CN107823638A

CN107823638A - A kind of B group meningitis coccis restructuring chimeric protein vaccine and preparation method thereof

Info

Publication number: CN107823638A
Application number: CN201711073721.5A
Authority: CN
Inventors: 苏桂民; 朱卫华; 高博; 郭通; 纪国存; 冀颖; 杜琳
Original assignee: Anhui Zhifei Longcom Biopharmaceutical Co Ltd; Chongqing Zhi Fei Biological Products Ltd By Share Ltd; Beijing Zhifei Lvzhu Biopharmaceutical Co Ltd
Current assignee: Anhui Zhifei Longcom Biopharmaceutical Co Ltd; Chongqing Zhi Fei Biological Products Ltd By Share Ltd; Chongqing Zhifei Biological Products Co Ltd; Beijing Zhifei Lvzhu Biopharmaceutical Co Ltd
Priority date: 2017-11-05
Filing date: 2017-11-05
Publication date: 2018-03-23
Anticipated expiration: 2037-11-05
Also published as: CN107823638B

Abstract

The present invention provides a kind of B group meningitis coccis restructuring chimeric protein vaccine and preparation method thereof, and the vaccine contains：Tri- kinds of P2 fHBP V1, P2 fHBP V2 and P2 fHBP V3 restructuring chimeric proteins; its amino acid sequence is respectively SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3; wherein every kind of protein content is respectively 30 50 μ g/ agent; optional, freeze drying protectant or vaccine adjuvant are also contained in the bacterin preparation；The present invention also provides the preparation method of three kinds of restructuring chimeric proteins, by the way that P2 is connected into the restructuring chimeric protein to be formed with B group meningitis cocci fHBP albumen, being capable of effective induction body fluid immune response, significantly improve fHBP immunogenicity, the vaccine of the present invention can effectively cover all B group meningitis coccis bacterial strains, so as to which the affecting conditions such as cerebrospinal meningitis, bacteremia, pneumonia and pericarditis as caused by B group meningitis coccis are provided with the prevention effect of wide spectrum.

Description

A kind of B group meningitis coccis restructuring chimeric protein vaccine and preparation method thereof

Technical field

The present invention relates to a kind of preparation of vaccine, more particularly to a kind of B group meningitis coccis restructuring chimeric protein vaccine and Its preparation method, the B group meningitis coccis restructuring chimeric protein vaccine include B group meningitis cocci people's H factor bindins (fHBP) the restructuring chimeric protein to be formed is connected with lockjaw versatility t cell epitope P2, the albumen there are three anomaly fHBP V1, fHBP V2 and fHBP V3, three kinds of anomalies are recombinated into bacterin preparation and its preparation that chimeric protein is formulated in proportion Method.

Background technology

Neisseria meningitidis (Neisseria meningitidis, Nm), is commonly called as meningococcus (Meningococcus) meningococal meningitis (abbreviation epidemic meningitis) and septicemia can, be caused, the disease has higher hair Sick rate and case fatality rate.Neisseria meningitidis is a kind of Pathogenic gram-negative diplococcus using the mankind as main host, once for several times Causing the outburst of global range, its prevention and control optimal strategy is vaccine inoculation with popular.

Nm virulence factor mainly includes capsular polysaccharide (capsular polysaccharide, CPS), fat oligosaccharides and outer Memebrane protein (outer membrane protein, OMP), wherein OMP mainly include pili, PFP (PorA and Por B), sticked Attached molecule Opc etc..Nm is divided into A, B, C, H, K, L, X, Y, Z, 29E and W135 totally 12 sero-groups according to capsular polysaccharide structure, A, B, C, Y and W135 are the common sero-group of aggressive cerebrospinal meningitis, and wherein case caused by A, B, C group meningitis cocci accounts for More than 90%.

A, the application of C, Y, W135 group polysaccharide vaccine and polysaccharide-protein combined vaccine so that corresponding sero-group popularity brain The scorching incidence of disease of film and case fatality rate are effectively controlled.Epidemic meningitis prevalence is noteworthy characterized by with obvious region, and Over time with the application of vaccine, it may occur that the conversion of sero-group.At present, B group meningitis Neisseria has become master The epidemic link wanted, according to statistics, B group meningitis Neisserias result in the meningococcal disease in the whole world about 50%；And B group is than A group, C The duration such as group are longer, up to 5-10.During B group's prevalence, invasion and attack rate can increase, and the age can also expand therewith, special It is not 4-19 year population of adolescent.

But the linear polymeric sialic acid contained in B group's Nm capsular polysaccharides and people's N-acetyl-neuraminate polymer architecture class Seemingly, therefore in human body do not possess immunogenicity, and have the danger for inducing spontaneous immunological diseases, so the strategy of polysaccharide vaccine It is not particularly suited for B group meningitis Neisserias.Therefore, B group meningitis coccis vaccine research and development concentrate on effective Outer membrane protein antigen On composition, such as H factor bindins (factor H-binding protein, fHBP), neisseria surface protein A (Neisseria surface protein A, NSPA), neisseria adhesin A (Neisseria adhesin A,

NadA), neisseria Heparin-binding antigen (Neisseria heparin binding antigen, NHBA) etc..

