CN108619506A - Anti- Hib-RSV- meningococcus-pneumococcus combined vaccine - Google Patents

Abstract

The invention discloses a kind of anti-Hib RSV meningococcus pneumococcus combined vaccine, the combined vaccine includes：Intermediate is immunized in Hib pneumococcus, intermediate is immunized in meningococcus and intermediate is immunized in Respiratory Syncytial Virus(RSV), the Hib pneumococcus is immunized the high conservative that intermediate is expressed using pneumococcus and has the albumen of immunogenicity as carrier protein, and Hib capsular polysaccharides are conjugated；It includes Δ fHbp that intermediate, which is immunized, in the meningococcus, the recombination Δ fHbp include fHbp can variant V1 VA, VB structural domain and fHbp can variant V3 VC, VD, VE structural domain；The antigen that intermediate is immunized in the Respiratory Syncytial Virus(RSV) is the RSV film surfaces fusion protein F and/or attachment protein G that can cause organism immune response, the nucleic acid sequence of wherein expression proteantigen is binned on nucleic acid carrier, and the nucleic acid sequence of recombinant protein is to be attenuated intracellular bacteria as bacteria carrier；The immune intermediate of said components is prepared as freeze dried powder respectively, faces with melting mixing again.

Description

Anti- Hib-RSV- meningococcus-pneumococcus combined vaccine

Technical field

The present invention relates to a kind of anti-Hib-RSV- meningococcus-pneumococcus combined vaccines, belong to field of biological pharmacy.

Background technology

1, streptococcus pneumonia and its epidemiology

Streptococcus pneumonia (Streptococcus pneumoniae) abbreviation pneumococcus (Pneumococcus), is lodged in It is the main pathogenic fungi of bacillary lobar pneumonia, meningitis, tympanitis, pneumonia, bronchitis in the nasopharyngeal cavity of normal person. Disease caused by pneumococcus is always the serious public health problem in the whole world, there is higher incidence in worldwide And case fatality rate, especially to 2 years old children and old man below.The S. pneumoniae capsular saccharide vaccine and capsular saccharides listed at present Protein combined vaccine, design are all based on S. pneumoniae capsular saccharide, cover the most common blood for leading to pneumococcal disease Clear type.But S. pneumoniae capsular saccharide is thymus independent antigen (Thymus independent antigen, TI-Ag), is resisted Precursor reactant depends on the linear epitope of its recurring unit composition, is directly drenched with B in the case where no T lymphocytes assist The IgM receptors crosslinking of bar cell surface, the antibody induced is mainly IgM and IgG2, lacks preferable complement activation ability, Antibody level cannot maintain the sufficiently long time, and be unable to inducing immunological memory, can not be generated in 2 years old or less child immune Protection.The structure of capsular saccharides complexity causes the immunogenicity of each serotype different, can not generate effective immune response. Pneumococcal conjugated vaccine includes that Serotypes are more, and specificity structure of each type for combination is different, leads to each of which type Combined method it is different.Modification to capsular saccharides and and carrier protein combination not lost ensureing the special group of capsular saccharides, It is carried out under the premise of antigenicity and immunogenicity are unaffected, while in order to avoid the excessive crosslinking and conjugate degerming of sugar chain The requirement of filtering should have certain control to capsular saccharides and conjugate bulk of molecule.7 valence vaccines are in 2 months 2000 in the U.S. It is approved to use.Since pneumococcus type is more, binding protein ingredient is needed in the manufacturing process of combined vaccine, because of protein ingredient It can cause local reaction, so producing comprising combined vaccines more than 12 types with regard to highly difficult.Combined vaccine is exempted from for the first time Antibody concentration living is only capable of maintaining some months after epidemic disease, will then drop to pre-immune levels；And the entire work of combined vaccine It needs that a variety of chemical reagent participation reactions are added during skill, and the serotype coverage rate of capsular glycoprotein combined vaccine is low Increase with nonvaccine serotype pneumococcal infection disease is so that more researchers begin to focus on the pneumonia ball in other directions Bacteria vaccine is developed.

Two, haemophilus influenzae and its epidemiology

The strongest serotype of b types haemophilus influenzae (Hib) invasiveness, is to cause children tight in haemophilus influenzae The important pathogenic bacteria of double infection dye, infection object is mainly the infant below of 5 years old or less children, especially 2 years old.Hib passes through Susceptible person's respiratory infectious, is colonized in pharynx nasalis, can behave as asymptomatic carrier, periods of months, and small part crowd then occurs Hib affecting conditions.It is meningitis and pneumonia that Hib, which infects most common disease, additionally includes osteomyelitis, septic joint Inflammation, epiglottiditis and septicemia etc..The most important virulence factors of Hib are its capsular polysaccharide PRP, can be in Hib infection and cloning procedure Middle offer survival advantage resists the phagocytosis of complement-mediated, inhibits serum bactericidal activity, escapes nasal mucosal immune, promotes thin Propagation of the bacterium in crowd.

Hib combined vaccines common are four kinds according to PRP length, carrier protein difference, and immunogenicity respectively has feature： (1) using diphtheria toxoid albumen as the combined vaccine (PRP-diphthena toxoid, PRP-D) of carrier：With Hib polysaccharide epidemic diseases Seedling immunogenicity is similar, has certain Age Characteristics, and the antibody of higher level can be generated after primary vaccination of being grown up, and>15 Though the children of the moon can generate higher level antibody, acted on without long-term holding after booster shot.(2) with the double balls of B groups meningitis Mycetocyte memebrane protein is the combined vaccine (PRP-OMP) of carrier:PRP-OMP has preferable immunogene to all age group crowds Property.It is most of to generate high-caliber antibody per capita after 1st dose of inoculation.Antibody titer after being inoculated with again is also more than PRP-D, The time that the antibody of Hb-OC, PRP-T, but PRP-OMP maintain is short, and antibody peak level, compared with Hb-OC, PRP-T is low.(3) with The diphtheria toxoid CRM197 of attenuation is the combined vaccine (Hb-OC, PRP-CRM197) of carrier：The 1st dose of inoculation of 2 month infants The antibody level of generation is relatively low, but 4 and 6 monthly ages respectively again inject after can induce out high-caliber antibody, antibody still may be used after 1 year Maintain certain level.(4) using tetanus toxoid as the combined vaccine (PRP-T) of carrier：It is similar with Hb-OC, in larger youngster Virgin and adult, the 1st dose of inoculation can show preferable immunogenicity.The 1st dose of immunoprophylaxis reaction of young infant pair is weaker, the 2nd dose And the 3rd dose be inoculated with again after can generate the antibody of higher level, and antibody level hold time it is longer.Currently, domestic and state PRP-CRM197, the Hib vaccines that two kinds of PRP-T, PRP-T are suitable for most of age bracket crowds is mainly used to connect on border Kind.

Three, Respiratory Syncytial Virus(RSV) and its epidemiology

Respiratory infectious disease is still one of the main reason for leading to death in the world so far, and influenza virus (Influenza virus, FLU) and Respiratory Syncytial Virus(RSV) (Respiratory Syncytial Virus, RSV) are then weights The respiratory pathogen wanted.Currently, influenza has safely and effectively different type vaccine, guarantee is provided for the prevention and control of influenza. And RSV there is no effective vaccine available due to autoimmune properties.RSV is to cause infant's lower respiratory tract infection most important Cause of disease, and cause the elderly and immune deficiency be grown up be hospitalized and because pneumonia death major reason.According to statistics, within 6 months Infant cause admission rate to be even as high as 99% up to the childhood infection rate within 70%, 2 one full year of life because of rsv infection.RSV is because of it Range of causing a disease is wide, and the state of an illness is occurred frequently, and serious complication can be caused etc., cause serious prestige to human health and life security The side of body.RSV vaccines are set to one of the vaccine first developed by the World Health Organization.

RSV is under the jurisdiction of Paramyxoviridae Pneumovirus, is sub-thread minus-stranded rna virus.RSV full-length genomes about 15Kb is compiled 10 kinds of major proteins of code, including three transmembrane proteins (G, F and SH), two stromatins (M and M2), three nucleocapsid proteins (N, P and L) and two non-structural proteins (NS1 and NS2) are constituted, wherein fusion protein F (Fusion protein, F) and attachment Protein G (Attchment protein, G) is that RSV excitating organisms generate the most important virus protein of protection antibody.G-protein Difference in height can be divided into two hypotypes of A and B according to G-protein antigenic specificity RSV；The F protein of RSV is highly conserved, mediates disease The fusion of malicious coating and host cell membrane so that virus is successfully invaded host cell and can also be caused between flanking cell plasma membrane Fusion promote the formation of external plasomidum.It can effectively prevent RSV for the neutralizing antibody of RSV F and G glycoprotein and infect again, Therefore RSV F and G-protein have been acknowledged as the pathogenic molecule of virulence and protective antigens of RSV.

For RSV, in the 1960s, the formalin-inactivated vaccine (FI-RSV) of the developments such as F μ Lginiti VA Due to induce Th2 types be immunized it is too drastic lead to 2 death of child, end in failure.The research of RSV vaccines is concentrated mainly at present Carrier bacterin, attenuated live vaccine, subunit vaccine, DNA vaccination, VLP vaccines, but it is available without granted RSV vaccines so far.RSV The research of vaccine is always the focus of international concern, from the RSV vaccines in existing development as it can be seen that injecting immune cannot It generates effective mucous membrane and cell immune response and immune protective effect is limited, there are potential safety and overall lengths for DNA vaccination F, there are the bottleneck problems such as potential Th1/Th2 loss of equilibrium are urgently to be resolved hurrily for G-protein vaccine.Hinder the major obstacle of RSV vaccine developments There is the following：First, it is half sensitive infection/reconstructed model that most animals model, which includes chimpanzee etc., therefore, it is difficult to complete It is complete to reproduce the pathogenic of RSV；Second is that the neonatal immune system developmental immaturity as vaccine primary target population, while body Interior existing female biography antibodies mediate immune interference；Third, there are two kinds of different antigenic subtypes by RSV, and natural infection is difficult to Generate effective immanoprotection action；Fourth, formalin-inactivated vaccine (FI-RSV) cannot not only prevent infant infection, In natural infection later, children's aggravation (i.e. disease humidification) of vaccine inoculation, even death.

