CN107961369A

CN107961369A - Multivalent meningococcal conjugate vaccines and preparation method thereof

Info

Publication number: CN107961369A
Application number: CN201710183434.3A
Authority: CN
Inventors: 史晋; 李津; 吴克; 王文灏
Original assignee: BRAVOVAX Co Ltd
Current assignee: BRAVOVAX Co Ltd
Priority date: 2017-03-22
Filing date: 2017-03-24
Publication date: 2018-04-27
Anticipated expiration: 2037-03-24
Also published as: CN107961369B; CN107151270A; CN107961370B; CN107961368B; CN107961370A; CN107961368A; CN107151270B

Abstract

The present invention provides a kind of multivalent meningococcal based on restructuring Δ fHbp NadA fusion protein carriers.By designing soft connection peptide chain by two activated protein amalgamation and expressions, keep allowing both to become single protein while respective activity again, thus in industrial production and immunity inoculation substantially reduced with more controllability, risk, and more polysaccharide protein binding sites can be provided.

Description

Multivalent meningococcal conjugate vaccines and preparation method thereof

Technical field

The invention belongs to biological technical field, more particularly to a kind of multivalent meningococcal conjugate vaccines and its preparation side Method.

Background technology

First, pathogenic microorganism and vaccine

It can cause human body or animal body that the microorganism of infectious disease occurs, be known as pathogenic microorganism or pathogenic microorganisms.Infect After referring to pathogenic microorganism intrusion body, in certain position growth, breeding, and cause a series of physiopathologic processes.When After pathogenic microorganism intrusion body, pathogenic microorganism interacts with body, changes the activity and function of other side, therefore energy mutually No to produce communicable diseases, on the one hand pathogenecity, that is, pathogenic or virulence depending on pathogenic microorganism, another aspect additionally depend on machine Resistance, that is, immunity of body.Pathogenic bacteria causes the capacity of water of infection, is exactly the virulence or pathogenic of bacterium.Bacterial poison The power of the presence or absence of power and virulence depends primarily upon its invasiveness, toxigenicity and the ability for causing hypersensitivity.

Bacteriogenic toxin can be divided into two major class of exotoxin and endotoxin.Exotoxin is pathogen during growth and breeding A kind of metabolite of ambient environment is secreted into, is mainly produced by gram-positive bacteria, a small number of Gram-negative bacterias can also produce It is raw.Its chemical composition is protein, and antigenicity is strong, and toxicity is also strong, but extremely unstable, sensitive to hot and some chemical substances, is held It is vulnerable to destruction.It is common as：The tetanus toxin, suddenly that diphtherotoxin that corynebacterium diphtheriae produces, clostridium tetani produce Botulinum toxin that enterotoxin, the clostridium botulinum of random vibrios generation produce etc..Most of gramnegative bacteriums can produce endotoxin, Actually it is present in the outer layer of bacteria cell wall, belongs to the part of cell membrane, is not secreted into environment under normal circumstances In, only just discharged after bacterolysis, thus referred to as endotoxin, its toxicity are lower than exotoxin, antigenicity is also weak.

The Different Individual of same organism, after they are contacted with pathogen, some illness, some is then safe and sound, reason It is that the immunity of Different Individual is different.It is immune just to refer to the one of body identification and exclusion antigen foreign matter (such as pathogenic microorganism) Kind aversion response.In general, it be to body favourable, in exception conditions, it is also possible to damage body.Human body is immunized It is divided into nospecific immunity and specific immunity.Wherein specific immunity refer to body for a certain or a certain quasi-microorganism or Special resistance caused by product.And to be scientist develop vaccine that body is produced specific immunity resistance cause of disease micro- The biological products of human body infringement are usually prepared in biology in itself by pathogenic microorganism.Bacterium, virus and rickettsia Vaccine is made in the pathogenic microorganisms such as family name's body, after injecting body, body is produced specificity or sensitization lymphocyte, secretion is anti- Body, reaches specific immunity effect.

And vaccine is divided into therapeutic and two kinds preventative, disease is treated by therapeutic vaccine, and passes through preventative epidemic disease Seedling protects infringement of the human body from invasive organism.By effort for many years, medical field has developed a variety of epidemic diseases Seedling is to prevent, bacterium, virus and fungi etc., various diseases caused by infection, drastically increases the healthy water of the mankind It is flat.The continuous development of biotechnology, promotes the variation of vaccine kind.There is inactivation disease to infectious disease caused by pre- anti-virus The vaccine that malicious technological development comes out, such as Vaccinum Encephalitidis Epidemicae, polio vaccine, influenza vaccines；Attenuated virus technological development goes out The attenuated live vaccine come, such as Rotavirus Vaccine, oral polio virus vaccine, measles virus vaccines, mumps virus Vaccine, rubella virus vaccine and chicken pox vaccine etc..To prevent big point of the biology such as the useful proteins of bacterial infectious disease and polysaccharide The bacterium class vaccine that sub- purification technique develops, as tetanus toxoid, diphtheria toxoid, pertussis toxoid and its Asia are thin Born of the same parents' component, epidemic meningitis Streptococcus polysaccharides and 23 valency pneumococal polysaccharides etc..More advanced useful half chemical combination technology is opened The prevention meningitis and the bacterial vaccine of pneumonia issued, such as popular influenzae type polysaccharide-protein conjugate vaccines, 7 valencys or 10 valency pneumococcal polysaccharide-protein conjugate vaccines and 4 valencys meningococcal polysacharide-albumen conjugate vaccines.By to biological skill Art is continuously improved, and can develop more new generation vaccine products and human health is chosen to deal with different pathogenic microorganisms War.

2nd, polysaccharide-protein conjugate vaccines and its protein carrier

Polysaccharide is a kind of important immune active ingredient in pathogen, have mycelial polysaccharides (OPS) and capsular polysaccharide (CPS) it Point.After pathogen invades body, they can stimulate body to produce protective immune response as immunogene.It is and many at present Can cause endanger serious disease pathogen such as haemophilus influenzae type b (Haemophilus influenzae type B, Hib), streptococcus pneumonia (Streptococcus pneumoniae, Spn) and Neisseria meningitidis (Neisseria Meningitidis, Nm) its surface all has polysaccharide, and these polysaccharide can assign killing and phagocytosis of the pathogen to complement-mediated Effect produces resistance, so as to promote the survival during their morbidities in blood.The vaccine that the first generation is directed to Hib, Spn and Nm is Using polysaccharide as antigen.But unfortunately, these polysaccharide vaccines are not immunogenicity in child, and cannot produce immune note Recall.This is because polysaccharide molecule belongs to the not dependent antigen of T cell (Ti-Ag), less immunogenic, is immunized after being inoculated with infant Effect is especially undesirable.In order to improve the immunogenicity of polysaccharide vaccine, the 1920s and the thirties by Landsteiner, Avery and Goebel with protein molecule by strengthening.1980, John Robbins and Rachel Schneerson describes the conjugate of Hib polysaccharide and diphtheria and tetanus toxoid albumen, greatly enhances animal model In antibody response.Final Hib polysaccharide-protein conjugates, trigger memory-type antibody response in human infant.A new generation Polysaccharide-protein conjugate vaccines, create recovery in vaccinology.This kind of conjugate is by past T cell dependent/non-dependent polysaccharide Vaccine, which is converted into children, has more strongly immunogenic T cell dependence vaccine, and display is with generation with high affinity Antibody ability, establish immunological memory, and produce group immunological effect.In addition, the prematurity that they improve young baby is exempted from The protective response of epidemic disease system and the aging immune system of the elderly, so as to reach optimal immune effect.

So far, 5 kinds of carrier proteins are had been used in the conjugate vaccines of license listing：Diphtheria toxin non-toxic variant (CRM197), tetanus toxoid (TT), meningococcal outer membrane albumen composition (OMP), diphtheria toxoid (DT) and influenza Influenzae Protein D (HiD).These verified conjugate vaccines of clinical test are in prophylaxis against infection diseases and change Hib, Spn The effect of with the medicine of Nm.All 5 kinds of carrier proteins are all effective in terms of vaccine immunogenicity is increased, but they The amount of caused antibody is different with affinity, and the ability of a variety of polysaccharide is carried in identical product and is made at the same time with other vaccines Ability is also different.

CRM197 is the non-toxic mutant of the separated diphtheria toxin from corynebacterium diphtheriae C7 (β 197) culture.CRM197 with The difference of wild type diphtheria toxin is that the point mutation at amino acid position 52 replaces glycine with glutamic acid, this disappears Except its enzymatic activity and toxicity.CRM197 and diphtheria toxin are undistinguishable on antigen, but have and be used as compound protein The advantages of：Nontoxic, and can be used for being conjugated with more lysyl side chains.Another form of CRM as conjugate It is the natural diphtheria toxin of purifying, it is then detoxified with formaldehyde, is known as diphtheria toxoid (DT), should not be obscured with CRM197.TT Be clostridium tetani culture produce tetanus toxin with formaldehyde removing toxic substances come be prepared into come.OMP is Nm bacterium serotypes B group's outer membranes Albumen composition.DT be corynebacterium diphtheriae culture produce diphtheria toxin, by formaldehyde removing toxic substances be prepared into come.HiD is initially to use It is ultrasonically treated in haemophilus influenzae, by SDS-PAGE step purifies and separates haemophilus influenzae surface proteins, but at present Vaccine be all that recombinant protein is obtained by genetic engineering.