FHBP is a kind of specific lipoprotein for being almost expressed in all Neisseria meningitidis surfaces, and relative molecular mass is about 29KD, it is Neisseria meningitidis important virulence factor and vaccine antigen.FHBP is according to antigenic cross-reaction and protein sequence Similitude is subdivided into 3 anomalies, i.e. V1, V2 and V3.Anomaly inner amino acid array homology is more than 90%, anomaly Between can as little as 62.8%.Sterilizing power experimental analysis shows that resistance abnormal shape V1 antibody is to anomaly V2, the sterilization of V3 meningitis separation strains Active cross reactivity very little, vice versa.

FH (the people H factors) is the important negative regulator during complement activation, and FI mediation C3b can be aided in be cracked into Fragment iC3b is inactivated, can also promote complement alternative route C3 convertase C3Bb half life.FHBP is combined with fH, covers fH Bacterium surface is placed on, so as to suppress the bactericidal action of complement system.Research finds that fHBP can trigger extensive cross reaction, and These reactions are non-PorA dependences bactericidal reactions, and the experiment of serum sterilizing power also indicates that fHBP has stronger immunogenicity.

Helper T lymphocyte (Th) epitope is to be combined in antigen with MHC class Ⅱmolecules, and jointly by helper T lymphocyte by Body (T Cell Receptor, TCR) specific recognition so as to induce corresponding Th cell clones to produce the peptide fragment of activation and propagation, Typically it is made up of 11-18 continuous amino acid residues.The activation of Th cells is to humoral immunity and immune response of cytotoxic T lymphocyte work( There can be important booster action；The most frequently used P2 and P30, Yi Jibai for tetanus toxoid (TT) of versatility t cell epitope The epitopes such as the DT1 or DT2 of larynx toxoid (DT).

Therefore, versatility t cell epitope and fHBP N-terminal are connected composition restructuring chimeric protein by the present invention, the chimeric egg Bai Jiyou fHBP effective bactericidal activity, there is the strong immune miscellaneous function of t cell epitope again, and being capable of solution expression with high efficiency.

The content of the invention

It is an object of the invention to provide a kind of restructuring containing fHBP V1, fHBP V2, fHBP V3 respectively to be fitted together to egg In vain, the epitope P2 or P30 of the restructuring chimeric protein and TT are combined, or are combined with DT DT1 or DT2 epitopes.

Another object of the present invention is to provide fHBP eggs chimeric with the restructuring of TT versatility t cell epitope P2 connections In vain, it is related to tri- anomalies of fHBP altogether, three anomalies are recombinated into chimeric protein is configured to after vaccine be B mass-brain films in proportion Scorching pneumococcal proteins vaccine, the restructuring chimeric protein expression quantity is high, is easy to purify, and the vaccine being configured to has very high immunogene Property.

Another object of the present invention is to provide the preparation method of B group meningitis coccis restructuring chimeric protein vaccine.

In order to realize the object of the invention, the invention provides a kind of restructuring chimeric protein epidemic disease of prevention B mass-brain perimyelitises Seedling preparation, the bacterin preparation contain：P2-fHBP V1, P2-fHBP V2 and tri- kinds of restructuring chimeric proteins of P2-fHBP V3, its Amino acid sequence is respectively SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, wherein every kind of protein content is respectively 30-50 μ g/ agent, optional, freeze drying protectant or vaccine adjuvant are also contained in the bacterin preparation.

Bacterin preparation of the present invention, it is preferred that wherein P2-fHBP V1, P2-fHBP V2 and tri- kinds of P2-fHBP V3 The content for recombinating chimeric protein is respectively 35-45 μ g/ agent, and the pH of bacterin preparation is 5.8-7.2；Contain adjuvant aluminium in bacterin preparation Adjuvant or CpG adjuvants, the aluminium content in adjuvant is 0.4-0.6mg/ml；Or the CpG contents in adjuvant are 120-180ug/ml.

Bacterin preparation of the present invention, for for the lyophilized or liquid preparation subcutaneously or intramuscularly injected.

Bacterin preparation of the present invention, it is preferred that be freeze-dried formulation, using lactose, gelatin, sucrose or human serum albumin Lyophilized formulations are prepared into Deng protective agent composition, it is lyophilized with cillin bottle packing；Diluent is sterile, pyrogen-free water for injection, life Salt solution or PBS etc. are managed, is dispensed with pre-filled syringe or ampoule bottle.

Bacterin preparation of the present invention, it is preferred that be liquid dosage form, helped using aluminium hydroxide, aluminum phosphate or other aluminium Agent etc. is prepared into aluminium adsorptive liquid vaccine, is dispensed with cillin bottle or pre-filled syringe；Or liquid is prepared by mixing into using CpG adjuvants Body vaccine, is dispensed with pre-filled syringe.

The present invention also provides the preparation method of the restructuring chimeric protein bacterin preparation, the described method comprises the following steps：

By P2-fHBP V1, P2-fHBP V2 and tri- anomaly restructuring chimeric proteins of P2-fHBP V3, dilute in proportion After preparation, optional, freeze drying protectant or adjuvant are added, B group meningitis cocci eggs are configured to by the way of lyophilized or absorption White bacterin preparation.

