune time increases and continues to raise.After carrying out 4 immunity, mice can produce the higher antibody of tiring, and so just helps realizing the conversion of HBeAg serum.
The specific embodiment
Below in conjunction with concrete embodiment the present invention is done further detailed description, said is to explanation of the present invention rather than qualification.
Embodiment 1
Proteic expression of HBeAg and purification
1.1 reorganization pcDNA-HBeAg Construction of eukaryotic
According to HBeAg gene order (1871-2347 gene order; Total length 477bp) and pcDNA3.1 carrier for expression of eukaryon design primer; In forward primer P1, introduce EcoR V restriction enzyme site, in downstream primer P2, introduce Xho I restriction enzyme site, institute's concrete sequence of designed primer is:
Forward primer P1:cagatatcca gggaatggtg tccaagctgt gccttgggtg g 41;
Downstream primer P2:gactcgagcg ttaaacaaca gtagtttccg g 31;
DNA with isolating HBV virus among hepatitis B " great three positive " patients serum is a template; With above-mentioned primer PCR amplification HBeAg gene order; After order-checking is correct the said gene product is cloned into the pcDNA3.1 carrier for expression of eukaryon through EcoRV and Xho I restriction enzyme site, makes up and obtain the pcDNA-HBeAg recombinant expression carrier; And it is correct with EcoRV/Xho I double digestion, PCR and gene sequencing confirmation construction of recombinant vector.
1.2 recombiant plasmid transfection CHO cell
Cultivate Chinese hamster ovary celI with the RP2MI1640 complete medium that contains 10% hyclone, carry out the test of G418 minimum lethal concentration preceding 2 weeks of transfection.18h before transfection, the Chinese hamster ovary celI that will be in increased logarithmic phase all is about 2 * 10 by every hole
5Individual cell inoculation is approximately at 60% o'clock to cell density in 6 well culture plates, abandon cell culture fluid, with serum-free RP2MI1640 culture medium washed cell 1 time, carries out transfection with Cellfectin.Transfection process is specially:
Be configured to down solution: A liquid (the pcDNA-HBeAg plasmid of 5 μ l reorganization adds 95 μ l sterilization deionized water) and B liquid (6 μ l Cellfectin add 94 μ l sterilization deionized water) is mixing gently, and room temperature is placed 30min; Behind the RPMI1640 culture medium washed cell of serum-free, antibiotic-free 3 times, dropwise add the AB mixed liquor, cultivate and change complete medium behind the 10h and cultivate 3 days (concrete grammar is with reference to the cellfectin of invitrogen company workbook) altogether.
Immunoblotting and immunocytochemical method detect the expression that detects HBeAg in culture supernatant and the Chinese hamster ovary celI respectively (anti-be mouse-anti-HBe monoclonal antibody).
1.2.1 SABC detects the expression of HBeAg
Collect the Chinese hamster ovary celI of pcDNA-HBeAg recombinant eukaryon expression vector transfection, conventional method prepares cell climbing sheet, and 4% paraformaldehyde is fixed, and 5% hydrogen peroxide is incubated; The sealing of 10% rabbit anteserum drips dilution in 1: 100 and resists-the HBe monoclonal antibody, and 4 ℃ are spent the night, and drips 40 μ l biotin labeled two anti-(sheep anti mouse); Hatch 30min for 37 ℃, drip Streptavidin-peroxide multienzyme complex 40 μ l, hatch 20min for 37 ℃; The DAB colour developing, haematoxylin is redyed, the neutral gum mounting; Observation by light microscope Expression of Fusion Protein and Subcellular Localization.
1.2.2 Western blot detects the proteic expression of HBeAg
1) Western blot detects the proteic expression of HBeAg in the culture supernatant
After collecting the culture supernatant thermal denaturation respectively, get the capable SDS-PAGE electrophoresis of 10 μ l supernatants, utilize Bio-Rad albumen transferring system to change film.The sealing of 5% defatted milk room temperature, the anti--HBeAg monoclonal antibody of dilution in 1: 200 is hatched, and the HRP-IgG of dilution in 1: 1000 is hatched, and TBST washes film, the DAB colour developing.