The toxoids such as TT, DT industrialized production already, is easier to obtain, and can be greatly enhanced really after being coupled with polysaccharide more The immunogenicity of sugar.But to obtain toxoid, chemical detoxication need to be carried out to bacteriotoxin.And it can produce in this course a little pair Point mutation or unnecessary modification, the physicochemical property of intoxicating element change, destroy important T cell antigen epitope, influence it and exempt from Epidemic focus.The above problem is avoided if using nontoxic variation toxin CRM197.But internal existing toxoid antibody (TT Largely used with DT) it may result in hypersensitivity or cause the problems such as suppressing anti-sugar reaction (immunosupress).Using identical The polysaccharide of kind bacterium can avoid these potential problems with carrier protein.Such as exploitation Nm vaccines, with the albumen or OMP of B groups Albumen for carrier prepare A, C, Y, W135 group polysaccharide-protein conjugate vaccines can yet be regarded as one it is good tactful, it can prevent in theory Nearly all pathogenic Nm, but on condition that the B groups of protein carriers be " wide spectrum " or there is very big cross reactivity.Cause This, develops new, and it is extremely urgent with the protein carrier of real protective immunity originality can to assign vaccine.

3rd, Neisseria meningitidis (Nm) and its epidemiology

Meningococal meningitis (epidemic cerebrospinal meningitis) is by Neisseria meningitdis Bacterium, by acute purulent meningitis caused by respiratory infectious, there is different degrees of prevalence all over the world.How is meningitis Plucked instrument bacterium is also referred to as meningococcus, is a kind of Grain stain negative diplococci, usually pharynx nasalis of the field planting in people.The mankind be its only One host, thus Nm shows the adaptability to human nasopharynx portion height, the healthy population of 10%-40% are all Nm without clinic Symptom carrier, in meningitis Outbreak period, Asymptomatic Carriers ratio more may be up to 70%.Under a few cases, Nm Can menses intrusion meninges, cause purulent inflammation, the cerebrospinal fluid change of meningismus and purulent meningitis occur.Epidemic disease Seedling prevention is to prevent the important means of meningococcosis, because even being interfered using antibiotic, this disease still has high morbidity Rate and the death rate.

With pathogenic Nm bacterial strains generally all with pod membrane structure, the bacterial strain that healthy population normally carries often lacks Pod membrane, becomes the bacterial strain that cannot divide group.According to the chemical composition of Nm capsular polysaccharides can be divided into A, B, C, D, H, I, K, L, X, Y, 13 sero-groups (Serogroup) of Z, 29E and w135, and according to its outer membrane protein (OMP), PorB (2 or 3 class OMp) and The antigenic specificity of PorA (1 class OMP), and it is divided into different serotype and blood serum subtype.Global annual about generation 1,200,000 Nm cases of infection, majority of cases are caused by A, B, C, W135, Y groups of bacterial strains.It is and popular as caused by B crowds of Nm in developed country Property cerebrospinal meningitis case is more up to the 80% of total case.In the popular mainly based on A groups of China, in recent years due to vaccine Extensive inoculation, infection caused by A group being effectively controlled, and the case as caused by B groups also increases relatively.Single sero-group, The Meningococcal Vaccine of serotype and blood serum subtype is difficult the generation for preventing meningococal meningitis on the whole.With to Nm surfaces Antigenic structure more in-depth study, Meningococcal Vaccine just develop towards diversification direction.

Epidemic meningitis polysaccharide-protein is conjugated vaccine and has developed, and Chiron and Wyem use detoxification diphtheria toxoid (CRM197) As protein carrier, Baxter develops A/C group meningitis cocci conjugate vaccines by the use of tetanus toxoid as carrier, in In November, 1999 introduces Britain.Hereafter Sanofi-Pasteur develops A/C/Y/W135 groups of polysaccharide covalent combination diphtheria class poison The tetravalence conjugate vaccines of plain (MCV4), China have being conjugated for several A, C group meningitis cocci capsular polysaccharides and carrier protein TT Vaccine approval listing.Due to meningococcal polysacharide and conjugate vaccines application, A, C, Y and W135 group meningitis are effectively prevented The infection of coccus.Different from A, C, W135, Y groups of bacterial strain capsular polysaccharides, the capsular polysaccharide immunogenicity of B groups of Nm bacterial strains is relatively low, and And the structure of MenB capsular polysaccharides and the nerve cell adhesion molecule in the nerve fiber developed and a small number of mature tissues are same Source, immunity inoculation are also easy to produce autoimmunity.Therefore, the capsular polysaccharide of B crowds of Nm cannot act as the antigen composition of vaccine.B groups of vaccines Research be concentrated mainly on non-capsular antigen at present, such as albumen, lipopolysaccharides (1ipopolysaccharide, LOS) etc..Screen non- The significant challenge of pod membrane surface antigen is its security, antigenic conservation and can trigger effective sterilizing power reaction extensively.It can make Need to possess following characteristics for the protein with immunogenicity of candidate vaccine：All expressed in most of bacterial strains and structure is protected Keep；Bactericidin or protection antibody can be induced.Certain progress is achieved in terms of the research of protein vaccine at present, Novartis is public Department passes through the clinical trial of III phase using the 4CMenB vaccines of reverse vaccinology development.There is PorA grinding hot spot candidate antigens, NhhA, GNA2132, NadA and fHBP etc., wherein fHBP are one of most promising candidate proteins.

FHBP is factor H associated proteins (Factor H-binding Protein, fHBP), also known as lipoprotein 2086, All Neisseria meningitidis surfaces are almost expressed in, are made of 255 amino acid residues, molecular weight 29000.Factor H is complement The key modulator of alternative route, it plays confactor during the C3b of factor I mediations is cracked into inactivation fragment iC3b and makees With.Also the decay of alternative route c3 invertases C3bBb is promoted.FHBP and factor H combinations can lower alternative route, so as to be conducive to Neisseria meningitidis survives.FHBP is for meningococcus in the presence of the blood of the mankind, serum and antibiotic property polypeptide Existence be particularly important.Its amino acid sequence is more conservative, and 91.6%-100% amino acid is identical in mutation.Restructuring fHBP resists Body can cause the bactericidal effect for the complement-mediated of same class mutation and the passive protection of inductive infection suckling mouse model.By complete Not only titre is high and insensitive to sequentially making a variation for the antibody that fHBP is obtained, even if there is a small amount of amino acid change, as long as determining space The key amino acid of conformation is constant, and the change of antibody bactericidal activity is little.All these features make fHBP become Neisseria meningitidis One of most effective antigen and most promising candidate's general vaccines.According to the antigenic cross-reaction of whole albumen and sequence phase Like property, meningococcus fHbp is divided into 3 antigenic variant groups.In general, fHbp (the also referred to as sub- families for variation V1 Race B) antibody for preparing has a bactericidal activity to the bacterial strain from variation V1 expression fHbp, but (is not also referred to as variation V2 and V3 For subtribe A) in expression fHbp bacterial strain, vice versa.There is also the sub- variation of fHbp in each variation group.Recently, fHbp Molecular structure have been shown as " modularization ", fHbp variations contain the various combination (V of five variable domains_A~V_E), Mei Geke Become section and be derived from subtribe A and subtribe B.Therefore, five domains being freely combined by genetic engineering means can be covered A, the restructuring Δ fHbp albumen of the antigen of two subtribes of B, three kinds of variations, becomes the vaccine antigen and protein carrier of wide spectrum.

But fHbp albumen, which is used alone, also the defects of certain, because the fHbp protein expressions in some MenB bacterial strains Horizontal relatively low, only the vaccine of the antigen of fHbp can not possibly be enough to be used in meningococcal infection is widely immunized, and merge table It can yet be regarded as a good selection up to a variety of antigens.Contain three kinds of components in a kind of pure protein vaccine (LP2086) ground, its In two components be fusion protein (GNA 2091 is merged with fHbp, and GNA 2132 is merged with GNA 1030), the 3rd component is Recombinate NadA.5 kinds of antigen negative staining applications can cause most of serum sterilizing responses in mouse, play good protection.Its In, GNA 2091, GNA 2132 and GNA 1030 are unknown function protein component, and NadA is Neisseria adhesion A, Adhesin/invasion of external junctional epithelium cell.The antigen be in MenB bacterial strains very it is conservative (>96% amino acid is same One property), but be not present in the bacterial strain of some genetic pedigrees, only 50% MenB pathogenic strains expression NadA albumen, but This 50% is all high pathogenic bacteria.Amalgamation and expression Δ fHbp albumen and NadA albumen, can undoubtedly improve its antigenicity, and with this antigen For protein carrier, with reference to the capsular polysaccharide of A, C, Y and W135 group meningitis cocci, in theory the multivalence conjugate vaccines can prevent A, B, the infection of five subgroup pathogenic bacteria of C, Y and W135, so as to prevent meningococal meningitis caused by most Nm infection.