In the present invention, all three restructuring chimeric proteins be brand-new albumen, for this present invention additionally comprises：

P2-fHBP V1 recombinate chimeric protein, it is characterised in that its preparation method passes through following steps：

According to P2 and fHBP V1 amino acid sequence and e. coli codon preferences, full length DNA sequence is designed and synthesized Row, the target gene fragment p2-fhbp V1 of synthesis are connected with plasmid pET43.1a with T4Ligase, and connection product conversion is big Enterobacteria E.coli BL21 (DE3), P2-fHBP V1/BL21 (DE3) bacterial strain is obtained, Spawn incubation is expanded to obtain thalline, passed through IPTG induced expressions, then obtain restructuring chimeric protein P2-fHBP V1 by protein extraction, protein purification.

P2-fHBP V2 recombinate chimeric protein, it is characterised in that its preparation method passes through following steps：

According to P2 and fHBP V2 amino acid sequence and e. coli codon preferences, full length DNA sequence is designed and synthesized Row, the target gene fragment p2-fhbp V2 of synthesis are connected with plasmid pET43.1a with T4Ligase, and connection product conversion is big Enterobacteria E.coli BL21 (DE3), P2-fHBP V2/BL21 (DE3) bacterial strain is obtained, Spawn incubation is expanded to obtain thalline, passed through IPTG induced expressions, then obtain restructuring chimeric protein P2-fHBP V2 by protein extraction, protein purification.

P2-fHBP V3 recombinate chimeric protein, it is characterised in that its preparation method passes through following steps：

According to P2 and fHBP V3 amino acid sequence and e. coli codon preferences, full length DNA sequence is designed and synthesized Row, the target gene fragment p2-fhbp V3 of synthesis are connected with plasmid pET43.1a with T4Ligase, and connection product conversion is big Enterobacteria E.coli BL21 (DE3), P2-fHBP V3/BL21 (DE3) bacterial strain is obtained, Spawn incubation is expanded to obtain thalline, passed through IPTG induced expressions, then obtain restructuring chimeric protein P2-fHBP V3 by protein extraction, protein purification.

Any restructuring chimeric protein of the present invention, equal and TT epitope P2 or P30 are combined, or with DT's DT1 or DT2 epitopes combine.

Preferable restructuring chimeric protein preparation method is as follows：

Recombinate the structure of chimeric protein colibacillus engineering strain：

FHBP albumen is divided into tri- anomalies of V1, V2 and V3, and it is special to screen fHBP V1, fHBP V2 and fHBP V3 respectively Anomaly protein sequence, V1 anomalies can select the V1 anomalies of International or National epidemic link, such as MC58, CDC1573 etc., For the present invention by taking CDC1573 strains fHBP as an example, its anomaly is V1.55, GenBank:AY330406.1；V2 anomalies can select 961-5945, M3153 etc., for the present invention by taking 961-5945 strains fHBP as an example, its anomaly is V2.16, GenBank: DQ523568.1；V3 anomalies can select M1239, M98250771 etc., and by taking M98250771 strains fHBP as an example, it becomes the present invention Abnormal shape is V3.45, GenBank:AY330361.1.

By each anomaly fHBP N-terminal connection universal t cell epitope, can be tetanus toxoid (TT) P2 and P30, or DT1 the or DT2 epitopes of diphtheria toxoid (DT), currently preferred is TT P2.

It is inclined according to the amino acid sequence for each anomalies of fHBP that P2 epitopes are connected in N-terminal, and e. coli codon Love property designs and synthesizes gene, and by gene chemical synthesis on plasmid vector pUC57-Amp or pUC57-Simple, the present invention preferably carries Body pUC57-Amp.

The selection of chimeric protein expression vector is recombinated as pBR322, pSC101, pET32a or pET43.1a etc., the present invention is preferably pET43.1a。

Resistant gene is using kanamycins, tetracycline or ampicillin (Ampicillin, Amp) etc., and the present invention is preferably Resistance is ampicillin.

Gene chemical synthesis product and plasmid are handled using the digestion such as restriction enzyme Hind III, Bam HI, NdeI, XhoI, Preferably restriction enzyme of the invention is NdeI, XhoI.

The big segments of DNA after digestion is handled are connected with expression vector plasmid pET43.1a with T4 ligases, connection product Convert bacillus coli DH 5 alpha, screening positive clone, after sequence verification DNA sequence dna is completely correct, extraction positive colony plasmid simultaneously turns Change e. coli bl21 (DE3), cultivated on the culture medium containing antibiotic, after picking Colony Culture, through electrophoresis or mass spectrum or After the methods of determined amino acid sequence is analyzed identification, it is defined as the expression of P2-fHBP chimeric proteins.