Recent studies indicate that the RSV vaccines built based on carrier are expected to be used for newborn.Carrier bacterin is main Including vector-viral vaccine and Bacterial vector vaccines.RSV vector-viral vaccines include mainly：Vaccinia virus (vaccinia Virus) carrier bacterin, adenovirus (adenovirus, Ad) carrier and paramyxovirus (paramyxovirus) carrier bacterin etc., In recent years, adenovirus carrier vaccine and paramyxovirus carrier bacterin receive significant attention.

Four, Neisseria meningitidis (Nm) and its epidemiology

Meningococal meningitis (epidemic cerebrospinal meningitis) is by Neisseria meningitdis Bacterium has different degrees of prevalence all over the world by acute purulent meningitis caused by respiratory infectious.Meningitis Neisseria 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 are Its unique host, thus Nm shows the adaptability to human nasopharynx portion height, the healthy population of 10%-40% is all Nm Without clinical symptoms carrier, in meningitis Outbreak period, Asymptomatic Carriers ratio more may be up to 70%.A small number of feelings Under condition, Nm can menses invade meninges, cause purulent inflammation, meningismus and the brain ridge of purulent meningitis occur Liquid changes.Vaccine prevention is the important means for preventing meningococcosis, because even being interfered using antibiotic, this disease Still there are high incidence 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, 13 sero-groups (Serogroup) of Y, Z, 29E and w135, and according to its outer membrane protein (OMP), PorB (2 or 3 class OMp) With the antigenic specificity of PorA (1 class OMP), and different serotype and blood serum subtype can be divided into.The whole world about occurs 120 every year Ten thousand Nm cases of infection, majority of cases are caused by A, B, C, W135, Y groups of bacterial strains.And caused by B crowds of Nm in developed country Meningococal meningitis case be more up to the 80% of total case.In the popular mainly based on A groups of China, in recent years Due to the extensive inoculation of vaccine, infection caused by A groups is effectively controlled, and the case caused by B groups also increases relatively.It is single The Meningococcal Vaccine of one sero-group, serotype and blood serum subtype is difficult the generation for preventing meningococal meningitis on the whole.With It to Nm antigenic surface structures more in-depth study, Meningococcal Vaccine just develops towards diversification direction.

The existing development of vaccine is conjugated in epidemic meningitis polysaccharide-protein, and Chiron and Wyem use detoxification diphtheria toxoid (CRM197) As protein carrier, Baxter uses tetanus toxoid as carrier, develops A/C group meningitis cocci conjugate vaccines, in In November, 1999 introduces Britain.Hereafter Sanofi-Pasteur develops A/C/Y/W135 groups of polysaccharide covalent combination diphtheria classes The tetravalence conjugate vaccines of toxin (MCV4), China have sewing for several A, C group meningitis cocci capsular polysaccharides and carrier protein TT Close vaccine approval listing.Due to meningococcal polysacharide and conjugate vaccines application, A, C, Y and W135 mass-brain are effectively prevented The infection of meningococcus.It is different from A, C, W135, Y groups of bacterial strain capsular polysaccharides, the capsular polysaccharide immunogenicities of B groups of Nm bacterial strains compared with It is low, and the nerve cell adhesion in the structure of MenB capsular polysaccharides and the nerve fiber developed and a small number of mature tissues Molecule homologous, 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 The research of group's vaccine is concentrated mainly on non-capsular antigen at present, such as albumen, lipopolysaccharides (1ipopolysaccharide, LOS) Deng.The significant challenge for screening non-pod membrane surface antigen is its safety, antigenic conservation and can cause extensive effective sterilizing power Reaction.Can need to have following characteristics as the protein with immunogenicity of candidate vaccine：It is all expressed in most of bacterial strains And structural conservation；Bactericidin or protection antibody can be induced.Achieved in terms of the research of protein vaccine at present centainly into Exhibition, Novartis Co., Ltd pass through the clinical trial of III phase using the 4CMenB vaccines of reverse vaccinology development.Grinding hot spot candidate antigens It is one of most promising candidate protein to have PorA, NhhA, GNA2132, NadA and fHBP etc., wherein fHBP.

FHBP is factor H binding 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, the C3b that it is mediated in factor I play confactor during being cracked into inactivation segment iC3b Effect.Also promote the decay of alternative route c3 invertases C3bBb.FHBP and factor H combinations can lower alternative route, to have It survives conducive to Neisseria meningitidis.FHBP is for meningococcus feelings existing for the blood of the mankind, serum and antibiotic property polypeptide Existence under condition is particularly important.Its amino acid sequence is more conservative, and 91.6%-100% amino acid is identical in mutation.Recombination FHBP antibody can cause the passive guarantor of the bactericidal effect and inductive infection suckling mouse model of the complement-mediated for same class mutation Shield.Not only titre is high and insensitive to sequentially making a variation for the antibody obtained by complete fHBP, even if there is a small amount of amino acid change, only Determine that the key amino acid of space conformation is constant, the variation of antibody bactericidal activity is little.All these features make fHBP become brain One of most effective antigen of film inflammation Neisseria and most promising candidate general vaccines.Antigen cross according to entire albumen is anti- Answering property and sequence similarity, meningococcus fHbp are divided into 3 antigenic variant groups.In general, for the fHbp of variant V1 Antibody prepared by (also referred to as subfamily B) has bactericidal activity to the bacterial strain for expressing fHbp from variant V1, but is not directed to and becomes The bacterial strain of expression fHbp in body V2 and V3 (also referred to as subtribe A), vice versa.There is also the Asias of fHbp in each variant group Variant.Recently, the molecular structure of fHbp has been shown as " modularization ", and fHbp variants are containing there are five different groups of variable domains It closes (VA~VE), each variable section is derived from subtribe A and subtribe B.Therefore, five are freely combined by genetic engineering means Structural domain can obtain the recombination Δ fHbp albumen of the antigen of covering A, B two subtribes, three kinds of variants, and the vaccine for becoming wide spectrum is anti- Former and protein carrier.

Lack the combined immunization medication for the various bacteria of the snout infection of the upper respiratory tract currently on the market, and upper breathing Road infection is easy to cause a series of complication, and contact is close between various diseases, is easy to influence each other, especially right For Susceptible population, joint illness has serious influence or even entail dangers to life for health.Therefore it needs to exhaling Desorption system class disease carries out combined immunization, to more efficiently avoid the generation of same type disease.

Invention content

In view of the above-mentioned problems existing in the prior art, the purpose of the present invention is obtain a kind of anti-Hib-RSV- meningitis ball Bacterium-pneumococcus combined vaccine.

For achieving the above object, anti-Hib-RSV- meningococcus-pneumococcus combined vaccine that the present invention uses Technical solution it is as follows：

The combined vaccine includes：

Intermediate is immunized in Hib- pneumococcus, and the high guarantor that intermediate is expressed with pneumococcus is immunized in the Hib- pneumococcus Keeping property simultaneously has the albumen of immunogenicity for carrier protein, and Hib capsular polysaccharides are conjugated；

Intermediate is immunized in meningococcus, and it includes Δ fHbp that intermediate, which is immunized, in the meningococcus, the recombination Δ FHbp include fHbp can variant V1 VA, VB structural domain and fHbp can variant V3 VC, VD, VE structural domain；

Intermediate is immunized in Respiratory Syncytial Virus(RSV), and the antigen that intermediate is immunized in the Respiratory Syncytial Virus(RSV) is that can cause machine The RSV film surfaces fusion protein F and/or attachment protein G of body immune response, wherein the nucleic acid sequence recombination of expression proteantigen On nucleic acid carrier, the nucleic acid sequence of recombinant protein is to be attenuated intracellular bacteria as bacteria carrier；

The immune intermediate of said components is prepared as freeze dried powder respectively, faces with melting mixing again.

Preferably, it further includes that the pneumococcal capsule conjugated with carrier protein is more that intermediate, which is immunized, in the Hib- pneumococcus Sugar.Although modified pneumococcus carrier protein has immunogenicity, in order to ensure that pneumococcus is stable and good exempts from Epidemic focus makes the pneumococcal capsular polysaccharide and lung of purifying and activation while Hib capsular polysaccharides and conjugated carrier protein Scorching coccus carrier protein is conjugated, forms new immune intermediate, ensures that the immunocompetence of intermediate is immunized in Hib- pneumococcus.

Preferably, the one kind or more of pneumococcal capsular polysaccharide in following serotype：1、2、3、4、5、6B、 7F、 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19F, 19A, 20,22F, 23F and/or 33F.

Preferably, pneumococcus carrier protein includes：Pneumococcus dissolved blood protein and its modification derivant, pneumococcus table Three histidine egg of face albumen and its modification derivant, pneumonia ball surface adhesion albumen and its modification derivant or pneumococcus White family fusion protein.

It is furthermore preferred that pneumococcus carrier protein is specially：Modified pneumococcus dissolved blood protein △ A146Ply, pneumonia ball Three groups of bacterium dissolved blood protein derivative dPly, Pneumococal surface protein A, Pneumococcal Surface adhesion protein A or pneumococcus ammonia Acid albumin PhtA, PhtB, PhtD, PhtE, PhtDE.