2) Western blot detects the proteic expression of HBeAg in the Chinese hamster ovary celI
Amplification culture is carried out in the positive cell strain of selecting screening to obtain, and (density is 10 to collecting cell
6Individual/as ml) to express each 25 μ l of supernatant with 24h, add equivalent 2 * sds gel sample loading buffer, row SDS-PAGE electrophoresis utilizes Bio-Rad albumen transferring system to change film.The sealing of 5% defatted milk room temperature, the anti--HBeAg monoclonal antibody of dilution in 1: 200 is hatched, and the HRP-IgG of dilution in 1: 1000 is hatched, and TBST washes film, the DAB colour developing.
1.3 the screening of the Chinese hamster ovary celI of stably express HBeAg
After confirming the transfection success,, cultivate with the complete medium that contains 800mg/mL G418 according to the test of G418 minimum lethal concentration; When the most of death of cellular control unit, use the selection screening of medium that contains 600mg/mLG418 instead behind the 3-4d, every 2-3d changes liquid and goes down to posterity; After the 14d cultivation, adopt limiting dilution assay, Chinese hamster ovary celI is moved in the culture plate; Continue screening with G418, change culture fluid in good time and go down to posterity.Until the Chinese hamster ovary celI clone who detects stably express HBeAg, the amplification culture and the cultivation of going down to posterity.
1.4 the proteic purification of reorganization HBeAg
Adopt the natural anti--HBe antibody of purification, utilize the magnetic bead co-immunoprecipitation method of SPA labelling to separate, collect the HBeAg albumen of cho cell expressing recombinant; And verify purity of protein with SDS-PAGE, the BCA method is measured protein concentration, packing frozen (80 ℃ of refrigerators).
The external synthetic and purification of embodiment 2.HBeAg proteantigen epitope peptide
HBeAg antigen epitope polypeptide shown in SEQ ID NO:2～11 is that length is the polypeptide of 8～32 amino acid residues, adopts Fmoc solid-phase polypeptide technology to accomplish its preparation (also can entrust special Synesis Company to accomplish):
Adopt Fmoc solid-phase polypeptide synthetic technology; On the full-automatic solid-phase polypeptide synthesizer of PS3; Aminoacid with N-α-Fmoc protection is raw material, and Rink amino resins is carrier, and 20% piperidines DMF solution is deprotection agent; HBTU is the peptide bond condensing agent, and coupling is synthetic successively to hold the order of N end by known array from C.
Take off reaction bulb from Peptide synthesizer PS3 behind the end of synthesis, be put in the fume hood.With DMF the peptide-resin in the reaction bulb all is flushed in the Poly-Prep chromatographic column.With washed with methanol resin 8 times, remove DMF.Nitrogen dries up the back, and (TFA: thioanisole: methyl phenyl ethers anisole: the volume ratio of dimercaptoethane is 90: 5: 2: 3) will synthesize polypeptide cracking from the resin and get off, and slough all blocking groups simultaneously with lysate.
Collection is dissolved with the lysate of synthetic peptide, with nitrogen carefully blow away TFA in the lysate until remaining lysate volume to about the 2mL, make the antigenic peptides deposition with-20 ℃ absolute ether then, centrifugal collecting precipitation is blown away ether with nitrogen, promptly gets to synthesize the peptide bullion.Synthetic peptide bullion is collected main peak through high-pressure liquid phase reversed phase chromatography post C18 separation and purification, after the lyophilization, obtains the synthetic peptide of purification.
Synthetic protein polypeptide is carried out mass spectrum identify, then the charge-mass ratio of mass spectral analysis and the theoretical molecular of prediction are compared.Through its purity of rp-hplc analysis, chromatographic condition is following again for synthetic peptide: and chromatographic column Jupiter Proteo (250mm * 4.6mm, 4IU), mobile phase A: 0.1%TFA/ water: Mobile phase B: 0.1%TFA/ acetonitrile; Linear gradient: 15%B → 40%B, 25min; Column temperature: 35 ℃; Flow velocity: 1.0mol/min; Detect wavelength: 220nm.