The content of the invention

In view of this, the object of the present invention is to provide a kind of multivalent meningococcal conjugate vaccines, to recombinate Δ fHbp- NadA fusion proteins are protein carrier, there is provided a kind of to cover the strongly immunogenic conjugated of five kinds of floras of A, B, C, Y and W135 Vaccine.

For achieving the above object, the technical solution for the multivalent meningococcal conjugate vaccines that the present invention uses is as follows：A Group, C crowds, Y crowds and the one or more of of W135 group meningitis cocci capsular polysaccharides recombinate Δ fHbp-NadA fusion proteins load with one A multivalence conjugate vaccines are conjugated into body, wherein, the restructuring Δ fHbp-NadA fusion protein carriers, including it is restructuring Δ fHbp, soft Property joining peptide and NadA, the restructuring Δ fHbp include fHbp can variation V1 V_A、V_BDomain and fHbp can variation V3 V_C、V_D、V_EDomain.

Preferably, the amino acid sequence such as sequence table SEQ ID NO of the restructuring Δ fHbp：Shown in 1, its nucleotide coding Sequence such as sequence table SEQ ID NO：Shown in 2.

Preferably, the nucleotide coding sequence such as sequence table SEQ ID NO of the flexible connection peptide fragment：Shown in 3.

Preferably, the amino acid sequence such as sequence table SEQ ID NO of the restructuring Δ fHbp-NadA fusion protein carriers：4 It is shown, its nucleotide coding sequence such as sequence table SEQ ID NO：Shown in 5.

It is a further object of the present invention to provide the preparation method of above-mentioned multivalent meningococcal conjugate vaccines, step bag Include：

S1, Prepare restructuring Δ fHbp-NadA albumen；

S2, prepare A groups, C groups, Y groups and W135 groups Nm bacterium capsular polysaccharides；

S3, combined using derivation after CDAP activated polysaccharides and to prepare multivalence capsular polysaccharide-Δ fHbp-NadA albumen epidemic disease is conjugated Seedling.

Preferably, prepared by the restructuring Δ fHbp-NadA fusion proteins carrier comprises the following steps：

S1a, design primer expand to obtain fHbp V1 genetic fragments, fHbp V3 genetic fragments respectively, then with this two sections of bases Because fragment is the Δ fHbp full length fragments that template carries out that bridging PCR amplification is recombinated, recycles Δ fhbp genetic fragments and pET is carried Body, Prepare restructuring plasmid pET- Δs fHbp；

S1b, design primer amplification obtain the total length CDS sequences of NadA albumen, recycle NadA genetic fragments and recombinant plasmid PET- Δ fHbp, Prepare restructuring plasmid pET- Δs fHbp-NadA；

S1c, by recombinant plasmid pET- Δ fHbp-NadA translation tables reach bacterial strain, obtains restructuring Δ fHbp-NadA fusion proteins Carrier.

Preferably, the A group meningitis coccis in step S2 use 29201 bacterial strains, and C group meningitis coccis use 29205 bacterium Strain, Y group meningitis coccis use 29028 bacterial strains, and W135 group meningitis coccis use 29037 bacterial strains.

The beneficial effects of the invention are as follows：1st, it is not suitable for use in vaccine for B group meningitis cocci capsular polysaccharide compositions, utilizes The technology of reverse genetics, expression B group meningitis cocci people H factor bindins recombinant proteins and Neisseria adhesion A egg White fusion product Δ fHbp-NadA.The Δ fHbp of the fusion protein contains fHbp A subtribes and fHbp B subtribes (V1~V3 tri- Kind of variation) conserved domain, B groups of bacterial strains of Nm of all expression fHbp albumen can be covered, also, Δ fHbp includes fHbp Complete V_A~V_EFive domains, its structure, function and antigen site maintain integrality；Meanwhile the NadA of the fusion protein Expressed in 50% B groups of bacterial strains of Nm, and these bacterial strains are essentially high pathogenic bacteria.Even if fHbp but expression are not therefore expressed The bacterial strain of NadA can also be covered by Δ fHbp-NadA albumen, be protected by protein vaccine energy wide spectrum made of antigen of this recombinant protein Protect the infection of nearly all Nm B groups of pathogenic bacteria.

2nd, restructuring Δ fHbp-NadA is a kind of brand-new carrier protein available for conjugate vaccines, different from existing conjugated In vaccine art commonly use protein carrier (such as CRM197, TT, OMP, DT, HiD), because without because same protein carrier it is inoculated more The immunogenicity of conjugate vaccines is caused to decline.

3rd, although Δ fHbp and the NadA component in Δ fHbp-NadA are the important factor that Nm survives and causes a disease, but its Body is non-toxic, so that detoxification treatment need not be carried out to the activated protein in the industrial production, will not change its structure and antigen Avtive spot, so as to obtain more preferably immune effect.

4th, Δ fHbp-NadA then keeps respective by designing soft connection peptide chain by two activated protein amalgamation and expressions Allow both to become single protein while active, thus be with more controllability, risk in industrial production and immunity inoculation Substantially reduce, and more polysaccharide-protein binding sites can be provided.

5th, it is provided by the invention to recombinate novel multivalent meningococcal conjugate vaccines of the Δ fHbp-NadA as protein carrier Immune effect with wide spectrum, it is anti-that its immune serum can produce crosslinking with the type strain of five groups of A, B, C, Y and W135 Should, while can cover and fHbp (variation in 3) is expressed in B groups and does not express fHbp but expresses the high pathogenic bacteria of NadA, and Wide spectrum and extremely strong bactericidal effect are shown in sterilization detection.

6th, it is provided by the invention that there is polysaccharide-protein to recombinate new conjugate vaccines of the Δ fHbp-NadA as protein carrier The general advantage of conjugate vaccines：T cell effect can be activated after immune, produces immunological memory, so as to really protect the baby of less than 2 years old Youngster.

7th, the present invention applies technique preparation multivalence capsular polysaccharide-Δ fHbp-NadA of derivation combination after CDAP activated polysaccharides Albumen conjugate vaccines.The toxicity of cyanogen bromide in conventional method is not only eliminated, and activates efficiency and significantly improves.

Brief description of the drawings

Fig. 1 is the variable binding domains of fHbp V1~V3 and restructuring Δ fHbp domains.

Fig. 2 expands electrophoresis pattern for Δ fHbp and identifies electrophoresis pattern with pET- Δs fHbp.

Fig. 3 expands electrophoresis pattern for NadA and identifies electrophoresis pattern with pET- Δs fhbp-NadA.

Fig. 4 induces recombinant bacterium to express Δ fHbp and Δ fHbp-NadA albumen for IPTG.

Fig. 5 identifies Δ fHbp and Δ fHbp-NadA recombinant proteins for Western-Blot.

Fig. 6 is the Δ fHbp and Δ fHbp-NadA recombinant proteins after SDS-PAGE purification Identifications.

Fig. 7 is that SDS-PAGE identifies the Δ fHbp-NadA recombinant proteins after large scale purification.

Embodiment

Below in conjunction with specific embodiment to a kind of multivalence brain based on Δ fHbp-NadA protein carriers provided by the invention Meningococcus conjugate vaccines are further described.The embodiments described below is exemplary, and is only used for explaining the present invention, and It is not considered as limiting the invention.

Experimental method in following embodiments, is conventional method unless otherwise specified.Reality used in following embodiments Test material unless otherwise specified, be that market is commercially available.

First, the clone and prokaryotic expression of Δ fHbp and Δ fHbp-NadA recombinant proteins

1st, the structure of Pet- Δs fHbp and Pet- Δs fHbp-NadA recombinant plasmids

FHbp is a kind of film surface lipoprotein, and many meningococcal bacterial strains all carry its gene, it has been found that do not carry The bacterial strain of the gene.According to the antigenic cross-reaction and sequence similarity of whole albumen, meningococcus fHbp can be divided For 3 antigenic variant group V1~V3.In general, for antibody prepared by the fHbp (also referred to as subfamily B) of variation V1 to from The bacterial strain of variation V1 expression fHbp has bactericidal activity, but not for expression fHbp in variation V2 and V3 (also referred to as subtribe A) Bacterial strain, vice versa.The protein molecular of fHbp contains the various combination (V of five variable domains_A~V_E), each variable section Derived from subtribe A and subtribe B.We devise the structure (as shown in Figure 1) of Δ fHbp according to its domain feature, it includes A, B structure domain (epitope containing V1 on B structure domain) of V1, and (C, D, E domain contain C, D, E domain of V3 The epitope of V2 and V3, and its 174-216 amino acids is highly conserved, the only difference of Individual amino acids), in theory, weight The Δ fHbp of group can cover the antigen site of all 3 variations of wild type fHbp, have the potentiality of broad-spectrum antiseptic.