As a preferred embodiment, it is preferred that modified nontoxic dissolved blood protein △ A146Ply and 7 valences, 13 valences or 23 valence pneumococcal capsular polysaccharides are conjugated.

Preferably, rsv protein is F protein, designs specific primer according to the nucleic acid sequence of expression albumen, primer sequence is such as Sequence table SEQ ID NO：6 and SEQ ID NO：Shown in 7.

Preferably, the rsv protein antigen of recombination is pcDNA3.1-F.

Preferably, attenuation intracellular bacteria carrier is selected from：L3261, SL7207 or ty21a.

Preferably, transfection to the RSV antigens in bacteria carrier are SL7207/pcDNA3.1-F.

Preferably, the amino acid sequence such as sequence table SEQ ID NO of the recombination Δ fHbp：Shown in 9, nucleotide coding Sequence such as sequence table SEQ ID NO：Shown in 10.

The MenB bacterial strains that the Δ fHbp albumen of recombination does not express low expression even fHbp lose protective capability, can be effective Infecting for the MenB bacterial strains of all expression fHbp (V1, V3 and V2 variant) is immunized in ground, therefore the immune of the albumen cannot achieve reason By upper all standing, but since the combined vaccine in the present invention needs to exist in hybrid form, between each antigen inevitably The relationship for generating competition and mutually inhibiting, the carrier as immune intermediate also should not be too large, experiments prove that, Δ fHbp Albumen can reach preset immune effect, therefore modification is not selected to couple all standing albumen of other albumen as carrier egg In vain.

Compared with prior art, the albumen of Pnu-Imune 23 of the invention-capsular saccharides combination can be in different serum Play the role of inducing Cross immunogenicity between type；And albumen itself can induce stronger immunoprotection as virulence factor Effect has gone up supplementary function to capsular saccharides immune, to enhance the immanoprotection action of vaccine, while pneumococcus Oneself protein can conflict to avoid generating to be immunized in vivo with other vaccines such as diphtheria vaccine, tetanus vaccine so that this combination Vaccine is implemented as in order to possible.This combined vaccine improves the immunological memory response of the pneumococcal infection of inoculator；Pneumonia The addition of pneumococcal proteins is so that the immune of pneumococcus needs dependence thymus gland immune, so that the immune realization of pneumococcus Full age bracket and whole the long-acting of serotype, full coverage type are immune；S. pneumoniae capsular saccharide is coupled it with pneumoprotein The speed of vivo immunization response is improved afterwards and extends its immune depth and range, although synergistic effect between the two It is good without the effect of each independent vaccine, but reduce inoculation times, inoculation efficiency is improved, the body of vaccinee is alleviated Body is born；

The RSV live vector vaccines that the Respiratory Syncytial Virus(RSV) intermediate of the present invention is built based on carrier can be thin in body Born of the same parents formed protein conformation it is identical with its native conformation, attenuation salmonella can both carry prokaryotic expression plasmid or Carry eukaryon expression plasmid, will not lead to the forfeiture or variation of epitope, the immunity of formation be more conducive to resist it is subsequent from So infection；Immune efficacy is high, at low cost and safety is good；Avoid that vitro propagation titre existing for RSV is low and stability difference Problem, recombinant attenuated salmonella vaccine simple production process, vaccine is easily stored and transports, therefore the cost of vaccine is opposite It is relatively low, be conducive to the extensive preparation of vaccine；Recombinant attenuated bacteria vaccine can be immunized by oral administration, intranasal immunisations and rectum are immune etc. Number of ways is immune, and antigen is by direct submission to APC cells, and effective activated cell is immunized and humoral immunity, and through oromucosal route It is immune not will produce disease humidification, and female interference for passing antibody can be broken through；

Meningococcus is immunized intermediate and 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 Three kinds of variants) conserved domain, B groups of bacterial strains of Nm of all expression fHbp albumen can be covered, also, Δ fHbp includes FHbp five structural domains of complete VA~VE, structure, function and antigen site maintain integrality；Meanwhile the fusion protein NadA expressed in 50% B groups of bacterial strains of Nm, and these bacterial strains are essentially high pathogenic bacteria.Even if therefore do not express fHbp but The bacterial strain of expression NadA can also be covered by Δ fHbp-NadA albumen, can be wide by protein vaccine made of antigen of this recombinant protein Spectrum protects just about the infection of all Nm B groups of pathogenic bacteria；

Three kinds of immune intermediates, which face, uses mixing as combined vaccine, can carry out broad spectrum protection, reduce inoculation times and inoculation After react, for inoculator have positive effect.

Description of the drawings

Fig. 1 is F protein pcr amplification product electrophoretogram provided in an embodiment of the present invention；

Fig. 2 is positive plasmid structure insertion point figure provided in an embodiment of the present invention；

Fig. 3 is the F protein pcr amplification product electrophoretogram of mouse In vivo infection provided in an embodiment of the present invention；

Fig. 4 is that fHbp V1~V3 can be changed binding domains and recombination Δ fHbp structural domains；

Fig. 5 is that Δ fHbp expands electrophoresis pattern and pET- Δs fHbp identification electrophoresis patterns；

Fig. 6 is that IPTG induces recombinant bacterium to express Δ fHbp albumen；

Fig. 7 is that Western-Blot identifies Δ fHbp recombinant proteins；

Fig. 8 is the Δ fHbp recombinant proteins after SDS-PAGE purification Identifications.

Specific implementation mode

With reference to embodiment to anti-Hib-RSV- meningococcus-pneumococcus combined vaccine provided by the invention make into One step is detailed, completely illustrates.The embodiments described below is exemplary, and is only used for explaining the present invention, and should not be understood as Limitation of the present invention.

Experimental method in following embodiments is unless otherwise specified conventional method.Reality as used in the following examples It tests material unless otherwise specified, is that market is commercially available.

1. preparing Hib- pneumococcus is immunized intermediate

1.1 prepare S. pneumoniae capsular saccharide

A. the common causative serotype 4 of existing 7 valence pneumococcus, the pneumonia ball of 6B, 9V, 14,18C, 19F and 23F are taken Bacterium is cultivated；

B. capsular polysaccharide 4,9V, 14,19F and the 23F that antigenicity is strong in any of the above Pneumococcal serotype are purified respectively, Oligosaccharides 18C and polysaccharide 6B；

C. supernatant is collected by centrifugation after pneumococcus inactivation, through being concentrated by ultrafiltration, according to each Pneumococcus serotypes characteristic point Not Jia Ru appropriate (volume fraction is 70%) pre-cooled ethanol, be collected by centrifugation, obtain crude capsular saccharides；Crude capsular saccharides are dissolved in In sodium acetate solution, 1 is then pressed：2 ratios remove removing protein with cold phenol mixing, centrifugation, and phenol carries 5-6 times repeatedly, collect supernatant, use Distilled water is dialysed, and liquid adds 2mol/L calcium chloride solutions after dialysis, and ethyl alcohol stirring is added, and centrifugation removal nucleic acid collects supernatant, Ethyl alcohol stirring (final concentration 80%) is added, precipitation is collected by centrifugation, washs precipitation with ethyl alcohol, acetone, pod must be refined after dehydration and drying Film sugar, sets -20 DEG C and saves backup.

The high conservative of 1.2 pneumococcus expression and the albumen △ A146Ply with immunogenicity

Ply gene (the sequence accession numbers announced according to GenBank：X52474), modified pneumococcus dissolved blood protein △ Design primer sequence such as the following table 1 of A146Ply：

Table 1

SEQ ID NO	Primer	Sequence (5 ' -3 ')
			1	F-△A146Ply-N	GGAATTCCATATGGCAAATAAAGCAGTA AAT
2	R-△A146Ply-N	CGAGCTCGTCATTTTCTACCTTATCCTCT
			3	F-△A146Ply-C	CGGCGGCCGCATGGCAAATAAAGCAGTAAATG
4	R-△A146Ply-C	CCCTCGAGTTACTAGTCATTTTCTACCTTATCCTCT

Wherein dashed part is corresponding restriction enzyme digestion sites.

After being expanded △ A146Ply gene PCRs according to routine experiment method, target DNA fragments are recycled in 1% gel electrophoresis, Target DNA fragments are connected in pET-28a plasmids, the conversion extracting recombinant DNA matter in competent escherichia coli cell BL21 , PAGE gel electroresis appraisal is carried out after IPTG induced expressions, and Ni-NTA trees are carried out after determining expression quantity and expression-form Fat purifies；Endotoxin, i.e. hemolytic activity are removed using 0.2% formalin solution, obtain the △ after removal endotoxin A146Ply albumen.△ A146Ply albumen is the modified protein of the 146th deletion of alanine of Ply albumen, and purity of protein reaches 80% or more, detection is proved with immunogenicity and remaining endotoxin content is less than 0.1EU/ μ g.

Pneumococcal surface protein and capsular saccharides are coupled using amino reduction method, specially obtain above-mentioned steps △ A146Ply albumen and a variety of capsular saccharides with 1：2~4 mass ratioes mix, and for preparing 10mL vaccinogen liquids, pod is added Film polysaccharide 4,9V, 14,0 μ g of 19F and 80 μ g, △ A146Ply protein 16s of 23F 2 μ g of each 40 μ g, oligosaccharides 18C and polysaccharide 6B.Through It crosses gel chromatography column and measures pod membrane sugared content after purification.