The preparation of embodiment 3 aluminium adjuvants-HBeAg protein vaccine
Get mass concentration and be 5% aluminum sulfate solution 250ml, under strong agitation, add 5% sodium hydroxide solution 100ml, precipitate 2 times, deposition is hung into makes cumulative volume reach 250ml in the normal saline again with the normal saline centrifuge washing.(also can directly adopt commercially available aluminium adjuvant (AL (OH)
3).
Get HBeAg albumen (20 μ g/mL) 5～100 μ g as immunogen, add equal-volume and contain aluminium adjuvant AL (OH)
3In the phosphate buffer (2mg/ml) (pH6.8), 4 ℃ of stirring and evenly mixing 2～3h add AL (OH) again
3At least a in the mannitol that quality is 0.1～2 times, sucrose, albumin, gelatin hydrolysate, lactose or the sorbitol and be prepared into the hepatitis B therapeutic protein vaccine.
The preparation of embodiment 4.PLEG-HBeAg vaccine
Adopt two emulsion (W1/O/W2) system solvent extraction processs:
Under aseptic condition; With polylactic acid one ethylene glycol copolymer (PLA-PEG copolymers; PLEG) copolymer is dissolved in dichloromethane, and (HBeAg is 1～1.5 with the mass ratio of PLEG: 3～4.5) and with ultrasonic emulsification become emulsion, obtain colostrum (W1/O) to add HBeAg then; Colostrum is added in the 0.3% polyvinyl alcohol aqueous solution, machinery stirs 12h fast and obtains stable emulsion (W1/O/W2) again.
At room temperature high-speed stirred is volatilized until dichloromethane fully, uses the microporous filter membrane sucking filtration then, separate centrifugal, with distilled water wash 3 times, lyophilization.Also available isopropanol water solution dissolving organic solvent (dichloromethane), centrifugalize is removed after the layering, and lyophilization obtains microsphere (being the HBeAg albumen of polylactic acid one ethylene glycol copolymer parcel).Application scanning electron microscope observation microsphere surface form, and calculate the package rate through detecting protein content, polylactic acid-polyglycol copolymer drug loading should be greater than 15%, envelop rate is greater than 80%.
Embodiment 5: the preparation of nano-class aluminum adjuvant-HBeAg vaccine
1) chemical preparation of nano-class aluminum adjuvant:
Get mass ratio and be 1: 1: 1～4 benzalkonium bromide, n-octyl alcohol, cyclohexane extraction in beaker; Under 25 ℃, magnetic agitation; Add isopyknic 1mol/L AlC13 solution stirring 10min again; Form benzalkonium bromide-n-octyl alcohol-cyclohexane extraction-AlC13 solution system, stir with the high speed dispersion homogenizer then, make its emulsifying become microemulsion.
Then microemulsion is poured into the reaction bulb heated and stirred of lid, slowly dropwise added pH value that excessive ammonia keeps system 8～10, cover lid; Behind reaction 2～3h; Add the acetone breakdown of emulsion, the centrifugal supernatant of abandoning is with the dehydrated alcohol of 950g/kg and the common washing precipitation of deionized water 3 times; Adopt freeze-drying (70 ℃) or boulton process (4 ℃) at last, with obtaining bulk nanometer Al (OH) 3 xerogel after the deposition drying.
Then with nanometer Al (OH)
3Xerogel is dissolved in the water, and uses ultrasonic dispersing, in aqueous solution, is dispersity; The method that electron microscope observation is measured is measured the nanoparticle warp, and obtaining particle diameter is the aluminium adjuvant of 50～100nm nanometer.
2) preparation of nano-class aluminum adjuvant vaccine
With water is solvent; Being made into concentration is the nano-class aluminum adjuvant solvent of 1～5mg/ml; After the ultrasonic dispersing nano-class aluminum adjuvant solution 20min, be that the HBeAg protein solution (using distilled water diluting) of 20 μ g/mL mixes with isopyknic concentration again, one week of absorption under 4 ℃ of conditions; With the centrifugal 30min of 15000r/min, resulting deposition is nano-class aluminum adjuvant-HBeAg vaccine again; Adopt albuminimetry to detect protein concentration in the supernatant, the package rate is greater than 90%, and the adsorption rate of nanometer Al (OH) 3 colloidal sols is greater than 10 μ g/1mg.