FHbp V1 and fHbp the V3 total length CDS sequences logged according to GeneBank, we design following 2 pairs of primers：Draw Thing introduces III restriction enzyme site of BamH I and Hind in P1 and P4 positions respectively.

P1：GGATTCATGAACCGAACTGCCTTCTGCTGCC

P2：GCCGGGCAGTTGGTTGAAGGCGGTA

P3：ACGGCATTCGGTTCAGACGATGCCA

P4：AAGCTTTTACTGCTTGGCGGCAAGACCGATA

Respectively using two plants of MenB groups of bacterium for expressing fHbp V1 and V3 as template (being purchased from U.S. ATCC), PCR amplification is carried out. C, D, E domain gene piece of the A of the amplifiable fHbp V1 of wherein P1, P2, B structure domain gene fragment, P3 and the amplifiable V3 of P4 Section.Again using this two sections of genetic fragments as template, P1, P4 are primer, carry out bridging PCR, and the amplifiable Δ fHbp that is recombinated is complete Long segment.With III double digestion target gene (PCR product) of BamH I and Hind and prokaryotic expression carrier pET32a (+), gel returns Receive box recycling Δ fhbp genetic fragments and linear pET32a carriers.After glue reclaim, with DNA ligase, 16 DEG C of connections overnight, convert Escherichia coli DH5a bacterial strain, monoclonal expand, after small upgrading grain, the about 800bp bands of III double digestion of BamH I and Hind identification, The screening positive clone under kalamycin resistance, obtains recombinant plasmid pET- Δ fHbp, and PCR amplification electrophoresis pattern is correct with identification Electrophoresis pattern is as shown in Figure 2.It will identify -70 DEG C in the form of the 15% glycerol stock preservations of correct recombinant plasmid, and sequencing identification is just Really, the amino acid sequence of its Δ fHbp such as sequence table SEQ ID NO：Shown in 1, its nucleotide coding sequence such as sequence table SEQ ID NO：Shown in 2 (826bp).

SEQ ID NO：2(826bp)：

ATGAACCGAACTGCCTTCTGCTGCCTTTTCCTGACCACCGCCCTGATTCTGACCGCCTGCAGCAGCGGA GGCGGCGGAAGCGGAAGCGGCGGTGTCGCCGCCGACATCGGCACGGGGCTTGCCGATGCACTAACTACGCCGCTCGA CCATAAAGACAAAGGTTTGAAATCTCTGACATTGGAAGACTCCATTCCCCAAAACGGAACACTAACCCTGTCGGCAC AAGGTGCGGAAAAAACTTTCAAAGCCGGCGACAAAGACAACAGCCTCAACACGGGCAAACTGAAGAACGACAAAATC AGCCGCTTCGACTTCGTGCAAAAAATCGAAGTGGACGGACAAACCATCACGCTGGCAAGCGGCGAATTTCAAATATA CAAACAGGACCACTCCGCCGTCGTTGCCCTACAGATTGAAAAAATCAACAACCCCGACAAAATCGACAGCCTGATAA ACCAACGCTCCTTCCTTGTCAGCGGTTTGGGCGGAGAACATACCGCCTTCAACCAACTGCCCGGCACGGCATTCGGT TCAGACGATGCCAGTGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGACACGGCAAAATCGAACATTT GAAATCGCCAGAACTCAATGTTGACCTGGCCGCCTCCGATATCAAGCCGGATAAAAAACGCCATGCCGTCATCAGCG GTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCTCTAGGCATCTTTGGCGGGCAAGCCCAGGAAGTTGCC GGCAGCGCAGAAGTGGAAACCGCAAACGGCATACGCCATATCGGTCTTGCCGCCAAGCAGTAA

Its amino acid sequence is SEQ ID NO：1：

MNRTAFCCLFLTTALILTACSSGGGGSGSGGVAADIGTGLADALTTPLDHKDKGLKSLTLEDSIPQNGT LTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDK IDSLINQRSFLVSGLGGEHTAFNQLPGTAFGSDDASGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAASDIKPDKKR HAVISGSVLYNQAEKGSYSLGIFGGQAQEVAGSAEVETANGIRHIGLAAKQ

But Δ fHbp albumen, which is used alone, also the defects of certain, because the fHbp protein expression water in some MenB bacterial strains Flat relatively low, only the vaccine of Δ fHbp antigens can not possibly be enough to be used in meningococcal infection is widely immunized.Neisseria Adhesin/invasion of adhesion A (NadA), in vitro junctional epithelium cell.The antigen is very conservative in MenB bacterial strains (>96% amino acid identities), it is not present in the bacterial strain of some genetic pedigrees, only 50% MenB pathogenic strains expression NadA albumen, but this 50% is all high pathogenic bacteria.Amalgamation and expression Δ fHbp albumen and NadA albumen, can undoubtedly improve its antigen Property, but flexible link peptide fragment is added between two albumen, that the most classical is (GGGGS) 3 of Huston design synthesis Sequence, being conducive to two albumen can correctly fold, so as to not influence its respective immune prototype.Logged according to GeneBank NadA total length CDS sequences, design primer：Primer P5 and P6 introduce I restriction enzyme site of Hind III and Xho, while the digestion of P5 respectively The sequence of the design expression connection albumen of (GGGGS) 3 behind site.

P5：AAGCTT GGCGGCGGCGGCAGT GGCGGCGGCGGCAGTGGCGGCGGCGGCAGT ATGAAACACTTTCCATCCAAAGTAC(SEQ ID NO：3)

P6：CTCGAGTTACCACTCGTAATTGACGCCGACA

To express the MenB group bacterium of NadA as template (being purchased from U.S. ATCC), P5, P6 are primer, and amplification obtains NadA albumen Total length CDS sequences, with Hind III and I double digestion target gene of Xho (NadA products) and recombinant plasmid pET- Δ fHbp, gel Recycling box recycles NadA genetic fragments and linear pET- Δs fHbp carriers.After glue reclaim, with DNA ligase, 16 DEG C of connections are stayed overnight, Escherichia coli DH5a bacterial strain is converted, monoclonal expands, after small upgrading grain, the about 1900bp bars of I double digestion of BamH I and Xho identification Band, the screening positive clone under kalamycin resistance, obtain recombinant plasmid pET- Δ fHbp-NadA, PCR amplification electrophoresis pattern with Identify that correct electrophoresis pattern is as shown in Figure 3.It will identify -70 DEG C in the form of the 15% glycerol stock preservations of correct recombinant plasmid, and survey Sequence identification is correct, recombinates the amino acid sequence such as sequence table SEQ ID NO of Δ fHbp-NadA fusion protein carriers：Shown in 4, its Nucleotide coding sequence such as sequence table SEQ ID NO：Shown in 5 (1089bp).

SEQ ID NO：5(1089bp)：

ATGAACCGAACTGCCTTCTGCTGCCTTTTCCTGACCACCGCCCTGATTCTGACCGCCTGCAGCAGCGGA GGCGGCGGAAGCGGAAGCGGCGGTGTCGCCGCCGACATCGGCACGGGGCTTGCCGATGCACTAACTACGCCGCTCGA CCATAAAGACAAAGGTTTGAAATCTCTGACATTGGAAGACTCCATTCCCCAAAACGGAACACTAACCCTGTCGGCAC AAGGTGCGGAAAAAACTTTCAAAGCCGGCGACAAAGACAACAGCCTCAACACGGGCAAACTGAAGAACGACAAAATC AGCCGCTTCGACTTCGTGCAAAAAATCGAAGTGGACGGACAAACCATCACGCTGGCAAGCGGCGAATTTCAAATATA CAAACAGGACCACTCCGCCGTCGTTGCCCTACAGATTGAAAAAATCAACAACCCCGACAAAATCGACAGCCTGATAA ACCAACGCTCCTTCCTTGTCAGCGGTTTGGGCGGAGAACATACCGCCTTCAACCAACTGCCCGGCACGGCATTCGGT TCAGACGATGCCAGTGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGACACGGCAAAATCGAACATTT GAAATCGCCAGAACTCAATGTTGACCTGGCCGCCTCCGATATCAAGCCGGATAAAAAACGCCATGCCGTCATCAGCG GTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCTCTAGGCATCTTTGGCGGGCAAGCCCAGGAAGTTGCC GGCAGCGCAGAAGTGGAAACCGCAAACGGCATACGCCATATCGGTCTTGCCGCCAAGCAGTAAGGCGGCGGCGGCAG T