1.3 prepare Hib b

A. it is fermented at 37 DEG C to Hib bacterial strains 1842 using CY culture mediums, zymotic fluid is through formalin-inactivated, centrifuging and taking After supernatant, precipitated using cetyl trimethylammonium bromide (CTAB).Then heavy to compound with the NaCl of 0.5~1M Shallow lake is dissociated, addition ice ethyl alcohol to final concentration 25% (v/v), precipitate nucleic acids, then ethyl alcohol on the rocks to 75% (v/ of final concentration V), precipitation obtains Thick many candies.

B. above-mentioned Thick many candies are dissolved with sodium acetate solution, and cold phenol extracts 5-6 times, and removal phenol residual is finally concentrated by ultrafiltration, Refined sugar is obtained through 75% (v/v) ethanol precipitation, -20 DEG C is set and saves backup.

1.4Hib capsular polysaccharides, pneumococcal capsular polysaccharide are combined with pneumoprotein

It will be combined, formed with pneumoprotein △ A146Ply after pneumococcal capsular polysaccharide, the activation of HIb capsular polysaccharides Intermediate is immunized in Hib- pneumococcus.

The immunological evaluation of intermediate is immunized in 1.5Hib- pneumococcus

In mouse experiment made on the living, every mouse muscle injection is spaced one month per injection amount 0.5mL, 3 needles is immunized altogether；Lung Scorching coccus-DTP vaccine dosage 0.5mL is spaced January, and 3 needles are immunized altogether.It is examined with ELISA method after the completion of inoculation It is horizontal to survey mice serum moderate resistance S. pneumoniae capsular saccharide PS IgG antibodies, using ELISA method detection anti-tetanus antibody GMT.It comments Estimate result such as the following table 2：

Table 2

2, it prepares Respiratory Syncytial Virus(RSV) and intermediate is immunized

Plasmid construction, expression and the purifying of 2.1 Respiratory Syncytial Virus(RSV) (RSV)

Select the areas the CDS gene (GeneID of RSV F proteins：1489825) it is expanded, the areas the CDS gene of F protein is such as Sequence table SEQ ID NO：Shown in 5, according to the specific designs upstream and downstream primer of F genes, upstream primer sequence such as sequence table SEQ ID NO：Shown in 6, downstream primer such as sequence table SEQ ID NO：Shown in 7, in conjunction with the segment to be amplified such as sequence after primer Table SEQ ID NO：Shown in 8.

F protein CDS region sequences are specific as follows：

ATGGAGCTGCTGATCCACAGGTTAAGTGCAATCTTCCTAACTCTTGCTATTAATGCA TTGTACCTCACCTCAAGTCAGAACATAACTGAGGAGTTTTACCAATCGACATGTAGTG CAGTTAGCAGAGGTTATTTTAGTGCTTTAAGAACAGGTTGGTATACCAGTGTCATAACA ATAGAATTAAGTAATATAAAAGAAACCAAATGCAATGGAACTGACACTAAAGTAAAAC TTATAAAACAAGAATTAGATAAGTATAAGAATGCAGTGACAGAATTACAGCTACTTATG CAAAACACACCAGCTGCCAACAACCGGGCCAGAAGAGAAGCACCACAGTATATGAA CTATACAATCAATACCACTAAAAACCTAAATGTATCAATAAGCAAGAAGAGGAAACGA AGATTTCTGGGCTTCTTGTTAGGTGTAGGATCTGCAATAGCAAGTGGTATAGCTGTATC CAAAGTTCTACACCTTGAAGGAGAAGTGAACAAGATCAAAAATGCTTTGTTATCTACA AACAAAGCTGTAGTCAGTCTATCAAATGGGGTCAGTGTTTTAACCAGCAAAGTGTTAG ATCTCAAGAATTACATAAATAACCAATTATTACCCATAGTAAATCAACAGAGCTGTCGC ATCTCCAACATTGAAACAGTTATAGAATTCCAGCAGAAGAACAGCAGATTGTTGGAAA TCAACAGAGAATTCAGTGTCAATGCAGGTGTAACAACACCTTTAAGCACTTACATGTT AACAAACAGTGAGTTACTATCATTGATCAATGATATGCCTATAACAAATGATCAGAAAA AATTAATGTCAAGCAATGTTCAGATAGTAAGGCAACAAAGTTATTCTATCATGTCTATAA TAAAGGAAGAAGTCCTTGCATATGTTGTACAGCTACCTATCTATGGTGTAATAGATACA CCTTGCTGGAAATTACACACATCACCTCTATGCACCACCAACATCAAAGAAGGATCAA ATATTTGTTTAACAAGGACTGATAGAGGATGGTATTGTGATAATGCAGGATCAGTATCC TTCTTTCCACAGGCTGACACTTGTAAAGTACAGTCCAATCGAGTATTTTGTGACACTAT GAACAGTTTGACATTACCAAGTGAAGTCAGCCTTTGTAACACTGACATATTCAATTCC AAGTATGACTGCAAAATTATGACATCAAAAACAGACATAAGCAGCTCAGTAATTACTT CTCTTGGAGCTATAGTGTCATGCTATGGTAAAACTAAATGCACTGCATCCAACAAAAAT CGTGGGATTATAAAGACATTTTCTAATGGTTGTGACTATGTGTCAAACAAAGGAGTAGA TACTGTGTCAGTGGGCAACACTTTATACTATGTAAACAAGCTGGAAGGCAAGAACCTT TATGTAAAAGGGGAACCTATAATAAATTACTATGACCCTCTAGTGTTTCCTTCTGATGA GTTTGATGCATCAATATCTCAAGTCAATGAAAAAATCAATCAAAGTTTAGCTTTTATTC GTAGATCTGATGAATTACTACATAATGTAAATACTGGCAAATCTACTACAAATATTATGA TAACTACAATTATTATAGTAATCATTGTAGTATTGTTATCATTAATAGCTATTGGTTTGCT GTTGTATTGCAAAGCCAAAAACACACCAGTTACACTAAGCAAAGACCAACTAAGTGG AATCAATAATATTGCATTCAGCAAATAG

It takes recovery and carries out the Vero cells of squamous subculture, diluted virus is accessed after cleaning, 37 DEG C adsorb 1h, at interval of 15min shakes once, makes the abundant infection cell of virus；It outwells the viral dilution for connecing poison cell bottle and does not connect poison cell bottle Culture solution adds the DMEM maintaining liquids that 6~8mL contains 2% serum, 37 DEG C of CO respectively₂It is cultivated in incubator, daily sight from second day Examine cytopathy situation.Received when 75% lesion occurs in cell malicious (about needing 7d), i.e., by culture solution in -80 DEG C/37 DEG C repeatedly After freeze thawing 3 times, 10000r/min centrifuges 10min, and sterile working takes supernatant, dispenses, in -80 DEG C of refrigerator long-term preservations.

Extraction RNA centrifuge tube, pipette tips and the PCR pipes needed is carried out at RNA enzymes using DEPC (coke diethyl phthalate) Reason, and using RNA/DNA kits extraction RSV RNA.The extracts kit used in the present embodiment is viral in a small amount for TaKaRa RNA/DNA extracts kits, by specification operation.The RNA extracted need to immediately using or be placed in -80 DEG C of refrigerators and preserve It is spare.

Reverse transcription system uses 25 μ L systems, and 12 μ L, universal primer Oligo (d) T18 of RNA, 1 μ L gently mixings are added, 70 DEG C are incubated 5min, ice bath 2min.AMV 1 μ L, AMV Buffer 5 μ L, dNTP 5 μ L, RNase are added into centrifuge tube 1 μ L of Inhibitor gently mixings, 42 DEG C of incubation 60min.70 DEG C of 10min inactivate transcriptase.Obtained product cDNA is needed immediately Using or in -20 DEG C of long-term preservations it is spare.

Specific primer is designed according to RSV F protein CDS segments, primer sequence is followed successively by protection alkali by 5 ' ends to 3 ' ends Base-restriction enzyme site-connection primer, upstream restriction enzyme site are Hind III digestions site, and downstream restriction enzyme site is Xho I digestions Site, primer sequence are specific as follows：

F:：5'-ccc-AAGCTT-CAGAAAACCGTGACCTATCAAG-3'

R：5'-cc-CTCGAG-ACATGAAGTTTTGCCTCACTAGTA-3'

PCR system uses 25 μ L systems, and 10 × rTaq buffer, 2.5 2 0.25 μ L of μ L, rTaq of μ L, dNTP are added, on Swim 1 μ L of primer, 1 μ L of downstream primer, 17.25 μ L, RT-PCR products cDNA of water, 1 μ L；Expand the reaction condition of F full length fragments： 94 DEG C of 5min, 94 DEG C of 50s, 55 DEG C of annealing 50s, 72 DEG C of extension 1.5min, 25 cycles, 72 DEG C of extension 10min, 4 DEG C are terminated； Expand the reaction condition of G full length fragments：94 DEG C of 5min, 94 DEG C of 45s, 58 DEG C of annealing 45s, 72 DEG C of extension 1min, 25 recycle, 72 DEG C of extension 7min, 4 DEG C of end.

Electrophoresis detection is carried out to PCR product using 1% agarose gel electrophoresis, confirms F genetic fragments size about after detection For the pcr amplified fragment of 2150bp.Amplified production electrophoretogram is as shown in Figure 1,1 F gene of band, band M1 are standard in figure DNA marker, band is clear, meets expected theoretical value.

Using DNA gel QIAquick Gel Extraction Kit recovery purifying PCR product.- 20 DEG C of preservations.

According to above-mentioned primer, pcDNA3.1-F is built using expression vector pcDNA3.1, F protein gene fragment clone is entered The CMV promoter downstream of pcDNA3.1, is built into eukaryon expression plasmid pcDNA3.1-F.Build successful part junction fragment As shown in Figure 2.