Embodiment 6: the preparation of water-in-oil type HBeAg nano vaccine
1) adopt the magnetic force ultrasonic method to prepare water-in-oil type HBeAg nano vaccine:
With HBeAg albumen 1.0mg, 80g/L Span-20 (Arlacel-20, sorbitol anhydride monolaurate), 180g/L poloxamer 188 (poloxamers
188) and the 0.6mL soybean oil, distilled water adjustment volume is 2.5mL, mix homogeneously obtains oil phase; Under the 3000r/min magnetic agitation, oil phase is dropwise added 7.5mL distilled water aqueous phase mix; Again mixture is moved on the vacuum high speed shear mulser, under the 0.7kpa vacuum, 23,000r/min, high speed shear 40min, temperature is controlled at below 80 ℃; The ultrasonic 5min of 40kHz under the ice bath makes the complete emulsifying of mixture repeatedly for 3 times, promptly obtains water-in-oil type HBeAg nano vaccine (0.1g/L).0.22 after the degerming of μ m membrane filtration, in 4 ℃ of preservations.
2) HBeAg receives the character of emulsion droplet and identifies:
The nanometer emulsion droplet of processing is diluted in the normal saline, gets 100 μ L and drip on 400 order copper mesh, phosphotungstic acid dyeing, transmission electron microscope is observed its form down, and the mean diameter that the image of gathering is tried to achieve the nanometer emulsion droplet through computer image analysis is 10～30nm.
3) encapsulation ratio, drug loading and stability are measured:
Adopt the Sephedex-G100 gel chromatography, collect free HBeAg with spectrophotometric determination A280 value.The HBeAg standard protein sample for preparing variable concentrations simultaneously with its shading value of spectrophotometric determination, and is drawn the standard curve of concentration and shading value.With the standard curve is foundation, calculates the content of free HBeAg in the nano-particle.
Encapsulation ratio by formula calculates: encapsulation ratio=(W total-W trip)/W is total * and 100%.
The total amount of used HBeAg when wherein W is always for preparation, the W trip is the amount of free complex antigen.The result shows encapsulation ratio greater than 80%, and is centrifugal in 4 ℃ of placements 6 months or 3000r/min 10min, no layering and deposited phenomenon.
4) water-in-oil type HBeAg nano vaccine biological safety detects:
Behind prepared vaccine intramuscular injection immune mouse, under naturalness, observe immune mouse vaccine soak time, have or not local response or systemic adverse reactions, injection site muscle injury situation.Require no muscular death, granuloma, the equivalent damage property of festering reaction.
The preparation of embodiment 7:PLGA-HBeAg nano vaccine
The concrete physical method that adopts prepares, and its concrete steps are:
Get 50mg polylactic acid-glycolic guanidine-acetic acid copolymer (PLGA) microsphere (LA: GA=50: 50; Relative molecular mass 15000) is dissolved in the 1mL dichloromethane (DCM); Add 1mL HBeAg protein solution (1mg/mL) then; Ultrasonic 1min (power is 100W), visible milky Water-In-Oil (W1/O) type colostrum forms; Add 2g/L polyvinyl alcohol (PVA) solution 2mL again; Ultrasonic once more 5～20min under condition of ice bath (power is 55W); Form W/O/W (W1/O/W2) type emulsion, then this emulsion is transferred in the 50mL deionized water (pH4.6), stir 4h under the room temperature organic solvent is volatilized fully; The centrifugal 20min of 10000 * g, and collecting precipitation.Resultant deposition is relaundered, disperses with distilled water, place the dry 40h of low-temperature freeze-drying machine (80 ℃), collect exsiccant powder (size is the microsphere of 100～400nm, and envelop rate is greater than 40.0%), obtain the PLGA-HBeAg nano vaccine, 4 ℃ of preservations.Carry out nanoparticle form, size, drug loading with reference to embodiment 6, the detection of biological safety.