The fermented and cultured and induced expression of engineering bacteria：

The soluble expression engineered strain of structure is taken, kind of a LB agar mediums (containing Amp) is drawn, 37 DEG C of overnight incubations, chooses Take single bacterium colony to be seeded in LB fluid nutrient mediums (containing Amp), and fermentation tank is progressively extended to 1.5-7.5% inoculative proportion Fermented and cultured, when OD600 reaches 18-22, using isopropyl-β-D-thiogalactoside (IPTG) Fiber differentiation 3-6 hours, Stop culture, coli somatic is collected by centrifugation.

Recombinate the extraction purification of chimeric protein：

The bacterium of fermented and cultured harvest is centrifuged, thalline is collected, after a certain proportion of broken bacterium buffer solution bacteriolyze, using anti- Multiple freeze thawing, ultrasonication, low-temperature ultrahigh-pressure homogeneous be broken or crushes microorganism the methods of osmotic shock, and bacterium is collected by centrifugation Broken supernatant, mesh is extracted by the methods of ammonium sulfate precipitation, alcohol fractional precipitation, isoelectric precipitation, aqueous two-phase extracting Albumen, then polishing purification is carried out using ion exchange or hydrophobic chromatography and molecular sieve etc., removes lipopolysaccharides, nucleic acid and other are big Enterobacteria host's foreign protein, obtain the restructuring P2-fHBP chimeric protein stostes that purity is more than 95%.

Bacterial endotoxin is not higher than 20EU/ml, and the present invention should be not higher than 10EU/ml, most preferably not higher than 5EU/ml.

The B group meningitis cocci protein vaccines of the present invention, its preparation method are as follows：

By restructuring fHBP chimeric proteins P2-fHBP V1, P2-fHBP V2 and the P2-fHBP V3 of three anomalies by corresponding After ratio is prepared, lyophilized formulations are prepared into using the protective agent composition such as lactose, gelatin, sucrose or human serum albumin, with cillin bottle Packing is lyophilized；Diluent is sterile, pyrogen-free water for injection, physiological saline or PBS etc., with pre-filled syringe or ampoule bottle Packing.

Or

By restructuring fHBP chimeric proteins P2-fHBP V1, P2-fHBP V2 and the P2-fHBP V3 of three anomalies by corresponding After ratio is prepared, adsorbed vaccine is prepared into using adjuvants such as aluminium hydroxide, aluminum phosphate or CpG, with pre-filled syringe or west Woods bottle dispenses.

By any of the above vaccine formulation mode, every dose is configured to final concentration V1 containing P2-fHBP, P2-fHBP V2 and P2- The bacterin preparation of each 30-50 μ g/ agent of fHBP V3.

It is an advantage of the invention that being not suitable for use in the feature of vaccine for B group meningitis cocci capsular polysaccharide compositions, utilize Reverse Genetics Technique, recombinantly expresses B group meningitis coccis people H factor bindins (fHBP) antigen, and the proteantigen contains Tri- anomalies of V1, V2 and V3, completely covers all bacterial strains of B group meningitis coccis, to caused by the infection of B group meningitis coccis The diseases such as cerebrospinal meningitis play effective and extensive prevention effect；Versatility t cell epitope is connected to fHBP N by the present invention End, the immunogenicity of fHBP albumen is significantly increased, and this is expressed as solubility expression.Three anomalies are recombinated into fHBP simultaneously Chimeric protein is configured to vaccine, by increasing capacitance it is possible to increase vaccine is to the preventive effects of all B group meningitis coccis wide spectrums, so as to provide more Extensively, the more efficiently vaccine protecting effect for B group meningitis coccis.

Brief description of the drawings

Fig. 1 is that the DNA sequence dna after p2-fhbp V1 genes are connected with pET43.1a identifies electrophoretogram.

Fig. 2 is that the DNA sequence dna after p2-fhbp V2 genes are connected with pET43.1a identifies electrophoretogram.

Fig. 3 is that the DNA sequence dna after p2-fhbp V3 genes are connected with pET43.1a identifies electrophoretogram.

Fig. 4 is the SDS-PAGE figures that P2-fHBP V1 recombinate chimeric protein expression.

Fig. 5 is the SDS-PAGE figures that P2-fHBP V2 recombinate chimeric protein expression.

Fig. 6 is the SDS-PAGE figures that P2-fHBP V3 recombinate chimeric protein expression.

Embodiment

In order to be more clearly understood that the present invention, the present invention is further described referring now to the following example and accompanying drawing.Embodiment It is only used for explaining without limiting the invention in any way.

The structure of embodiment 1P2-fHBP V1 engineered strains：

According to fHBP V1 and P2 amino acid sequence and e. coli codon preferences, full length DNA sequence is designed and synthesized Row p2-fhbp V1, p2 are connected to fhbp V1N ends, are gene chemical synthesis plasmid, gene p2-fhbp V1 are blended into plasmid On pUC57, gene chemical synthesis plasmid pUC57 and carrier pET43.1a is handled with restriction enzyme NdeI and XhoI digestion, obtained Plasmid pET43.1a after target gene fragment and digestion；By target gene fragment p2-fhbp V1 and plasmid pET43.1a with T4ligase connections, connection product Transformed E .coli DH5 α, 37 DEG C of overnight incubations.Picking positive colony is inoculated with LB Liquid Cultures Base, 37 DEG C of shaken cultivations are stayed overnight.With PCR method amplifying target genes segment, analyze and identify result such as Fig. 1 and show, it is amplifiable to arrive About 800bp or so the big segment of p2-fhbp V1 genes.Target gene segment is sequenced, as a result shows genetic fragment DNA sequence dna (p2-fhbp V1) is completely correct.