GGCGGCGGCGGCAGT GGCGGCGGCGGCAGTATGAAACACTTTCCATCCAAAGTACTGACCACAGCCAT CCTTGCCACTTTCTGTAGCGGCGCACTGGCAGCCACAAGCGACGACGATGTTAAAAAAGCTGCCACTGTGGCCATTG TTGCTGCCTACAACAATGGCCAAGAAATCAACGGTTTCAAAGCTGGAGAGACCATCTACGACATTGGTGAAGACGGC ACAATTACCCAAAAAGACGCAACTGCAGCCGATGTTGAAGCCGACGACTTTAAAGGTCTGGGTCTGAAAAAAGTCGT GACTAACCTGACCAAAACCGTCAATGAAAACAAACAAAACGTCGATGCCAAAGTAAAAGCTGCAGAATCTGAAATAG AAAAGTTAACAACCAAGTTAGCAGACACTGATGCCGCTTTAGCAGATACTGATGCCGCTCTGGATGAAACCACCAAC GCCTTGAATAAATTGGGAGAAAATATAACGACATTTGCTGAAGAGACTAAGACAAATATCGTAAAAATTGATGAAAA ATTAGAAGCCGTGGCTGATACCGTCGACAAGCATGCCGAAGCATTCAACGATATCGCCGATTCATTGGATGAAACCA ACACTAAGGCAGACGAAGCCGTCAAAACCGCCAATGAAGCCAAACAGACGGCCGAAGAAACCAAACAAAACGTCGAT GCCAAAGTAAAAGCTGCAGAAACTGCAGCAGGCAAAGCCGAAGCTGCCGCTGGCACAGCTAATACTGCAGCCGACAA GGCCGAAGCTGTCGCTGCAAAAGTTACCGACATCAAAGCTGATATCGCTACGAACAAAGCTGATATTGCTAAAAACT CAGCACGCATCGACAGCTTGGACAAAAACGTAGCTAATCTGCGCAAAGAAACCCGCCAAGGCCTTGCAGAACAAGCC GCGCTCTCCGGCCTGTTCCAACCTTACAACGTGGGTCGGTTCAATGTAACGGCTGCAGTCGGCGGCTACAAATCCGA ATCGGCAGTCGCCATCGGTACCGGCTTCCGCTTTACCGAAAACTTTGCCGCCAAAGCAGGCGTGGCAGTCGGCACTT CGTCCGGTTCTTCCGCAGCCTACCATGTCGGCGTCAATTACGAGTGGTAA

Its amino acid sequence is SEQ ID NO：4：

MNRTAFCCLFLTTALILTACSSGGGGSGSGGVAADIGTGLADALTTPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGA EKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQR SFLVSGLGGEHTAFNQLPGTAFGSDDASGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAASDIKPDKKRHAVISGSV LYNQAEKGSYSLGIFGGQAQEVAGSAEVETANGIRHIGLAAKQGGGGS GGGGS GGGGSMKHFPSKVLTTAILATFCSGALAATSDDDVKKAATVAIVAAYNNGQEINGFKAGETIYDIGEDGTITQKDAT AADVEADDFKGLGLKKVVTNLTKTVNENKQNVDAKVKAAESEIEKLTTKLADTDAALADTDAALDETTNALNKLGEN ITTFAEETKTNIVKIDEKLEAVADTVDKHAEAFNDIADSLDETNTKADEAVKTANEAKQTAEETKQNVDAKVKAAET AAGKAEAAAGTANTAADKAEAVAAKVTDIKADIATNKADIAKNSARIDSLDKNVANLRKETRQGLAEQAALSGLFQP YNVGRFNVTAAVGGYKSESAVAIGTGFRFTENFAAKAGVAVGTSSGSSAAYHVGVNYEW

2nd, the prokaryotic expression and identification of Δ fHbp and Δ fHbp-NadA recombinant proteins

By recombinant plasmid pET- Δ fHbp and pET- Δ fHbp-NadA Transformed E .coli BL21 (DE3), 37 DEG C of trainings overnight Support and (receive penicillin containing 50 μ g/ml cards), obtain Δ fHbp/BL21 (DE3) and Δ fHbp-NadA/BL21 (DE3) and express bacterium, choose Monoclonal is taken, 37 DEG C of shaken cultivations to strain density reach OD600 about 0.5~1.0, add the isopropyl-β-D- sulphur of final concentration 0.5mM For galactoside (IPTG), when 37 DEG C of vibration Fiber differentiation 2-6 are small, identified through SDS-PAGE, as a result such as Fig. 4 is shown, Δ fHbp/ Have an obvious band of expression after BL21 (DE3) and Δ fHbp-NadA/BL21 (DE3) inductions, molecular weight be respectively 30kD and 72kD.Δ fHbp and Δ fHbp-NadA recombinant proteins are accredited as through Western-Blot, as shown in Figure 5：1,3 swimming lane is Δ FHbp/BL21 (DE3) induced expression thing, 2,4 swimming lanes are Δ fHbp-NadA/BL21 (DE3) induced expression thing, and 1,2 is mono- with fHbp Anti- detection, there is the expection band of 30kD and 72kD respectively, and 3,4 are detected with NadA monoclonal antibodies, and only 4 swimming lanes have 72kD bands, as a result Meet expection.Most latter two recombinant protein is determined as Δ fHbp and Δ fHbp-NadA recombinant proteins through Mass Spectrometric Identification.

2nd, the small scale purification and its Efficacy evaluation of Δ fHbp and Δ fHbp-NadA recombinant proteins

By Δ fHbp/BL21 (DE3) and Δ fHbp-NadA/BL21 (DE3) inoculations LB (receiving mycin containing 50 μ g/ml cards) liquid Body culture medium, 37 DEG C, 200rpm culture 12 it is small when, with 1% inoculative proportion expand cultivate, 37 DEG C, 200rpm culture 3-6 it is small when Afterwards, when OD600 to 16~20, IPTG (0.5mM) inductions 4 are small.Centrifuge collects thalline.Carrying out ultrasonic bacteria breaking, SDS- before PAGE shows, expression of recombinant proteins in the form of inclusion body (Fig. 3).Successively use TE+300mM Nacl, TE+1%Triton-100 After washing inclusion body, 8000rpm centrifugations 20min obtains inclusion body, after washing, is dissolved in 3mol/L urea, 20mmol/L In Tris-Cl, 1mmol/L EDTA, pH4.0 solution, through CM column cation exchange chromatographies, it can obtain and target protein size Two destination proteins being consistent.Last albumen dilution refolding to be reorganized is to after 24h, ultrafilter low temperature rapid concentration to original volume 1.2 times, anion-exchange column Q SepharoseF.F are crossed, collect destination protein peak, detection method is the same as Fig. 4 and Fig. 5, gel detection Protein content reaches more than 95.3%, and purification effect is as shown in fig. 6, WB detections are really purpose albumen.Dialysed with PBS (pH7.4) After desalination, BCA methods survey protein concentration content about 0.5mg/mL.

Mouse immune：Respectively with SPF grades of Δ fHbp after purification and 6 week old of Δ fHbp-NadA recombinant proteins subcutaneous inoculation NIH mouse 10, immunizing dose only (are dissolved in for Δ fHbp every time after purification or Δ fHbp-NadA recombinant proteins 50ug/ In 0.2ml physiological saline), immune programme 0,2,4 weeks, takes a blood sample, serum is collected by centrifugation for 2 weeks after being immunized；Separately set 10 small Mouse compares, same procedure injecting normal saline.The serum ELISA method gathered surveys the titre of serum antibody, and specific method is as follows： Using Δ fHbp after purification or Δ fHbp-NadA recombinant proteins, respectively with optimal dose coated elisa plate, 37 DEG C were incubated At night, add the mice serum to be measured being serially diluted after board-washing, 37 DEG C be incubated after board-washing, add horseradish peroxidase-labeled goat-anti Mouse IgG secondary antibodies develop the color, and microplate reader measure, reads 492nm (630nm is reference wavelength) absorbance (A values).Negative control group blood The standard deviation of clear+3 times of A averages is Cutoff values, and test serum A values are judged to the positive more than Cutoff values, are more than with A values The greatest dilution of Cutoff values calculates the geometric mean titer of each type mouse, is mice serum IgG antibody potency (table 1).The serum antibody that height is generated after mouse is immunized in two kinds of recombinant proteins.

The titer determination (geometrical mean) of serum antibody after mouse is immunized in 1. each group antigen of table three times

The evaluation of two kinds of recombinant protein immune effects, takes two methods, and one measures full cell for whole cell coating ELISA, another sterilizing power for epidemic strain are tested.Whole cell coating selects MenB crowds of bacterial strain MC58, and (height expression fHbp V1 are variable Body), 8047 (height expression fHbp V2 can variation), M1239 high expression fHbp V3 can variation), 961-5945 (middle amount expression fHbp Albumen) and 67/00 (low amounts expression fHbp albumen), after these bacterial strains are expanded culture propagation, bacterium lawn is scraped, with physiology salt Water dissolves, and after formaldehyde sterilization, is diluted to 2 × 10⁸A/ml, is coated with 96 hole elisa Plates, 100ul/ holes, 4 DEG C were coated with this concentration At night, after board-washing, be detected, the results are shown in Table 2.Equally, cultivate the MenB group bacterial strain MC58 (high express fHbp V1 can variation), 8047 (height expression fHbp V2 can variation), M1239 high expression fHbp V3 can variations), 961-5945 (middle amount expression fHbp eggs In vain) and 67/00 (low amounts expression fHbp albumen) carries out sterilization detection with corresponding serum antibody afterwards, measures and calculates bactericidin drop Spend (compared with complement negative control hole, the serum highest dilution of more than 50% sterilizing rate is serum antibody bactericidal titre), knot Fruit is shown in Table 3.