Embodiment 8: the preparation of chitosan-HBeAg nano vaccine
88.5% deacetylation chitosan and normal saline are made into the solution of 10g/L; Handle 2 times each 5min, 1min at interval again through ultrasonic (output 80Hz); To get its supernatant behind the centrifugal 10min of 50 * g; And, to collect its precipitate behind the centrifugal 10min of 1400 * g, be the chitosan granule again with the filtration of 400 order mesh screens.
Get mass concentration and be the normal saline solution (β-(1-4) the amino 2-deoxidation of 2--D-glucosan of 1% chitosan; CTS); Be diluted to the NaAC-HAC buffer of 0.1mol/L 0.2% for use, again with chitosan solution through 0.22 μ m membrane filtration, it is for use in dry test-tube to get 10mL.
Get 3mL HBeAg albumen stock solution (concentration is 20 μ g/mL) and 3mL Na2SO4 solution (concentration is 50mmol/L) mixing; Under the vibrations of suspendible vibrator, in the chitosan solution that 10mL 0.2% is housed, add the mixed vaccine diluent of 3mLNa2SO4, dropwise add sodium tripolyphosphate 550 μ L again, this moment, solution presented outstanding turbid shape; Behind the stirring at room 10min, carry out ultrasonic (output frequency 80Hz) and handle 2 times, each 5min, 1min at interval) handle after, with the speed high speed centrifugation 20min of 10000r/min, gained is precipitated as chitosan-HBeAg nano vaccine, and (particle diameter is 100～200nm).It is resuspended subsequent use with 0.9%NaCl solution, carry out nanoparticle form, size with reference to embodiment 6, drug loading, biological safety detects.
In the middle of the foregoing description 3～8, those skilled in the art also can replace with the HBeAg antigen epitope polypeptide with HBeAg albumen wherein, to prepare various corresponding HBeAg antigen epitope polypeptide vaccines.
The preparation of embodiment 9:KLH-HBeAg antigen epitope polypeptide vaccine
The coupling of HBeAg antigen epitope polypeptide and carrier protein:
A liquid: 15mg key hole limpet hemocyanin (KLH) is dissolved in 2ml glycerol;
B liquid: 5～100 μ g HBeAg antigen epitope polypeptides (SEQ ID NO:2～11 a kind of, perhaps several kinds arbitrary proportion mixes) are dissolved in the 1ml dimethyl sulfoxide;
Behind A liquid and B liquid mixing back adding coupling agent carbodiimide (EDS) 5mg vibration 1h; Place stirred overnight on the magnetic stirring apparatus, the link coupled HBeAg antigen epitope polypeptide of chromatography purification KLH next day is collected the protein-contg eluent of merging; Measure protein concentration; According to the antigen concentration of 5～100 μ g/ml ,-20 ℃ of preservations are put in the packing of 1ml dosage.
The immune effect of vaccine
HBeAg albumen (SEQ ID NO:1) that the present invention obtains and HBeAg antigen epitope polypeptide (SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11) are as immunogen and adopt vaccine production method or prepared by other therapeutic hepatitis B vaccine suitable in the above-mentioned embodiment; This vaccine is used for the biological immune treatment of the positive hepatitis B patient of HBeAg; Can bring out the patient and produce anti--HBe antibody; Reality hepatitis B patient HBeAg serum is changed, and reaches the purpose of final healing hepatitis B.
The immune effect of the hepatitis B therapeutic HBeAg albumen/polypeptide vaccine of the method preparation that the foregoing description is described; Be the immune effect of hepatitis B therapeutic HBeAg albumen (polypeptide) vaccine of the method preparation described of Detection of antigen embodiment 3-9 with the HBeAg albumen (SEQ ID NO:1) of expressing cho cell and purification wherein, concrete grammar is following:
1) animal immune
BALB/c mouse is divided into experimental group (each vaccine can be divided into 1 group) and matched group at random; Every group 4, adopt back subcutaneous multi-point injection mode immune animal, per injection 100 μ L; The immunogen total amount that is comprised in the middle of the vaccine is 5 μ g, and the matched group inoculation contains the PBS solution of corresponding adjuvant.