Correct plasmid Transformed E .coli BL21 (DE3) competent cell will be connected, (contains 100 μ g/ml in LB culture mediums Ampicillin) 37 DEG C be incubated overnight, obtain positive P2-fHBP V1/BL21 (DE3) expression bacterial strain, the single clone of picking, 37 DEG C shaken cultivation is to bacterial density to OD₆₀₀When about 0.5~1.0, final concentration of 0.5mM isopropyl-beta D-thio gala is added Glucosides (IPTG), 37 DEG C of vibration Fiber differentiation 2-6 hours, identified through SDS-PAGE, as a result such as Fig. 4 is shown, P2-fHBP V1/ There are obvious band of expression, molecular weight 32KD after BL21 (DE3) inductions.Through Mass Spectrometric Identification it is P2- by expressing protein band FHBP V1 recombinate chimeric protein, and sequence table is up to correctly.

The fermentation of embodiment 2P2-fHBP V1 engineering bacterias

By strain be inoculated with LB fluid nutrient mediums, 37 DEG C, 200rpm cultivate 12 hours.Expanded with 5% ratio and be seeded to fermentation Tank, after 37 DEG C of culture 4-8 hours, when OD600 rises to 18~22,4-8 hours are induced with final concentration 0.05mM IPTG.Greatly Thalline is collected by centrifugation in capacity centrifugation machine.

Embodiment 3P2-fHBP V1 recombinate the extraction purification of chimeric protein

Bacterial cell disruption：Thalline is taken to add broken bacterium buffer solution (20mM PB, 2mM EDTA, pH7.0), low temperature (4 DEG C) super-pressure After the broken bacterium of (1100-1400bar) homogeneous two times, high speed centrifugation (10000rpm, 60min, 4 DEG C) takes bacteria breaking supernatant.

Protein extraction：Using the method for ammonium sulfate precipitation, precipitated 1 hour under 15% 4 DEG C of ammonium sulfate stirring, 6500rpm8 DEG C centrifuges 60 minutes, takes centrifugation supernatant；By supernatant with 4 DEG C of 45% ammonium sulfate stirring precipitation 1 hour, albumen precipitation is taken Dissolved with 20mM PB PH7.4,10KD film bag ultrafiltration is simultaneously concentrated into the 1/5-1/3 of original volume.

Albumen polishing purification：Using Sepharose QFF, with 20mM PB PH7.4 equilibrium liquid loadings, with 20mMPB+1M NaCl PH7.4 eluent 0-100% gradient elutions, collect 25% eluting peak.25% gradient eluent is taken with 10KD milipore filter bags It is concentrated by ultrafiltration to the 1/3 of original volume, is chromatographed through GE Sepharose 6FF, collect V₀Locate protein peak.

It is prepared by embodiment 4P2-fHBP V1 restructuring chimeric protein stostes：

The V collected will be chromatographed through GE Sepharose 4FF₀Locate protein peak purifyingization liquid, with 0.15mol/L sodium chloride, 10KD film bags ultrafiltration more than 5 times, and it is 1-2mg/ml to be concentrated into protein content, 0.22 μm of aseptic filtration.Through sterility test, albumen After content inspection, molecular weight and purity test, protein-specific inspection, baterial endotoxin test are qualified, as P2-fHBP V1 Albumen stoste.

Embodiment 5P2-fHBP V1 molecular weight of albumen and purity test：SDS-PAGE is carried out using 15% separation gel, on Sample amount is 10ug, and with unconverted e. coli bl21 (DE3) for negative control；After electrophoresis film coomassie brilliant blue staining, With Gel Doc^TMXR+ gel imagers carry out gel image scanning, calculate molecular weight and purity analysis with Image Lab softwares, as a result As shown in figure 4, molecular weight is 32.3KD, purity 98.2%.

The protein-specific of embodiment 6 checks Western Blot：Sample and unconverted e. coli bl21 (DE3) warp After SDS-PAGE, electricity is transferred on PVDF or NC films, and rabbit-anti fHBP specific antibodies are combined after being closed with skimmed milk power, and room temperature is incubated Educate 2 hours, washed 5 times with PBST, with reference to goat-anti rabbit secondary antibody, be incubated at room temperature 1 hour, washed 5 times with PBST, DAB colour developings.As a result It has been shown that, P2-fHBP V1 show specific protein leukorrhea in 32KD or so, and unconverted Escherichia coli then show without specific protein leukorrhea It is existing, it was demonstrated that to be expressed for specific fHBP V1.

Embodiment 7 is built according to embodiment 1-6 strain and protein preparation method, and the spy of P2-fHBP V2 albumen in itself Property build and prepare P2-fHBP V2 stostes, specific embodiment is improved or changed in embodiment 1-6 method, warp Identification and analysis albumen stoste is that P2-fHBP V2 are correctly expressed, and molecular weight 31.8, purity reaches 97.6%.