Each MenB strain whole-cells ELISA of table 2.

Each MenB bacterial strains sterilizing power experiment of table 3.:BC50titer(1：)

Remarks：Sterilizing power is tested with more than 1：8 be with protectiveness.

It can be obtained by the result comprehensive analysis of table 2 and table 3, all expression fHbp can be effectively immunized in Δ fHbp protein vaccines The MenB bacterial strains of (V1, V3 and V2 variation) infect, but the MenB bacterial strains for not expressing low expression fHbp even lose protection energy Power；And Δ fHbp-NadA protein vaccines are in the base infected for the MenB bacterial strains that all expression fHbp (V1, V3 and V2 variation) are immunized On plinth, while the MenB bacterial strains that can not even express low expression fHbp still have protective effect.Meanwhile Δ fHbp-NadA Protein vaccine can produce the antibody titer of higher and have stronger sterilizing ability, illustrate the fusion table of two species specificity albumen Danone enough plays the role of collaboration and promotes immune response, and can provide the vaccine defencive function of more wide spectrum.Therefore, after us Continuous process choice Δ fHbp-NadA recombinant proteins are carried as the albumen of novel multivalent meningococcus and pneumococcus conjugate vaccines Body.

3rd, the fermented and cultured of engineering bacteria, induced expression and extensive antigen protein concentrate and purify

Original species are made according to the requirement of pharmacopeia in correct Δ fHbp-NadA/BL21 (DE3) recombinant bacterial strains of above-mentioned identification Word bank and work seed bank.Technological process is as follows：

1. take engineered strain to work seed bank strain, kind of a LB agar medium (receiving mycin containing card) is drawn, 37 DEG C of overnight incubations, Picking single bacterium colony is seeded in LB fluid nutrient mediums (receiving mycin containing 50 μ g/ml cards), and is progressively expanded with the inoculative proportion of 1-2% Greatly to ferment tank culture, when OD600 reaches 16-20, induced and trained using isopropyl-β-D-thiogalactoside (IPTG) Support 4-8 it is small when, stop culture, large capacity centrifuge collect thalline.

2. thalline is taken to add broken bacterium buffer solution (50mM PB, 2mM EDTA, pH7.5), high pressure (800-1000bar) homogeneous After broken bacterium two times, centrifugation (10000rpm, 60min, 8 DEG C) takes supernatant.Using 30-60% ammonium sulfate precipitations, 50mM PB PH7.5 dissolves, 50kD ultrafiltration 5 times, and is concentrated into the 1/5-1/3 of original volume.

3. using Sepharose QFF chromatographies, equilibrium liquid：50mM PB PH7.2, eluent：50mM PB+1M NaCl PH7.2.Elution process：0-100% gradient elutions, collect 10% eluent.

4. 10% gradient eluent is taken to be concentrated by ultrafiltration through 50KD ultrafiltration membranes bag, using Sepharose 4FF Gel filtrations Analysis, collects protein peak at V0, with 0.15mol/L sodium chloride, 10KD ultrafiltration more than 5 times, and it is 1-2mg/ to be concentrated into protein content Ml, 0.22 μm of aseptic filtration.Through sterility test, protein content inspection, molecular weight and purity test, protein-specific inspection, thin Bacterium tiny electrolytic cell is Δ fHbp-NadA albumen stostes.

Technical requirements are to obtain the restructuring Δ fHbp-NadA albumen stostes that purity is more than 95%.Bacterial endotoxin is not higher than 20EU/ml, the present invention should be not higher than 10EU/ml, most preferably not higher than 5EU/ml.Molecular weight and purity test are shown in Fig. 7, and purity is 96.3%.Protein-specific detection is as shown in Figure 5, and WB is detected as target protein.

4th, the preparation of multivalent meningococcal conjugate vaccines and Efficacy evaluation

The preparation of 1.A groups, C groups, Y groups and W135 groups Nm bacterium capsular polysaccharides

A group meningitis coccis use 29201 bacterial strains, and C group meningitis coccis use 29205 bacterial strains, and Y group meningitis coccis are adopted With 29028 bacterial strains, W135 group meningitis coccis are used 29037 bacterial strains, after the fermented culture of meningococcus, are centrifuged using sand culture Machine, disc centrifuge or other large capacity centrifuges are separately cultured liquid, collect centrifugation supernatant；Supernatant will be centrifuged with 100KD films Bag is concentrated by ultrafiltration, and carries out fractional precipitation using the ethanol of 25-80%, collects and precipitate and washed respectively with absolute ethyl alcohol and acetone, Obtain rough polysaccharide；Sterilized water for injection dissolves polysaccharide and after NaTDC is handled, using GE filler Capto adhere With Capto DEAE or the ion-exchange packing of other producer's identical function properties, with series connection chromatography or step chromatography method into Row raw sugar refines, and collection flows through peak, and desalination, ethanol precipitation or jelly are carried out to flowing through peak using GE Sephadex G25Coarse Dry recycling polysaccharide, is placed in -20 DEG C and saves backup.

2.A/C/Y/W135 the preparation of group's polysaccharide-Δ fHbp-NadA albumen conjugate vaccines

The common method of the industry has two kinds, i.e. Amine reduction or cyanogen bromide activation prepares polysaccharide protein conjugate.Amine Reduction method is to aoxidize A/C/Y/W135 groups of polysaccharide through sodium metaperiodate lucifuge, then adds adipic dihydrazide (ADH) and boron Cymag to prepare Polysaccharide derivates, ADH and boron Cymag etc. are removed, then add the Δ fHbp-NadA carriers prepared using hyperfiltration process Albumen, under carbodiimide (EDAC) effect, forms polysaccharide protein conjugate.Cyanogen bromide activation is by A/C/Y/W135 mass-brains Meningococcus polysaccharide is activated through cyanogen bromide (CNBr), the adipic dihydrazide in connection on the sugar chain of activation, will using the method for ultrafiltration Cyanogen bromide, free adipic dihydrazide remove, and the Δ fHbp-NadA carrier proteins prepared are then added, in the company of carbodiimide Connect under effect, polysaccharide protein combines to form polysaccharide protein complex.

Wherein, cyanogen bromide has the function that stronger activated polysaccharide hydroxyl, at present the A groups of domestic listing and A, C mass-brain film Scorching Streptococcus polysaccharides conjugate vaccines are prepared using the method for cyanogen bromide-activated polysaccharide.But due to the use of hypertoxic chemical reagent bromination Cyanogen, preparation process are respectively provided with personnel and environment larger potential risk, and conjugate yield is not high.With chemical synthesising technology Development, successfully have developed a kind of new type water-solubility cyanide, i.e. 1- cyano group -4-dimethylaminopyridine-tetrafluoride boron (1- Cyano-4-dimethylamin-opyridinium tetrafluoroborate, CDAP).Not only eliminated using the reagent The toxicity of cyanogen bromide, and activate efficiency and significantly improve.There is researcher by cyanogen bromide compared with two methods of CDAP, CDAP methods Polysaccharide yield up to 35%-40%, and cyanogen bromide only has 5%-20%, and conjugate still has good immunogenicity.

Therefore, the present invention applies technique preparation multivalence capsular polysaccharide-Δ fHbp- of derivation combination after CDAP activated polysaccharides NadA albumen conjugate vaccines.Specific method is：A/C/Y/W135 meningococcal polysaccharides are dissolved to 5-15mg/ml, are adjusted PH value to 10.8 or so, in polysaccharide solution add 1/10 (g/g) CDAP, room temperature, maintain pH value 10.8 alkaline environment under Activated polysaccharide 8min.By 1：The ratio of 1 (volume ratio with activated polysaccharide) adds the adipoyl hydrazine of 0.5mol/L, room temperature reaction 50-70min.The ultrafiltration of 50KD film bags removes cyanogen bromide and adipoyl hydrazine, obtains polysaccharide derivates.By polysaccharide derivates and carrier egg White Δ fHbp-NadA is with 1：The ratio of 1 (g/g) is mixed and stirred for, by carbodiimide：Polysaccharide=1：It is sub- that 10 ratio adds carbon two Amine, reacts 60min under room temperature, sour environment (pH5.6).Impurity is removed through the ultrafiltration of 100KD ultrafiltration membrane bags, and is concentrated.Finally adopt Gel chromatography is carried out with GE Sepharose 4FF, collects the eluent near V0.With 0.22 μm of filter membrane aseptic filtration, obtain To A/C/Y/W135 meningococcal polysaccharides-Δ fHbp-NadA albumen conjugate vaccines stostes.