F protein gene order, digestion with restriction enzyme PCR product and pcDNA3.1 are expanded, is connected after double digestion product, Complete plasmid construction.

Segment to be amplified after primer connection is specific as follows：

CTTAGTTATTCAAAAACTACATCTTAGCAGAAAACCGTGACCTATCAAGCAAGAA CGAAATTAAACCTGGGGCAAATAACCATGGAGCTGCTGATCCACAGGTTAAGTGCAAT CTTCCTAACTCTTGCTATTAATGCATTGTACCTCACCTCAAGTCAGAACATAACTGAGG AGTTTTACCAATCGACATGTAGTGCAGTTAGCAGAGGTTATTTTAGTGCTTTAAGAACA GGTTGGTATACCAGTGTCATAACAATAGAATTAAGTAATATAAAAGAAACCAAATGCAA TGGAACTGACACTAAAGTAAAACTTATAAAACAAGAATTAGATAAGTATAAGAATGCA GTGACAGAATTACAGCTACTTATGCAAAACACACCAGCTGCCAACAACCGGGCCAGA AGAGAAGCACCACAGTATATGAACTATACAATCAATACCACTAAAAACCTAAATGTATC AATAAGCAAGAAGAGGAAACGAAGATTTCTGGGCTTCTTGTTAGGTGTAGGATCTGC AATAGCAAGTGGTATAGCTGTATCCAAAGTTCTACACCTTGAAGGAGAAGTGAACAAG ATCAAAAATGCTTTGTTATCTACAAACAAAGCTGTAGTCAGTCTATCAAATGGGGTCA GTGTTTTAACCAGCAAAGTGTTAGATCTCAAGAATTACATAAATAACCAATTATTACCC ATAGTAAATCAACAGAGCTGTCGCATCTCCAACATTGAAACAGTTATAGAATTCCAGC AGAAGAACAGCAGATTGTTGGAAATCAACAGAGAATTCAGTGTCAATGCAGGTGTAA CAACACCTTTAAGCACTTACATGTTAACAAACAGTGAGTTACTATCATTGATCAATGAT ATGCCTATAACAAATGATCAGAAAAAATTAATGTCAAGCAATGTTCAGATAGTAAGGC AACAAAGTTATTCTATCATGTCTATAATAAAGGAAGAAGTCCTTGCATATGTTGTACAG CTACCTATCTATGGTGTAATAGATACACCTTGCTGGAAATTACACACATCACCTCTATGC ACCACCAACATCAAAGAAGGATCAAATATTTGTTTAACAAGGACTGATAGAGGATGGT ATTGTGATAATGCAGGATCAGTATCCTTCTTTCCACAGGCTGACACTTGTAAAGTACAG TCCAATCGAGTATTTTGTGACACTATGAACAGTTTGACATTACCAAGTGAAGTCAGCC TTTGTAACACTGACATATTCAATTCCAAGTATGACTGCAAAATTATGACATCAAAAACA GACATAAGCAGCTCAGTAATTACTTCTCTTGGAGCTATAGTGTCATGCTATGGTAAAAC TAAATGCACTGCATCCAACAAAAATCGTGGGATTATAAAGACATTTTCTAATGGTTGTG ACTATGTGTCAAACAAAGGAGTAGATACTGTGTCAGTGGGCAACACTTTATACTATGTA AACAAGCTGGAAGGCAAGAACCTTTATGTAAAAGGGGAACCTATAATAAATTACTATG ACCCTCTAGTGTTTCCTTCTGATGAGTTTGATGCATCAATATCTCAAGTCAATGAAAAA ATCAATCAAAGTTTAGCTTTTATTCGTAGATCTGATGAATTACTACATAATGTAAATACT GGCAAATCTACTACAAATATTATGATAACTACAATTATTATAGTAATCATTGTAGTATTGT TATCATTAATAGCTATTGGTTTGCTGTTGTATTGCAAAGCCAAAAACACACCAGTTACA CTAAGCAAAGACCAACTAAGTGGAATCAATAATATTGCATTCAGCAAATAGACAAAAA ACCACCTGATCATGTTTCAACAACAGTCTGCTGATCACCAATCCCAAATCAACCCATA ACAAACACTTCAACATCACAGTACAGGCTGAATCATTTCTTCACATCATGCTACCCAC ACAACTAAGCTAGATCCTTAACTCATAGTTACATAAAAACCTCAAGTATCACAATCAAA CACTAAATCAACACATCATTCACAAAATTAACAGCTGGGGCAAATATGTCGCGAAGAA ATCCTTGTAAATTTGAGATTAGAGGTCATTGCTTGAATGGTAGAAGATGTCACTACAGT CATAATTACTTTGAATGGCCTCCTCATGCCTTACTAGTGAGGCAAAACTTCATGTTAAA CAAGATACTCAAGTCAATGGACAAAAGCATAGACACTTTGTCTGAAATAAGTGGAGCT GCTGAACTGGACAGAACAGAAGAATATGCTCTTGGTATAGTTGGAGTGCTAGAGAGTT ACATAGGATCTATAAACAACATAACA

Underline position is the promoter and terminator of F protein in segment.

PCR plasmid PCR product F segments are about 2150bp, consistent with expected PCR product.Positive plasmid sequencing results It is completely the same with former F gene orders.Double digestion carrier and target fragment position are also consistent with expection.Prove positive plasmid structure Success.

2.2 eukaryotic expression recombinant plasmids import attenuation salmonella

In the flat lining out culture salmonella typhimurium SL7207 of LB；Picking single bacterium colony is inoculated with 3ml LB Liquid Cultures Base, 37 DEG C of overnight shaking cultures；100 μ L overnight cultures are inoculated with per 5ml LB liquid mediums, 37 DEG C, 225prm oscillations are trained It supports, until OD_600nm=0.6；Culture ice bath 30min, 4000rpm centrifuge 10min；All nutrient solutions are removed, with isometric, ice Bacterium, 4000prm, centrifugation 10min (WB=10% glycerine, 90% distilled water, filtering) is resuspended in the WB solution of bath；Repeat upper one Step 2 times：Incline most of WB, and it is that 50 μ L (prepare 50 μ L of competent cell, i.e., per 10ml primary cultures to make residual volume 0.5%), mixing is competent cell.

5 μ L plasmids pcDNA3.1-F, pcDNA3.1 and luciferase plasmid pEGFP are added in every 40 μ L competent cells, mix It is even；It draws 40 μ L to be added in the pole cup of ice precooling, carries out electrotransformation.Parameter is set as 2.5Kv, 200 Ω, 25 μ F, t ≈ 4.5-5.0ms.Separately the competent cell electrotransformation for being not added with plasmid is taken to compare：After electric shock, 1ml is added into pole cup immediately LB, mixing, 37 DEG C of shaken cultivation 1h；200 μ L layings are taken to have Amp^RThe LB tablets of resistance, 37 DEG C are incubated overnight.

The bacterium colony that picking is converted to is with Amp^RLB liquid medium in culture to OD_600nm=1.0；After extracting plasmid Electroresis appraisal, it was demonstrated that identical as known plasmid size.

2.3 carry the salmonella immunological evaluation of RSV recombinant proteins

2.3.1 abdominal cavity primary macrophage trial test is infected

Mouse peritoneal primary macrophage is taken, after extracing eyeball bloodletting, the neck that breaks puts to death mouse, is impregnated with 75% ethyl alcohol small Mouse；Mouse is fixed, the sterile skin for opening mouse web portion, exposure peritonaeum；By the serum free medium of 37 DEG C of water-baths of 10ml RPMI1640 is injected into the abdominal cavity of mouse along pubis forward position, ventrimeson side, not extract syringe needle, is then gently pressed with finger Rub abdomen；Sterile collection ascites, and detach macrophage.

The macrophage of separation is subjected to cell count, 24 porocyte plates of bed board, 5 × 10⁵~1 × 10⁶A holes cell/, 37 DEG C of absorption 2h in cell bottle containing RPMI culture mediums；Cell 2 is washed with the incomplete culture medium RPMI1640 of antibiotic-free It is secondary, to remove unadsorbed cell；Thalline were collected by centrifugation by 5000rpm, is resuspended with 0.01mol/L PBS (pH7.4)；It will attenuation After salmonella counting 1 is pressed with cell number:1、5:1、10:1、20:1、50:1、100:1、500:1 and 1000:1 different ratio It is added in 24 porocyte plates each ratio and repeats 3 holes, 37 DEG C of incubation 30min；It is washed carefully with 0.01mol/L PBS (pH7.4) Born of the same parents 3 times, then with the RPMI1640 culture solution culture cell 2h of the gentamicin sulphate containing 100mg/L, to kill extracellular bacteria； The tetracycline of 10mg/L, 37 DEG C of incubation 2h, to block the breeding of intracellular bacterium are added into cell culture again；Use instead again containing The RPMI1640 (containing 10%FCS) of the gentamicin sulphate of 10mg/L continues to cultivate 48-96h, and every 12h observations, whether there is or not dirts It dyes now, to determine infective dose.

Recombinant attenuated salmonella and macrophage are acted on by MOI=20,48~72h is seen with fluorescence microscope after infection Examine in cell that whether there is or not green fluorescence expression.48h and 72h, which can be seen in about 34/ cell, after infection has green glimmering Light is expressed, and without finding fluorescence in compareing.

2.3.2 recombinant attenuated salmonella In vivo infection experiment

The present embodiment carries out In vivo infection experiment, oral medication using mouse.

6-8 week old female mices are taken, are divided into 4 groups, mono- group of SL7207/pcDNA3.1-F, compare SL7207/pcDNA3.l One group, FI-RSV vaccines are as one group of vaccine positive control, and PBS groups are as negative control.30min is first with 7.5% before immune NaHCO₃In gavage and hydrochloric acid in gastric juice, 100 μ L/ only, then oral immunity recombinant bacterium (10⁸Cfu/, 200 μ L).