After the 1st inoculation, 2 week back reinforcements 1 time; Later on per 4 week inoculations 1 time are inoculated 4 times altogether.Since the 2nd inoculation, each inoculation back 1 all tail veins are got blood, and the variation of antibody content is observed in 1 all eyeball posterior vein blood samplings after the 4th immunity inoculation.
2) the ELISA method detects antibody titer
The conventional method separation of serum, adding has encapsulated the proteic 96 hole elisa plates of HBeAg (adopting conventional method is antigen with the HBeAg albumen that embodiment 1 described method prepares purification, encapsulates elisa plate) behind the doubling dilution.37 ℃ add horseradish peroxidase (HRP) the anti-mice IgG of labelled goat (1: 2000) after hatching 2h, hatch 1h for 37 ℃, add TMB liquid reaction 5min behind the thorough washing, 2mol/LH
2SO
4Cessation reaction is measured every hole A value down in wavelength 490nm.
Through comparing with matched group, calculating antibody is tired, and the result is as shown in table 1.The result shows that what adopt method preparation that the foregoing description provided is that immunogenic therapeutic hepatitis B vaccine can bring out BALB/c mouse and produces the height anti--HBe antibody of tiring with HeAg albumen or HBeAg epitope peptide.Antibody titer promptly began to raise after immunity in 3 weeks, and after this institute's immune time increases and continues to raise, and in a week after the 4th immunity, serum anti-HBe antibody titer ELISA testing result is as shown in table 1.Can find out that mice can produce the higher antibody of tiring after carrying out 4 immunity, so just help realizing the conversion of HBeAg serum, produce protectiveness and resist-HBs antibody.
Serum antibody titer after table 1 HBeAg albumen/polypeptide vaccine immunity
Claims (8)
- With HBeAg albumen and/or HBeAg antigen epitope polypeptide as immunogenic vaccine, it is characterized in that, be by as immunogenic HBeAg albumen and/or HBeAg antigen epitope polypeptide, mix with adjuvant and process;The proteic aminoacid sequence of described HBeAg is shown in SEQ ID NO:1;Described HBeAg antigen epitope polypeptide is one or more shown in SEQ ID NO:2～SEQ ID NO:11.
- 2. as claimed in claim 1 with HBeAg albumen and/or HBeAg antigen epitope polypeptide as immunogenic vaccine, it is characterized in that, be AL (OH) with 0.1～10 times of HBeAg albumen and/or HBeAg antigen epitope polypeptide and its quality 3, mixing in the phosphate buffer of pH6.8 adds AL (OH) again 3The mannitol that quality is 0.1～2 times, sucrose, albumin, gelatin hydrolysate, lactose or sorbitol mixing.
- 3. as claimed in claim 1 with HBeAg albumen and/or HBeAg antigen epitope polypeptide as immunogenic vaccine; It is characterized in that; Be that HBeAg albumen and/or HBeAg antigen epitope polypeptide are adsorbed in polylactic acid-polyglycol copolymer (PLA-PEG) through two emulsion systems; Forming particle diameter is the Nano microsphere of 0.5～10 μ m, polylactic acid-polyglycol copolymer drug loading greater than 15%, envelop rate is greater than 80%.
- 4. as claimed in claim 1 with HBeAg albumen and/or HBeAg antigen epitope polypeptide as immunogenic vaccine, it is characterized in that, be that HBeAg albumen and/or HBeAg antigen epitope polypeptide are adsorbed in the nanometer Al that particle diameter is 50～100nm (OH) 3In the middle of the colloidal sol, HBeAg albumen and/or HBeAg antigen epitope polypeptide: nanometer Al (OH) 3The mass ratio of colloidal sol=10～20 μ g: 1mg, nanometer Al (OH) 3The adsorption rate of colloidal sol is greater than 10 μ g/1mg.