This stoste is used into Aluminium phosphate adjuvant stirring and adsorbing, is diluted and prepared with 0.15mol/L sodium chloride, to multivalence polysaccharide egg White conjugate final concentration in terms of polyoses content is respectively 20 μ g/ml, and the final concentration of 0.45-0.6mg/ml of aluminium content, stirs evenly, Dispensed with pre-filled syringe, 0.5ml/ branch, A/C/Y/W135 meningococcal polysaccharides-Δ fHbp-NadA albumen is made and sews Close bacterin preparation, 2-8 DEG C of preservation.Also 80mg/ml sucrose can be added in vaccinogen liquid as excipient, every polyoses content 20 Protein vaccine preparation, 2-8 DEG C of preservation is made in μ g/ml after freezing.

3.A/C/Y/W135 group's polysaccharide-Δ fHbp-NadA albumen conjugate vaccines effect assessments

Mouse immune：A/C/Y/W135 groups of polysaccharide-Δ fHbp-NadA albumen conjugate vaccines albumen stoste is used respectively, is freezed For each 0.2ml/ only, journey is immunized in preparation and adjuvant sorbent formulation subcutaneous inoculation SPF grades of 6 week old NIH mouse 10, immunizing dose Sequence is 0,2,4 weeks, takes a blood sample within 2 weeks after being immunized, serum is collected by centrifugation；It is another to set 10 control mices, same procedure injection physiology Brine.The serum ELISA method gathered surveys the titre of serum antibody, and specific method is as follows：Using Δ fHbp-NadA after purification Recombinant protein, respectively with optimal dose coated elisa plate, 37 DEG C are incubated overnight, and the mouse blood to be measured being serially diluted is added after board-washing Clearly, board-washing after 37 DEG C of incubations, adds the colour developing of horseradish peroxidase-labeled sheep anti-mouse igg secondary antibody, microplate reader measure, reads 492nm (630nm is reference wavelength) absorbance (A values).The standard deviation of+3 times of negative control group serum A averages is Cutoff Value, test serum A values are judged to the positive more than Cutoff values, and each type of greatest dilution calculating that Cutoff values are more than with A values is small The geometric mean titer of mouse, is mice serum IgG antibody potency (table 4).Mouse is immunized in conjugate vaccines stoste and two kinds of formulations The serum antibody of height is generated afterwards.

The titer determination (geometrical mean) of serum antibody after mouse is immunized in 4. each group antigen of table three times

The evaluation of each formulation immune effect of conjugate vaccines, equally takes two methods, and whole cell coating measures full cell The sterilizing power of ELISA and epidemic strain is tested.29201 bacterial strain of whole cell coating selection A group meningitis coccis, B group meningitis coccis MC58 and 67/00 bacterial strain, 29205 bacterial strain of C group meningitis coccis, 29028 bacterial strain of Y group meningitis coccis, W135 group meningitis balls 29037 bacterial strain of bacterium, after these bacterial strains are expanded culture propagation, scrapes bacterium lawn, with physiological saline solution, after formaldehyde sterilization, It is diluted to 2 × 10⁸A/ml, is coated with 96 hole elisa Plates, 100ul/ holes, 4 DEG C of coatings are stayed overnight, after board-washing, with corresponding epidemic disease with this concentration Seedling serum antibody carries out ELISA detections, the results are shown in Table 5.

Each group meningitis cocci strain whole-cell ELISA of table 5.

Equally, 29201 bacterial strain of A group meningitis coccis, B group meningitis coccis MC58 and 67/00 bacterial strain, C group meningitis balls 29205 bacterial strain of bacterium, 29028 bacterial strain of Y group meningitis coccis, with corresponding vaccine serum after 29037 bacterial strain of W135 group meningitis coccis Antibody carries out sterilization detection, measure and calculate bactericidin titre (compared with complement negative control hole, more than 50% sterilizing rate Serum highest dilution is serum antibody bactericidal titre), it the results are shown in Table 6.

Each group meningitis cocci bacterial strain sterilizing power experiment of table 6.:BC50titer(1：)

It can be obtained by result above comprehensive analysis, to recombinate novel multivalent meningitis balls of the Δ fHbp-NadA as protein carrier Bacterium conjugate vaccines have the immune effect of wide spectrum, its immune serum can be with the type strain of five groups of A, B, C, Y and W135 Cross-linking reaction is produced, while can cover and fHbp (3 kinds of variations) is expressed in B groups and does not express fHbp but expresses the high cause of NadA Germ, and wide spectrum and extremely strong bactericidal effect are shown in sterilization detects, so as to protect people (especially infant and old age Weakling) endangered from spinal cord meningitis caused by the infringement of most Nm pathogenic bacteria.

The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the present invention spirit and Within principle, any modification, equivalent replacement, improvement and so on, should all be included in the protection scope of the present invention.

Sequence table

<110>Wuhan Bo Wo bio tech ltd

<120>Multivalent meningococcal conjugate vaccines and preparation method thereof

<130> 2017

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 274

<212> PRT

<213>It is artificial synthesized

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Met Asn Arg Thr Ala Phe Cys Cys Leu Phe Leu Thr Thr Ala Leu Ile

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Val Asp Leu Ala Ala Ser Asp Ile Lys Pro Asp Lys Lys Arg His

210 215 220

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

225 230 235 240

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

245 250 255

Ala Glu Val Glu Thr Ala Asn Gly Ile Arg His Ile Gly Leu Ala Ala

260 265 270

Lys Gln

<210> 2

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<213>It is artificial synthesized

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atgaaccgaa ctgccttctg ctgccttttc ctgaccaccg ccctgattct gaccgcctgc 60

agcagcggag gcggcggaag cggaagcggc ggtgtcgccg ccgacatcgg cacggggctt 120

gccgatgcac taactacgcc gctcgaccat aaagacaaag gtttgaaatc tctgacattg 180

gaagactcca ttccccaaaa cggaacacta accctgtcgg cacaaggtgc ggaaaaaact 240

ttcaaagccg gcgacaaaga caacagcctc aacacgggca aactgaagaa cgacaaaatc 300

agccgcttcg acttcgtgca aaaaatcgaa gtggacggac aaaccatcac gctggcaagc 360

ggcgaatttc aaatatacaa acaggaccac tccgccgtcg ttgccctaca gattgaaaaa 420

atcaacaacc ccgacaaaat cgacagcctg ataaaccaac gctccttcct tgtcagcggt 480

ttgggcggag aacataccgc cttcaaccaa ctgcccggca cggcattcgg ttcagacgat 540

gccagtggaa aactgaccta caccatagat ttcgccgcca agcagggaca cggcaaaatc 600

gaacatttga aatcgccaga actcaatgtt gacctggccg cctccgatat caagccggat 660

aaaaaacgcc atgccgtcat cagcggttcc gtcctttaca accaagccga gaaaggcagt 720

tactctctag gcatctttgg cgggcaagcc caggaagttg ccggcagcgc agaagtggaa 780

accgcaaacg gcatacgcca tatcggtctt gccgccaagc agtaa 825

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<213>It is artificial synthesized

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aagcttggcg gcggcggcag tggcggcggc ggcagtggcg gcggcggcag tatgaaacac 60

tttccatcca aagtac 76

<210> 4

<211> 651

<212> PRT

<213>It is artificial synthesized

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Met Asn Arg Thr Ala Phe Cys Cys Leu Phe Leu Thr Thr Ala Leu Ile

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Val Asp Leu Ala Ala Ser Asp Ile Lys Pro Asp Lys Lys Arg His