Respectively after oral immunity 0h, for 24 hours, 48h and 72h, extract the total serum IgE of intestinal mucosa, 2 every time.Clip small intestine Mucous membrane about 100mg is added 1.0mol TRIzol reagents, is homogenized with tissue mashing machine after shredding, 5min is incubated at 15~30 DEG C； The chloroform of 200 μ L is added, covers tightly pipe lid, acutely shakes 15s；15~30 DEG C of incubation 2-3mni；2-8 DEG C of 12000g centrifuges 15min； It shifts in water phase to a new centrifuge tube, 0.5ml isopropanols, 15i-30 DEG C of effect 10min, 2-8 DEG C of 12000g centrifugation is added 10min；Liquid is discarded supernatant, 75% ethyl alcohol is added l.0ml, shaking, 2-8 DEG C of 7500g centrifuges 5min；It discards supernatant, it is drying precipitated Object (room temperature about 5min)；20 μ L DEPC water dissolutions, -20 DEG C of preservations are added.

After extracting total serum IgE, carry out reverse transcription cDNA synthesis and PCR amplification, amplifying target genes F whether transcriptional expression. Use β-actin as internal reference when amplification.

β-actin specific primers：P β l sequences are 5 '-GTGGGCCGCTcTAGGCACCAA-3 '；2 sequences of P β are 5 '- CTCTTTGATGTCACGCACGATTTC-3’；

F protein gene：94 DEG C of pre-degenerations 3min, 35 cycle, 72 DEG C of extension 10min, it is to be measured that product sets refrigerator.

β-actin：Negate 2.0 μ L of transcription product, 48.0 μ L of PCR mixed liquors are added, including P β 1, P β 2 each 40pmol, 94 DEG C pre-degeneration 5min, 35 cycle, 72 DEG C of extension 5min, it is to be measured that product sets refrigerator.

PCR product is examined with 2% agarose gel electrophoresis, and inspection result is as shown in Figure 3.Band 1 is F protein base in figure Cause, band 2 are β-actin genes.Inspection result shows that pillar location is correctly clear, and illustration purpose gene F is correctly transcribed. 2.3.3 the expression of indirect immunofluorescence assay (IFA) detection F protein in vivo

Respectively after oral immunity for 24 hours, 48h and 72h acquisition mouse peritoneal macrophage, after counting, 96 hole of bed board is thin Born of the same parents' plate, 5 × 10⁵~1 × 10⁶A holes cell/；It washed once with PBS after incubating 2h in 37 DEG C, 5% carbon dioxide incubator, Then PBS is discarded；It is added in 150 μ L, 4 DEG C of pre- cold acetones to 96 porocyte plates, 4 DEG C of incubation 30min；Acetone is discarded, room temperature is dry It is dry；The immune mice serum 1 of the eukaryon expression plasmid of F protein:80 dilutions, each group repeat two holes；It is diluted that 50 μ L are added Serum, 37 DEG C of incubation 30min；Dilute serum is removed, 200 μ L PBS are washed 6 times；50 μ L 1 are added:100 diluted fluorescence marks Remember sheep anti-mouse igg, 37 DEG C of incubation 30min；Liquid is removed, so that cell plates is dried on paper handkerchief, is washed 4 times with PBS；In fluorescence Microscopically observation.

High pressure electrotransformation to SL7207 plants of the attenuated salmonella typhimurium that aroA is mutated is built into recombinant salmonella SL7207/pcDNA3.1-F prepares Turnover of Mouse Peritoneal Macrophages, infects SL7207/pcDNA3.1-F, can through fluorescence microscope To have detected fluorescent protein expression.By SL7207/pcDNA3.1-F oral immunity mouse, extraction intestinal mucosa is total after 2d RNA can detect the transcript and expression of target gene；The peritoneal macrophage that mouse is immunized is prepared simultaneously, is marked with FITC Sheep anti-mouse igg carry out indirect immunofluorescence assay can detect specificity yellow-green fluorescence.The result shows that the attenuation is husky Target gene can be not only presented to Mouse Somatic Cells by door Salmonella, moreover, the target gene of transhipment can obtain transcription and table It reaches.

Oral medication takes blood after a week, through mouse orbit, and separation serum is with PBS by each group mice serum from 1：10000 open Beginning doubling dilution is to 1：5120000, indirect ELISA detects antibody titer, i.e. IgG titres (geometrical mean).Testing result It is shown in Table 3.

3 mouse IgG antibody titre table of table

	Parallel 1	Parallel 2	Parallel 3	Parallel 4
					SL7207/pcDNA3.1-F	10523	14962	13328	17385
SL7207/pcDNA3.1	0	0	0	0
					FI-RSV	29618	28619	21309	24661
PBS negative controls	0	0	0	0

The Respiratory Syncytial Virus(RSV) recombinant protein used in the present invention it can be seen from above-mentioned experiment using bacterium as carrier, After in into Mice Body, mouse survival rate is normal, and stool examination is normal；The normal immunological response of mouse is excited, and passes through Mucous membrane inspection by sampling detects a certain amount of IgA antibody in the sample, and illustrating can be with the recombinant vector bacterium that is obtained in the present invention As antigen to Respiratory Syncytial Virus(RSV) carry out immune response, and can further experiment verify its validity.

3, it prepares meningococcus and intermediate is immunized

The clone of 3.1 Δ fHbp recombinant proteins and prokaryotic expression

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 entire albumen, meningococcus fHbp can be divided For 3 antigenic variant group V1~V3.In general, antibody prepared by the fHbp (also referred to as subfamily B) for being directed to variant V1 is to coming The bacterial strain that fHbp is expressed from variant V1 has bactericidal activity, but is not directed in variant V2 and V3 (also referred to as subtribe A) and expresses fHbp Bacterial strain, vice versa.The protein molecular of fHbp contains the various combination (V there are five variable domains_A~V_E), each variable region Section is derived from subtribe A and subtribe B.We devise the structure (such as Fig. 4 shows) of Δ fHbp according to its structural domain feature, it includes (C, D, E structural domain contain for the A of V1, B structure domain (epitope containing V1 on B structure domain) and C, D, E structural 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), theoretically, weight The Δ fHbp of group can cover the antigen site of all 3 variants of wild type fHbp, the potentiality with broad-spectrum antiseptic.

According to fHbp V1 and fHbp the V3 overall length CDS sequences that GeneBank is logged in, we design following 2 pairs of primers：Draw Object introduces III restriction enzyme site of BamH I and Hind respectively in the positions P1 and P4.

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 segment, P3 and the amplifiable V3 of P4 Section.Again using this two sections of genetic fragments as template, P1, P4 are primer, and it is complete to carry out bridging PCR, the amplifiable Δ fHbp recombinated 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 recycling, with DNA ligase, 16 DEG C of connections overnight, turn Change escherichia coli DH5a bacterial strain, monoclonal expands, after small upgrading grain, the about 800bp items of III double digestion of BamH I and Hind identification Band, the screening positive clone under kalamycin resistance obtain recombinant plasmid pET- Δ fHbp, PCR amplification electrophoresis pattern and identification Correct electrophoresis pattern such as Fig. 5 shows.It will identify -70 DEG C in the form of the 15% glycerol stock preservations of correct recombinant plasmid, and identification is sequenced Correctly, the amino acid sequence of Δ fHbp such as sequence table SEQ ID NO：9 show, nucleotide coding sequence such as sequence table SEQ ID NO：Shown in 10 (826bp).

SEQ ID NO：10(826bp)：

ATGAACCGAACTGCCTTCTGCTGCCTTTTCCTGACCACCGCCCTGATTCTGACCGCCT GCAGCAGCGGAGGCGGCGGAAGCGGAAGCGGCGGTGTCGCCGCCGACATCGGCACG GGGCTTGCCGATGCACTAACTACGCCGCTCGACCATAAAGACAAAGGTTTGAAATCTC TGACATTGGAAGACTCCATTCCCCAAAACGGAACACTAACCCTGTCGGCACAAGGTG CGGAAAAAACTTTCAAAGCCGGCGACAAAGACAACAGCCTCAACACGGGCAAACTG AAGAACGACAAAATCAGCCGCTTCGACTTCGTGCAAAAAATCGAAGTGGACGGACA AACCATCACGCTGGCAAGCGGCGAATTTCAAATATACAAACAGGACCACTCCGCCGTC GTTGCCCTACAGATTGAAAAAATCAACAACCCCGACAAAATCGACAGCCTGATAAAC CAACGCTCCTTCCTTGTCAGCGGTTTGGGCGGAGAACATACCGCCTTCAACCAACTGC CCGGCACGGCATTCGGTTCAGACGATGCCAGTGGAAAACTGACCTACACCATAGATTT CGCCGCCAAGCAGGGACACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGT TGACCTGGCCGCCTCCGATATCAAGCCGGATAAAAAACGCCATGCCGTCATCAGCGGT TCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCTCTAGGCATCTTTGGCGGGC AAGCCCAGGAAGTTGCCGGCAGCGCAGAAGTGGAAACCGCAAACGGCATACGCCAT ATCGGTCTTGCCGCCAAGCAGTAA

Its amino acid sequence is SEQ ID NO：9：

MNRTAFCCLFLTTALILTACSSGGGGSGSGGVAADIGTGLADALTTPLDHKDKGLKSLTLE DSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASG EFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPGTAFGSDDASG KLTYTIDFAAKQGHGKIEHLKSPELNVDLAASDIKPDKKRHAVISGSVLYNQAEKGSYSL GIFGGQAQEVAGSAEVETANGIRHIGLAAKQ

By recombinant plasmid pET- Δ fHbp Transformed E .coli BL21 (DE3), 37 DEG C are incubated overnight and (receive blueness containing 50 μ g/ml cards Mycin), Δ fHbp/BL21 (DE3) expression bacterium, picking monoclonal are obtained, 37 DEG C of shaken cultivations to strain density reach OD600 about 0.5 ~1.0, the isopropyl-β-D-thiogalactoside (IPTG) of final concentration 0.5mM is added, 37 DEG C of oscillation Fiber differentiation 2-6 are small When, it is identified through SDS-PAGE, as a result such as Fig. 6 is shown, has apparent band of expression, molecule after Δ fHbp/BL21 (DE3) inductions Amount is respectively 30kD.Δ fHbp recombinant proteins are accredited as through Western-Blot, as shown in Figure 7：1,2 swimming lane is Δ fHbp/ BL21 (DE3) induced expression object, 1,2 is detected with fHbp monoclonal antibodies, and 1 has the expection band of 30kD, as a result meets expection.Last two A recombinant protein is determined as Δ fHbp recombinant proteins through Mass Spectrometric Identification.