Embodiment 8 is built according to embodiment 1-6 strain and protein preparation method, and the spy of P2-fHBP V3 albumen in itself Property build and prepare P2-fHBP V3 stostes, specific embodiment is improved or changed in embodiment 1-6 method, warp Identification and analysis albumen stoste is that P2-fHBP V3 are correctly expressed, and molecular weight 32.1, purity reaches 98.5%.

The preparation of the vaccine of embodiment 9：

The preparation of trivalent fHBP protein vaccines

Three kinds of anomalies are recombinated into B group's fHBP chimeric proteins P2-fHBP V1, P2-fHBP V2 and P2-fHBP V3, respectively Adsorbed with hydrogen-oxygen aluminium adjuvant, 4 DEG C are stirred overnight, and adsorption rate is more than 95%.Diluted and prepared with 0.15mol/L sodium chloride, to egg White final concentration is respectively 240 μ g/ml, aluminium content final concentration of 0.45-0.6mg/ml, pH value 5.8-7.2.

In the present embodiment, by three kinds of restructuring chimeric proteins absorption stostes, by being hybridly prepared into trivalent vaccine finished product in equal volume. Dispensed with Prefilled syringe, as absorbent-type liquid vaccine shots finished product.

Embodiment 10B group recombinates the immunogenicity research of chimeric protein vaccine

Immune formulation is：Aluminum hydroxide adjuvant absorption P2-fHBPV1/V2/V3 unit prices and trivalent vaccine finished product, and helped with aluminium Agent absorption physiological saline is negative control.

Mouse immune：12-14 grams of SPF level NIH mouse 10 of subcutaneous inoculation respectively, immunizing dose are 0.1ml/, are immunized Program is 0,2,4 weeks, takes a blood sample within 2 weeks after immune programme for children terminates, serum is collected by centrifugation；Separately set 10 mouse negative controls, phase Tongfang Method injection aluminium adjuvant absorption physiological saline.

Sterilizing power (SBA) experiment of epidemic strain：

Using the bacterial strain of B group meningitis coccis 440902, the bacterial strain is ST4821 sequence types, category ST4821 sequences group (clone Group), for domestic B group meningitis cocci epidemic links in the recent period, fHBP partings are V2 anomalies.

The preparation of target bacterium：The bacterial strain of epidemic meningitis coccus 440902 is cultivated, on 8-12% sheep blood agar flat boards, 37 DEG C 6-10% carbon dioxide culture 16-18 hours, scraping lawn is into physiological saline, and bacterium turbidimetry counts, according to counting, by target Bacterium is diluted to 1 × 10⁶。

Mice serum to be checked is inactivated 1 hour in 56 DEG C, to inactivate the intrinsic complement activity of mice serum.Will in experimentation Pel-Freez children's rabbit complement is added in mice serum to be checked, while sets inactivation complement, complement control control, doubling dilution To 96 well culture plates, and the target bacterium 10ul of Fresh is added dropwise.Concussion mixes cultivates 2-4 hours after 37 DEG C.

Point sample：The mixed bacteria liquid after culture is taken, is added dropwise to 10ul amount on Solid nutritional agar synthetic medium, 37 DEG C 5% carbon dioxide overnight incubation.

Colour developing：The soft agar of TTC containing 150-300ug/ml is covered in the Solid nutritional agar synthesis being incubated overnight On culture medium, developed the color with preference temperature, suitable time.

Count：High-definition shooting colour developing bacterium colony photo, using image scanning techniques, with dedicated analysis software analysis and calculate thin Bacterium bacterium colony number, bactericidal titre is calculated with sterilizing power result software for calculation.As a result it is as follows：

Mice serum sterilizing power titre (BC Titer 1：)

Sequence table

110 Beijing Zhifei Lvzhu Biopharmaceutical Co., Ltd.

120 a kind of B group meningitis coccis restructuring chimeric protein vaccines and preparation method thereof