210 215 220

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

225 230 235 240

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

245 250 255

Ala Glu Val Glu Thr Ala Asn Gly Ile Arg His Ile Gly Leu Ala Ala

260 265 270

Lys Gln Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

275 280 285

Ser Met Lys His Phe Pro Ser Lys Val Leu Thr Thr Ala Ile Leu Ala

290 295 300

Thr Phe Cys Ser Gly Ala Leu Ala Ala Thr Ser Asp Asp Asp Val Lys

305 310 315 320

Lys Ala Ala Thr Val Ala Ile Val Ala Ala Tyr Asn Asn Gly Gln Glu

325 330 335

Ile Asn Gly Phe Lys Ala Gly Glu Thr Ile Tyr Asp Ile Gly Glu Asp

340 345 350

Gly Thr Ile Thr Gln Lys Asp Ala Thr Ala Ala Asp Val Glu Ala Asp

355 360 365

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

370 375 380

Thr Val Asn Glu Asn Lys Gln Asn Val Asp Ala Lys Val Lys Ala Ala

385 390 395 400

Glu Ser Glu Ile Glu Lys Leu Thr Thr Lys Leu Ala Asp Thr Asp Ala

405 410 415

Ala Leu Ala Asp Thr Asp Ala Ala Leu Asp Glu Thr Thr Asn Ala Leu

420 425 430

Asn Lys Leu Gly Glu Asn Ile Thr Thr Phe Ala Glu Glu Thr Lys Thr

435 440 445

Asn Ile Val Lys Ile Asp Glu Lys Leu Glu Ala Val Ala Asp Thr Val

450 455 460

Asp Lys His Ala Glu Ala Phe Asn Asp Ile Ala Asp Ser Leu Asp Glu

465 470 475 480

Thr Asn Thr Lys Ala Asp Glu Ala Val Lys Thr Ala Asn Glu Ala Lys

485 490 495

Gln Thr Ala Glu Glu Thr Lys Gln Asn Val Asp Ala Lys Val Lys Ala

500 505 510

Ala Glu Thr Ala Ala Gly Lys Ala Glu Ala Ala Ala Gly Thr Ala Asn

515 520 525

Thr Ala Ala Asp Lys Ala Glu Ala Val Ala Ala Lys Val Thr Asp Ile

530 535 540

Lys Ala Asp Ile Ala Thr Asn Lys Ala Asp Ile Ala Lys Asn Ser Ala

545 550 555 560

Arg Ile Asp Ser Leu Asp Lys Asn Val Ala Asn Leu Arg Lys Glu Thr

565 570 575

Arg Gln Gly Leu Ala Glu Gln Ala Ala Leu Ser Gly Leu Phe Gln Pro

580 585 590

Tyr Asn Val Gly Arg Phe Asn Val Thr Ala Ala Val Gly Gly Tyr Lys

595 600 605

Ser Glu Ser Ala Val Ala Ile Gly Thr Gly Phe Arg Phe Thr Glu Asn

610 615 620

Phe Ala Ala Lys Ala Gly Val Ala Val Gly Thr Ser Ser Gly Ser Ser

625 630 635 640

Ala Ala Tyr His Val Gly Val Asn Tyr Glu Trp

645 650

<210> 5

<211> 1959

<212> DNA

<213>It is artificial synthesized

<400> 5

atgaaccgaa ctgccttctg ctgccttttc ctgaccaccg ccctgattct gaccgcctgc 60

agcagcggag gcggcggaag cggaagcggc ggtgtcgccg ccgacatcgg cacggggctt 120

gccgatgcac taactacgcc gctcgaccat aaagacaaag gtttgaaatc tctgacattg 180

gaagactcca ttccccaaaa cggaacacta accctgtcgg cacaaggtgc ggaaaaaact 240

ttcaaagccg gcgacaaaga caacagcctc aacacgggca aactgaagaa cgacaaaatc 300

agccgcttcg acttcgtgca aaaaatcgaa gtggacggac aaaccatcac gctggcaagc 360

ggcgaatttc aaatatacaa acaggaccac tccgccgtcg ttgccctaca gattgaaaaa 420

atcaacaacc ccgacaaaat cgacagcctg ataaaccaac gctccttcct tgtcagcggt 480

ttgggcggag aacataccgc cttcaaccaa ctgcccggca cggcattcgg ttcagacgat 540

gccagtggaa aactgaccta caccatagat ttcgccgcca agcagggaca cggcaaaatc 600

gaacatttga aatcgccaga actcaatgtt gacctggccg cctccgatat caagccggat 660

aaaaaacgcc atgccgtcat cagcggttcc gtcctttaca accaagccga gaaaggcagt 720

tactctctag gcatctttgg cgggcaagcc caggaagttg ccggcagcgc agaagtggaa 780

accgcaaacg gcatacgcca tatcggtctt gccgccaagc agtaaggcgg cggcggcagt 840

ggcggcggcg gcagtggcgg cggcggcagt atgaaacact ttccatccaa agtactgacc 900

acagccatcc ttgccacttt ctgtagcggc gcactggcag ccacaagcga cgacgatgtt 960

aaaaaagctg ccactgtggc cattgttgct gcctacaaca atggccaaga aatcaacggt 1020

ttcaaagctg gagagaccat ctacgacatt ggtgaagacg gcacaattac ccaaaaagac 1080

gcaactgcag ccgatgttga agccgacgac tttaaaggtc tgggtctgaa aaaagtcgtg 1140

actaacctga ccaaaaccgt caatgaaaac aaacaaaacg tcgatgccaa agtaaaagct 1200

gcagaatctg aaatagaaaa gttaacaacc aagttagcag acactgatgc cgctttagca 1260

gatactgatg ccgctctgga tgaaaccacc aacgccttga ataaattggg agaaaatata 1320

acgacatttg ctgaagagac taagacaaat atcgtaaaaa ttgatgaaaa attagaagcc 1380

gtggctgata ccgtcgacaa gcatgccgaa gcattcaacg atatcgccga ttcattggat 1440

gaaaccaaca ctaaggcaga cgaagccgtc aaaaccgcca atgaagccaa acagacggcc 1500

gaagaaacca aacaaaacgt cgatgccaaa gtaaaagctg cagaaactgc agcaggcaaa 1560

gccgaagctg ccgctggcac agctaatact gcagccgaca aggccgaagc tgtcgctgca 1620

aaagttaccg acatcaaagc tgatatcgct acgaacaaag ctgatattgc taaaaactca 1680

gcacgcatcg acagcttgga caaaaacgta gctaatctgc gcaaagaaac ccgccaaggc 1740

cttgcagaac aagccgcgct ctccggcctg ttccaacctt acaacgtggg tcggttcaat 1800

gtaacggctg cagtcggcgg ctacaaatcc gaatcggcag tcgccatcgg taccggcttc 1860

cgctttaccg aaaactttgc cgccaaagca ggcgtggcag tcggcacttc gtccggttct 1920

tccgcagcct accatgtcgg cgtcaattac gagtggtaa 1959

Claims

A kind of 1. multivalent meningococcal conjugate vaccines, it is characterised in that：By A groups, C groups, Y groups and W135 group meningitis cocci pods One or more of be conjugated with a restructuring Δ fHbp-NadA fusion protein carriers of film polysaccharide forms, wherein, the restructuring Δ FHbp-NadA fusion protein carriers, including restructuring Δ fHbp, flexible connection peptide fragment and NadA, the restructuring Δ fHbp are included FHbp can variation V1 V_A、V_BDomain and fHbp can variation V3 V_C、V_D、V_EDomain.
2. multivalent meningococcal conjugate vaccines according to claim 1, it is characterised in that：The ammonia of the restructuring Δ fHbp Base acid sequence such as sequence table SEQ ID NO：Shown in 1, its nucleotide coding sequence such as sequence table SEQ ID NO：Shown in 2.
3. multivalent meningococcal conjugate vaccines according to claim 1, it is characterised in that：The flexible connection peptide fragment Nucleotide coding sequence such as sequence table SEQ ID NO：Shown in 3.
4. multivalent meningococcal conjugate vaccines according to claim 1, it is characterised in that：The restructuring Δ fHbp- The amino acid sequence of NadA fusion protein carriers such as sequence table SEQ ID NO：Shown in 4, such as sequence table of its nucleotide coding sequence SEQ ID NO：Shown in 5.
5. the preparation method of any multivalent meningococcal conjugate vaccines of claim 1-4, it is characterised in that step bag Include：

S1, Prepare restructuring Δ fHbp-NadA albumen；

S2, prepare A groups, C groups, Y groups and W135 groups Nm bacterium capsular polysaccharides；

S3, combine preparation multivalence capsular polysaccharide-Δ fHbp-NadA albumen conjugate vaccines using derivation after CDAP activated polysaccharides.
6. the preparation method of the multivalent meningococcal conjugate vaccines described in claim 5, it is characterised in that weight described in step S1 Group Δ fHbp-NadA fusion protein carriers prepare further comprising the steps of：

S1a, design primer expand to obtain fHbp V1 genetic fragments, fHbp V3 genetic fragments respectively, then with this two sections of gene pieces Section carries out the Δ fHbp full length fragments that bridging PCR amplification is recombinated for template, recycles Δ fhbp genetic fragments and pET carriers, Prepare restructuring plasmid pET- Δs fHbp；

S1b, design primer amplification obtain the total length CDS sequences of NadA albumen, recycle NadA genetic fragments and recombinant plasmid pET- Δ fHbp, Prepare restructuring plasmid pET- Δs fHbp-NadA；

S1c, by recombinant plasmid pET- Δ fHbp-NadA translation tables reach bacterial strain, obtains restructuring Δ fHbp-NadA fusion proteins and carries Body.
7. the preparation method of the multivalent meningococcal conjugate vaccines described in claim 5, it is characterised in that the A in step S2 Group meningitis cocci uses 29201 bacterial strains, and C group meningitis coccis use 29205 bacterial strains, and Y group meningitis coccis use 29028 bacterium Strain, W135 group meningitis coccis use 29037 bacterial strains.