The small scale purification and its Efficacy evaluation of 3.2 Δ fHbp recombinant proteins

Δ fHbp/BL21 (DE3) is inoculated with LB (receiving mycin containing 50 μ g/ml cards) fluid nutrient medium, 37 DEG C, 200rpm trains Support 12 hours, with 1% inoculative proportion expand cultivate, 37 DEG C, 200rpm cultivate 3-6 hours after, OD600 to 16~20, IPTG (0.5mM) is induced 4 hours.Centrifuge collects thalline.Carrying out ultrasonic bacteria breaking, SDS-PAGE before show that recombinant protein is expressed In the form of inclusion body (Fig. 3).Successively with TE+300mM Nacl, after TE+1%Triton-100 washs inclusion body, 8000rpm It centrifuges 20min and obtains inclusion body, after washing, be dissolved in 3mol/L urea, 20mmol/L Tris-Cl, 1mmol/L EDTA, In pH4.0 solution, through CM column cation exchange chromatographies, available two destination proteins being consistent with target protein size.Most Albumen dilution refolding to be reorganized 1.2 times of ultrafilter low temperature rapid concentration to original volume, crosses anion-exchange column to after for 24 hours afterwards Q SepharoseF.F collect destination protein peak, the same Fig. 7 of detection method, and gel detection protein content reaches 95.3% or more, Purification effect is as shown in figure 8, WB detections are really purpose albumen.After PBS (pH7.4) dialysis desalinations, BCA methods survey albumen concentration Content about 0.5mg/mL.

Mouse immune：Δ fHbp recombinant proteins subcutaneous inoculation SPF grades of 6 week old NIH mouse 10 after purification are used respectively, Immunizing dose is Δ fHbp recombinant proteins 50ug/ (being dissolved in 0.2ml physiological saline) every time after purification, immune programme It is 0,2,4 weeks, takes a blood sample within 2 weeks after being immunized, serum is collected by centrifugation；Another to set 10 control mices, same procedure injects physiology salt Water.The serum ELISA method gathered surveys the titre of serum antibody, and the specific method is as follows：Egg is recombinated using Δ fHbp after purification In vain, respectively with optimal dose coated elisa plate, 37 DEG C of overnight incubations are added the mice serum to be measured being serially diluted after board-washing, and 37 DEG C be incubated after board-washing, be added horseradish peroxidase-labeled sheep anti-mouse igg secondary antibody colour developing, microplate reader measure, read 492nm (630nm is reference wavelength) absorbance value (A values).The standard deviation of+3 times of negative control group serum A mean values is Cutoff values, Test serum A values are judged to the positive more than Cutoff values, and the greatest dilution that Cutoff values are more than with A values calculates each type mouse Geometric mean titer, as mice serum IgG antibody potency (table 4).Two kinds of recombinant proteins produce height after mouse is immunized Low serum antibody.

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

Immunizing antigen	Serum IgG antibody titre (GMT)
		ΔfHbp	1:25489
Negative control (physiological saline)	0

Two methods are taken in the evaluation of recombinant protein immune effect, and one measures full cell ELISA for whole cell coating, separately One tests for the sterilizing power of epidemic strain.Whole cell coating select the MenB group bacterial strain MC58 (high express fHbp V1 can variant), 8047 (height expression fHbp V2 can variant), M1239 high expression fHbp V3 can variants), (middle amount expresses fHbp eggs to 961-5945 In vain) and 67/00 (low amounts expresses fHbp albumen) scrapes bacterium lawn, with physiology salt after these bacterial strains are expanded culture proliferation Water dissolution is diluted to 2 × 10 after formaldehyde sterilization⁸A/ml is coated with 96 hole elisa Plates, the holes 100ul/, 4 DEG C of coatings with this concentration Overnight, it after board-washing, is detected, the results are shown in Table 5.Equally, cultivate the MenB group bacterial strain MC58 (high express fHbp V1 can variant), 8047 (height expression fHbp V2 can variant), M1239 high expression fHbp V3 can variants), (middle amount expresses fHbp eggs to 961-5945 In vain) and 67/00 (low amounts expression fHbp albumen) carries out sterilization detection with corresponding serum antibody afterwards, measures and calculates bactericidin Titre (compared with complement negative control hole, the serum highest dilution of 50% or more sterilizing rate is serum antibody bactericidal titre), It the results are shown in Table 6.

5 each MenB strain whole-cells ELISA of table

Bacterial strain	Δ fHbp serum	Physiological saline serum
			MC58	979956	<200
8047	534678	<200
			M1239	997854	<200
961-5945	328940	<200
			67/00	25679	<200

6 each MenB bacterial strains sterilizing power of table is tested:BC50titer(1：)

Bacterial strain	Δ fHbp serum	Physiological saline serum
			MC58	1756	1
8047	1023	2
			M1239	1872	-2
961-5945	154	-1
			67/00	6	1

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

It can be obtained by the result comprehensive analysis of table 4 and table 5, all expression fHbp can be effectively immunized in Δ fHbp protein vaccines The MenB bacterial strains of (V1, V3 and V2 variant) infect, but the MenB bacterial strains for not expressing low expression fHbp even lose protection Ability, but since combined vaccine antigen type is more, excessive carrier should not be selected to carry out subsequent experimental, therefore select immune Protein carriers of the stronger Δ fHbp of ability as immune intermediate, without being carried out again to carrier protein because pursuing protection comprehensively It is coupled.

The preparation of 3.4 multivalence Nm polysaccharide-Δ fHbp albumen conjugate vaccines and Efficacy evaluation

3.4.1.A the preparation of group, C groups, the Y groups and W135 groups Nm bacterium capsular polysaccharides

A group meningitis coccis use 29201 bacterial strains, C group meningitis coccis to use 29205 bacterial strains, Y group meningitis coccis Using 29028 bacterial strains, W135 group meningitis coccis use 29037 bacterial strains, after the fermented culture of meningococcus, using sand culture Centrifuge, disc centrifuge or other large capacity centrifuges are separately cultured liquid, collect centrifugation supernatant；Will centrifugation supernatant with 100KD film packets are concentrated by ultrafiltration, and fractional precipitation is carried out using the ethyl alcohol of 25-80%, collect precipitation and are divided with absolute ethyl alcohol and acetone It does not wash, obtains crude polysaccharide；Sterilized water for injection dissolves polysaccharide and after NaTDC is handled, using GE fillers Capto The ion-exchange packing of adhere and Capto DEAE or other producer's identical function properties, with series connection chromatography or step chromatography Method carries out raw sugar and refines, and collection flows through peak, carries out desalination to flowing through peak using GE Sephadex G25Coarse, ethyl alcohol is heavy It forms sediment or polysaccharide is recycled in freeze-drying, be placed in -20 DEG C and save backup.

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

The present invention prepares multivalence capsular polysaccharide-Δ fHbp albumen using the technique that derivation after CDAP activated polysaccharides combines and is conjugated Vaccine.Specific method is：A/C/Y/W135 meningococcal polysaccharides are dissolved to 5-15mg/ml, adjust pH value to 10.8 Left and right, in polysaccharide solution be added 1/10 (g/g) CDAP, room temperature, maintain pH value 10.8 alkaline environment under activated polysaccharide 8min.By 1：The adipoyl hydrazine of 0.5mol/L is added in the ratio of 1 (volume ratio with activated polysaccharide), reacts at room temperature 50-70min. 50KD film packet ultrafiltration removes cyanogen bromide and adipoyl hydrazine, obtains polysaccharide derivates.By polysaccharide derivates and carrier protein Δ fHbp With 1：The ratio of 1 (g/g) is mixed and stirred for, by carbodiimide：Polysaccharide=1：Carbodiimide, room temperature, acidity is added in 10 ratio 60min is reacted under environment (pH5.6).Impurity is removed through 100KD ultrafiltration membrane packet ultrafiltration, and is concentrated.Finally use GE Sepharose 4FF carry out gel chromatography, collect the eluent near V0.With 0.22 μm of filter membrane aseptic filtration, A/ is obtained The conjugated immune intermediate stoste of C/Y/W135 meningococcal polysaccharides-Δ fHbp albumen.