160 3

210 1

211 276

212 PRT

213 artificial sequences

220

221

222

223

400 1

Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu

5 10 15

Leu Cys Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Val Thr

20 25 30

Ala Asp Ile Gly Thr Gly Leu Ala Asp Ala Leu Thr Ala Pro Leu

35 40 45

Asp His Lys Asp Lys Gly Leu Lys Ser Leu Thr Leu Glu Asp Ser

50 55 60

Ile Ser Gln Asn Gly Thr Leu Thr Leu Ser Ala Gln Gly Ala Glu

65 70 75

Lys Thr Tyr Gly Asn Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys

80 85 90

Asn Asp Lys Val Ser Arg Phe Asp Phe Ile Arg Gln Ile Glu Val

95 100 105

Asp Gly Gln Leu Ile Thr Leu Glu Ser Gly Glu Phe Gln Val Tyr

110 115 120

Lys Gln Ser His Ser Ala Leu Thr Ala Leu Gln Thr Glu Gln Glu

125 130 135

Gln Asp Pro Glu His Ser Glu Lys Met Val Ala Lys Arg Arg Phe

140 145 150

Arg Ile Gly Asp Ile Ala Gly Glu His Thr Ser Phe Asp Lys Leu

155 160 165

Pro Lys Asp Val Met Ala Thr Tyr Arg Gly Thr Ala Phe Gly Ser

170 175 180

Asp Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala Ala

185 190 195

Lys Gln Gly His Gly Lys Ile Glu His Leu Lys Ser Pro Glu Leu

200 205 210

Asn Val Asp Leu Ala Val Ala Tyr Ile Lys Pro Asp Glu Lys His

215 220 225

His Ala Val Ile Ser Gly Ser Val Leu Tyr Asn Gln Asp Glu Lys

230 235 240

Gly Ser Tyr Ser Leu Gly Ile Phe Gly Glu Lys Ala Gln Glu Val

245 250 255

Ala Gly Ser Ala Glu Val Glu Thr Ala Asn Gly Ile His His Ile

260 265 270

Gly Leu Ala Ala Lys Gln

275

210 2

211 270

212 PRT

213 artificial sequences

220

223

400 2

Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu

5 10 15

Leu Cys Ser Ser Gly Gly Gly Gly Val Ala Ala Asp Ile Gly Ala

20 25 30

Gly Leu Ala Asp Ala Leu Thr Ala Pro Leu Asp His Lys Asp Lys

35 40 45

Ser Leu Gln Ser Leu Thr Leu Asp Gln Ser Val Arg Lys Asn Glu

50 55 60

Lys Leu Lys Leu Ala Ala Gln Gly Ala Glu Lys Thr Tyr Gly Asn

65 70 75

Gly Asp Ser Leu Asn Thr Gly Lys Leu Lys Asn Asp Lys Val Ser

80 85 90

Arg Phe Asp Phe Ile Arg Gln Ile Glu Val Asp Gly Gln Leu Ile

95 100 105

Thr Leu Glu Ser Gly Glu Phe Gln Ile Tyr Lys Gln Asp His Ser

110 115 120

Ala Val Val Ala Leu Gln Ile Glu Lys Ile Asn Asn Pro Asp Lys

125 130 135

Ile Asp Ser Leu Ile Asn Gln Arg Ser Phe Leu Val Ser Gly Leu

140 145 150

Gly Gly Glu His Thr Ala Phe Asn Gln Leu Pro Asp Gly Lys Ala

155 160 165

Glu Tyr His Gly Lys Ala Phe Ser Ser Asp Asp Ala Gly Gly Lys

170 175 180

Leu Thr Tyr Thr Ile Asp Phe Ala Ala Lys Gln Gly His Gly Lys

185 190 195

Ile Glu His Leu Lys Thr Pro Glu Gln Asn Val Glu Leu Ala Ala

200 205 210

Ala Glu Leu Lys Ala Asp Glu Lys Ser His Ala Val Ile Leu Gly

215 220 225

Asp Thr Arg Tyr Gly Ser Glu Glu Lys Gly Thr Tyr His Leu Ala

230 235 240

Leu Phe Gly Asp Arg Ala Gln Glu Ile Ala Gly Ser Ala Thr Val

245 250 255

Lys Ile Gly Glu Lys Val His Glu Ile Gly Ile Ala Gly Lys Gln

260 265 270

211 270

210 3

211 277

212 PRT

213 artificial sequences

220

223

400 3

Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu

5 10 15

Leu Cys Ser Ser Gly Ser Gly Ser Gly Gly Gly Gly Val Ala Ala

20 25 30

Asp Ile Gly Thr Gly Leu Ala Asp Ala Leu Thr Ala Pro Leu Asp

35 40 45

His Lys Asp Lys Gly Leu Lys Ser Leu Thr Leu Glu Asp Ser Ile

50 55 60

Ser Gln Asn Gly Thr Leu Thr Leu Ser Ala Gln Gly Ala Glu Lys

65 70 75

Thr Phe Lys Val Gly Asp Lys Asp Asn Ser Leu Asn Thr Gly Lys

80 85 90

Leu Lys Asn Asp Lys Ile Ser Arg Phe Asp Phe Val Gln Lys Ile

95 100 105

Glu Val Asp Gly Gln Thr Ile Thr Leu Ala Ser Gly Glu Phe Gln

110 115 120

Ile Tyr Lys Gln Asp His Ser Ala Val Val Ala Leu Gln Ile Glu

125 130 135

Lys Ile Asn Asn Pro Asp Lys Ile Asp Ser Leu Ile Asn Gln Arg

140 145 150

Ser Phe Leu Val Ser Gly Leu Gly Gly Glu His Thr Ala Phe Asn

155 160 165

Gln Leu Pro Ser Gly Lys Ala Glu Tyr His Gly Lys Ala Phe Ser

170 175 180

Ser Asp Asp Ala Gly Gly Lys Leu Thr Tyr Thr Ile Asp Phe Ala

185 190 195

Ala Lys Gln Gly His Gly Lys Ile Glu His Leu Lys Thr Pro Glu

200 205 210

Gln Asn Val Glu Leu Ala Ser Ala Glu Leu Lys Ala Asp Glu Lys

215 220 225

Ser His Ala Val Ile Leu Gly Asp Thr Arg Tyr Gly Ser Glu Glu

230 235 240

Lys Gly Thr Tyr His Leu Ala Leu Phe Gly Asp Arg Ala Gln Glu

245 250 255

Ile Ala Gly Ser Ala Thr Val Lys Ile Arg Glu Lys Val His Glu

260 265 270

Ile Gly Ile Ala Gly Lys Gln

275

Claims