- 5. as claimed in claim 1 with HBeAg albumen and/or HBeAg antigen epitope polypeptide as immunogenic vaccine; It is characterized in that; Be that HBeAg albumen and/or HBeAg antigen epitope polypeptide are scattered in the oil phase; Processing particle diameter then is the water-in-oil type nano-particle of 10～30nm, and the volume ratio of oil phase and water is 1: 3～5; Oil phase is Span-20: poloxamer 188: the mass ratio of soybean oil=80: 180: 100 mixes, and water is a distilled water; The mass ratio of HBeAg albumen and oil phase is 1: 200～300.
- 6. as claimed in claim 1 with HBeAg albumen and/or HBeAg antigen epitope polypeptide as immunogenic vaccine; It is characterized in that; Be HBeAg albumen and/or HBeAg antigen epitope polypeptide and polylactic acid-glycolic guanidine-acetic acid copolymer (PLGA) according to mass ratio stirring and emulsifying in organic solvent of 1: 20～50, form W/O/W type emulsion, remove organic solvent under the room temperature then; The resulting particle diameter of lyophilization is the microsphere of 100～400nm after the reuse water washing, and envelop rate is greater than 40.0%.
- 7. as claimed in claim 1 with HBeAg albumen and/or HBeAg antigen epitope polypeptide as immunogenic vaccine; It is characterized in that; Be that HBeAg albumen and/or HBeAg antigen epitope polypeptide are adsorbed in the chitosan particle; Forming particle diameter is the nano-particle of 100～200nm, HBeAg albumen and/or HBeAg antigen epitope polypeptide: the mass ratio of chitosan 1: 3～5.
- 8. as claimed in claim 1 with HBeAg albumen and/or HBeAg antigen epitope polypeptide as immunogenic vaccine; It is characterized in that; Be that the HBeAg antigen epitope polypeptide is coupled to key hole limpet hemocyanin, the HBeAg antigen epitope polypeptide: the mass ratio of key hole limpet hemocyanin 1: 3000～2: 30; The HBeAg antigen epitope polypeptide is one or more in SEQ ID NO:2～11.
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| CN105311631A (en) * | 2014-07-02 | 2016-02-10 | 北京亦科为生物技术有限公司 | CaP-PLA/PLGA nano-microsphere, and preparation method and application thereof |
| CN106031794A (en) * | 2015-03-20 | 2016-10-19 | 中国科学院过程工程研究所 | Intracellular pH-response polylactic-acid nanometer microspheres and preparing method thereof |
| CN108421035A (en) * | 2018-02-09 | 2018-08-21 | 中山大学 | A kind of nano particle vaccine and its preparation method and application based on chitosan |
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| CN105311631A (en) * | 2014-07-02 | 2016-02-10 | 北京亦科为生物技术有限公司 | CaP-PLA/PLGA nano-microsphere, and preparation method and application thereof |
| CN106031794A (en) * | 2015-03-20 | 2016-10-19 | 中国科学院过程工程研究所 | Intracellular pH-response polylactic-acid nanometer microspheres and preparing method thereof |
| CN106031794B (en) * | 2015-03-20 | 2020-10-02 | 中国科学院过程工程研究所 | Intracellular pH response polylactic acid nano microsphere and preparation method thereof |
| CN105194662A (en) * | 2015-09-16 | 2015-12-30 | 华北制药金坦生物技术股份有限公司 | Sustained-release microsphere preparation containing recombinant hepatitis b virus surface antigen and preparation method of preparation |
| CN105194662B (en) * | 2015-09-16 | 2018-08-28 | 华北制药金坦生物技术股份有限公司 | Sustained release microsphere agents and preparation method thereof containing recombination hepatitis B surface antigen |
| CN105288613A (en) * | 2015-11-25 | 2016-02-03 | 河北师范大学 | Nanoparticle vaccine preparation containing recombinant hepatitis B surface antigen and preparation method thereof |
| CN105288613B (en) * | 2015-11-25 | 2018-08-31 | 河北师范大学 | A kind of nano particle vaccine preparation and preparation method thereof containing recombination hepatitis B surface antigen |
| CN108421035A (en) * | 2018-02-09 | 2018-08-21 | 中山大学 | A kind of nano particle vaccine and its preparation method and application based on chitosan |
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