1. a kind of restructuring chimeric protein bacterin preparation of prevention B mass-brain perimyelitises, it is characterised in that the bacterin preparation contains Have：P2-fHBP V1, P2-fHBP V2 and tri- kinds of restructuring chimeric proteins of P2-fHBP V3, its amino acid sequence is respectively SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, wherein every kind of protein content is respectively 30-50 μ g/ agent, optional, the epidemic disease Also contain freeze drying protectant or vaccine adjuvant in seedling preparation.

2. bacterin preparation according to claim 1, it is characterised in that wherein P2-fHBP V1, P2-fHBP V2 and P2- The content of tri- kinds of restructuring chimeric proteins of fHBP V3 is respectively 35-45 μ g/ agent, and the pH of bacterin preparation is 5.8-7.2；Bacterin preparation In contain adjuvant aluminium adjuvant or CpG adjuvants, the aluminium content in adjuvant is 0.4-0.6mg/ml；Or the CpG contents in adjuvant are 120-180ug/ml。

3. bacterin preparation according to claim 1, it is characterised in that be the lyophilized or liquid system for subcutaneously or intramuscularly injecting Agent.

4. bacterin preparation according to claim 3, it is characterised in that be freeze-dried formulation, using lactose, gelatin, sucrose or The protective agent composition such as human serum albumin is prepared into lyophilized formulations, lyophilized with cillin bottle packing；Diluent is sterile, pyrogen-free note Penetrate and use water, physiological saline or PBS etc., dispensed with pre-filled syringe or ampoule bottle.

5. bacterin preparation according to claim 3, it is characterised in that be liquid dosage form, using aluminium hydroxide, aluminum phosphate or Other aluminium adjuvants etc. are prepared into aluminium adsorptive liquid vaccine, are dispensed with cillin bottle or pre-filled syringe；Or using the mixing of CpG adjuvants Aqueous vaccine is prepared into, is dispensed with pre-filled syringe.

6. the preparation method of chimeric protein bacterin preparation is recombinated described in claim 1, it is characterised in that comprise the following steps：

By P2-fHBP V1, P2-fHBP V2 and tri- anomaly restructuring chimeric proteins of P2-fHBP V3, dilution in proportion is prepared Afterwards, optionally, freeze drying protectant or adjuvant are added, B group meningitis cocci albumen epidemic diseases are configured to by the way of lyophilized or absorption Seedling preparation.

7.P2-fHBP V1 recombinate chimeric protein, it is characterised in that its preparation method passes through following steps：

According to P2 and fHBP V1 amino acid sequence and e. coli codon preferences, full length DNA sequence is designed and synthesized, The target gene fragment p2-fhbp V1 of synthesis are connected with plasmid pET43.1a with T4Ligase, connection product conversion large intestine bar Bacterium E.coli BL21 (DE3), P2-fHBP V1/BL21 (DE3) bacterial strain is obtained, Spawn incubation is expanded to obtain thalline, through IPTG Induced expression, then obtain restructuring chimeric protein P2-fHBP V1 by protein extraction, protein purification.

8.P2-fHBP V2 recombinate chimeric protein, it is characterised in that its preparation method passes through following steps：

According to P2 and fHBP V2 amino acid sequence and e. coli codon preferences, full length DNA sequence is designed and synthesized, The target gene fragment p2-fhbp V2 of synthesis are connected with plasmid pET43.1a with T4Ligase, connection product conversion large intestine bar Bacterium E.coli BL21 (DE3), P2-fHBP V2/BL21 (DE3) bacterial strain is obtained, Spawn incubation is expanded to obtain thalline, through IPTG Induced expression, then obtain restructuring chimeric protein P2-fHBP V2 by protein extraction, protein purification.

9.P2-fHBP V3 recombinate chimeric protein, it is characterised in that its preparation method passes through following steps：

According to P2 and fHBP V3 amino acid sequence and e. coli codon preferences, full length DNA sequence is designed and synthesized, The target gene fragment p2-fhbp V3 of synthesis are connected with plasmid pET43.1a with T4Ligase, connection product conversion large intestine bar Bacterium E.coli BL21 (DE3), P2-fHBP V3/BL21 (DE3) bacterial strain is obtained, Spawn incubation is expanded to obtain thalline, through IPTG Induced expression, then obtain restructuring chimeric protein P2-fHBP V3 by protein extraction, protein purification.

10. claim 7,8,9 any one restructuring chimeric protein, it is characterised in that the restructuring chimeric protein and TT resist Former epitope P2 or P30 are combined, or are combined with DT DT1 or DT2 epitopes.