This stoste is used into Aluminium phosphate adjuvant stirring and adsorbing, is diluted and is prepared with 0.15mol/L sodium chloride, until multivalence polysaccharide egg White conjugate final concentration in terms of polyoses content is respectively 20 μ g/ml, the final concentration of 0.45-0.6mg/ml of aluminium content, and stirring is equal It is even, it is dispensed with pre-filled syringe, 0.5ml/ branch, it is conjugated that A/C/Y/W135 meningococcal polysaccharides-Δ fHbp albumen is made Vaccine preparation, 2-8 DEG C of preservation.Also 80mg/ml sucrose can be added in vaccinogen liquid as excipient, every 20 μ of polyoses content Protein vaccine preparation, 2-8 DEG C of preservation are made after g/ml freeze-dryings.

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

Mouse immune：Respectively use A/C/Y/W135 groups of polysaccharide-Δ fHbp albumen conjugate vaccines albumen stoste, lyophilized preparation and Adjuvant sorbent formulation subcutaneous inoculation SPF grades of 6 week old NIH mouse 10, immunizing dose are each 0.2ml/, and immune programme is It 0,2,4 week, takes a blood sample within 2 weeks after being immunized, serum is collected by centrifugation；Another to set 10 control mices, same procedure injects physiology salt Water.The serum ELISA method gathered surveys the titre of serum antibody, and the specific method is as follows：Egg is recombinated using Δ fHbp after purification In vain, respectively with optimal dose coated elisa plate, 37 DEG C of overnight incubations are added the mice serum to be measured being serially diluted after board-washing, and 37 DEG C be incubated after board-washing, be added horseradish peroxidase-labeled sheep anti-mouse igg secondary antibody colour developing, microplate reader measure, read 492nm (630nm is reference wavelength) absorbance value (A values).The standard deviation of+3 times of negative control group serum A mean values is Cutoff values, Test serum A values are judged to the positive more than Cutoff values, and the greatest dilution that Cutoff values are more than with A values calculates each type mouse Geometric mean titer, as mice serum IgG antibody potency (table 7).After mouse is immunized in conjugate vaccines stoste and two kinds of dosage forms Produce the serum antibody of height.

The titer determination (geometrical mean) of serum antibody after mouse is immunized in 7. each group antigen of table three times

Immune object	Serum IgG antibody titre (GMT)
		Vaccinogen liquid	1:22449
Vaccine freeze-drying preparation	1:25376
		Vaccine adjuvant sorbent formulation	1:24936
Negative control (physiological saline)	0

The evaluation of each dosage form immune effect of conjugate vaccines equally takes two methods, whole cell coating to measure 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 29037 bacterial strain of coccus scrapes bacterium lawn after these bacterial strains are expanded culture proliferation, with physiological saline solution, formaldehyde sterilization Afterwards, 2 × 10 are diluted to⁸A/ml is coated with 96 hole elisa Plates, the holes 100ul/ with this concentration, and 4 DEG C of coatings overnight, after board-washing, use phase It answers vaccine serum antibody to carry out ELISA detections, the results are shown in Table 8.

8 each group meningitis cocci strain whole-cell ELISA of table

Bacterial strain	Vaccinogen liquid	Vaccine freeze-drying preparation	Vaccine adjuvant sorbent formulation	Physiological saline
					A groups 29201	1059456	1174856	1298936	<200
C groups 29205	996543	1025467	1145793	<200
					Y groups 29028	998359	1104574	1221453	<200
W groups 29037	1032579	1124694	1308945	<200
					B crowds of MC58	1125690	1231245	1324760	<200
B groups 67/00	678947	797320	895680	<200

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, 50% or more sterilizing rate Serum highest dilution be serum antibody bactericidal titre), the results are shown in Table 9.

9 each group meningitis cocci bacterial strain sterilizing power of table is tested:BC50titer(1：)

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

It can be obtained by result above comprehensive analysis, to recombinate novel multivalent meningococcal conjugates of the Δ fHbp as protein carrier There is vaccine the immune effect of wide spectrum, immune serum can be generated with the type strain of five groups of A, B, C, Y and W135 Cross-linking reaction, at the same can cover in B groups express fHbp (3 kinds of variants) and do not express fHbp but express NadA height cause a disease Bacterium, and show wide spectrum and extremely strong bactericidal effect in sterilization detects, to protect people (especially infant and old age Weakling) from spinal cord meningitis harm caused by the infringement of most Nm pathogenic bacteria.

4, the effect assessment of Respiratory Syncytial Virus(RSV)-RSV- epidemic meningitis-pneumonia combined vaccine

After the SL7207/pcDNA3.1-F recoveries of Respiratory Syncytial Virus(RSV) recombinant vector bacterium, with A/C/Y/W135 mass-brain films The conjugated immune intermediate freeze-dried powder of scorching Streptococcus polysaccharides-Δ fHbp albumen, the immune intermediate of Hib- pneumococcus, which face, uses mixing, into Row mouse immune.

SPF grades of 6 week old NIH mouse 10 are immunized in selection, and immunizing dose is each 0.2ml/, immune programme 0,2,4 It week takes a blood sample within 2 weeks after being immunized, serum is collected by centrifugation；Separately set 10 control mices, same procedure injecting normal saline.It collects Good serum ELISA method surveys the titre of serum antibody, and testing result is as shown in table 10.

Table 10

It is immune	GMT	Physiological saline
			Hib	1265	0
RSV	801	0
			A groups 29201	1354	0
C groups 29205	1246	0
			Y groups 29028	1221	0
W groups 29037	1129	0
			B crowds of MC58	1362	0
B groups 67/00	561	0
			4 type serum polysaccharide of pneumococcus	3901	0
Pneumococcus 6B type serum polysaccharide	4167	0
			Pneumococcus 9V type serum polysaccharide	5279	0
14 type serum polysaccharide of pneumococcus	3298	0
			18 type serum polysaccharide of pneumococcus	4116	0
19 type serum polysaccharide of pneumococcus	4879	0
			Pneumococcus 23F type serum polysaccharide	5238	0

As shown in Table 10, the antibody titer experiment after combined immunization be individually immunized electrodeless significant difference (with it is above-mentioned individually It is immune to compare, P<0.005, statistics positive control data is shown in each intermediate volume data in above-mentioned preparation method.), it can effectively carry out Hib, RSV and the protection of the Multiple immunizations of epidemic meningitis, but immune effect relative reduction, it is contemplated that repeatedly immune to have ensured effectively to be immunized.

It is it is necessary to described herein finally：Above example is served only for making technical scheme of the present invention further detailed Ground illustrates, should not be understood as limiting the scope of the invention, those skilled in the art's the above according to the present invention Some the nonessential modifications and adaptations made all belong to the scope of protection of the present invention.

Sequence table

<110>The Wuhan bio tech ltd Bo Wo

<120>Anti- Hib-RSV- meningococcus-pneumococcus combined vaccine

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 31

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 1

ggaattccat atggcaaata aagcagtaaa t 31

<210> 2

<211> 29

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 2

cgagctcgtc attttctacc ttatcctct 29

<210> 3

<211> 32

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

cggcggccgc atggcaaata aagcagtaaa tg 32

<210> 4

<211> 36

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

ccctcgagtt actagtcatt ttctacctta tcctct 36

<210> 5

<211> 1725

<212> DNA

<213> respiratory syncytial virus

<400> 5

atggagctgc tgatccacag gttaagtgca atcttcctaa ctcttgctat taatgcattg 60

tacctcacct caagtcagaa cataactgag gagttttacc aatcgacatg tagtgcagtt 120

agcagaggtt attttagtgc tttaagaaca ggttggtata ccagtgtcat aacaatagaa 180

ttaagtaata taaaagaaac caaatgcaat ggaactgaca ctaaagtaaa acttataaaa 240

caagaattag ataagtataa gaatgcagtg acagaattac agctacttat gcaaaacaca 300

ccagctgcca acaaccgggc cagaagagaa gcaccacagt atatgaacta tacaatcaat 360

accactaaaa acctaaatgt atcaataagc aagaagagga aacgaagatt tctgggcttc 420

ttgttaggtg taggatctgc aatagcaagt ggtatagctg tatccaaagt tctacacctt 480

gaaggagaag tgaacaagat caaaaatgct ttgttatcta caaacaaagc tgtagtcagt 540

ctatcaaatg gggtcagtgt tttaaccagc aaagtgttag atctcaagaa ttacataaat 600

aaccaattat tacccatagt aaatcaacag agctgtcgca tctccaacat tgaaacagtt 660

atagaattcc agcagaagaa cagcagattg ttggaaatca acagagaatt cagtgtcaat 720

gcaggtgtaa caacaccttt aagcacttac atgttaacaa acagtgagtt actatcattg 780

atcaatgata tgcctataac aaatgatcag aaaaaattaa tgtcaagcaa tgttcagata 840

gtaaggcaac aaagttattc tatcatgtct ataataaagg aagaagtcct tgcatatgtt 900

gtacagctac ctatctatgg tgtaatagat acaccttgct ggaaattaca cacatcacct 960

ctatgcacca ccaacatcaa agaaggatca aatatttgtt taacaaggac tgatagagga 1020

tggtattgtg ataatgcagg atcagtatcc ttctttccac aggctgacac ttgtaaagta 1080

cagtccaatc gagtattttg tgacactatg aacagtttga cattaccaag tgaagtcagc 1140

ctttgtaaca ctgacatatt caattccaag tatgactgca aaattatgac atcaaaaaca 1200

gacataagca gctcagtaat tacttctctt ggagctatag tgtcatgcta tggtaaaact 1260

aaatgcactg catccaacaa aaatcgtggg attataaaga cattttctaa tggttgtgac 1320

tatgtgtcaa acaaaggagt agatactgtg tcagtgggca acactttata ctatgtaaac 1380

aagctggaag gcaagaacct ttatgtaaaa ggggaaccta taataaatta ctatgaccct 1440

ctagtgtttc cttctgatga gtttgatgca tcaatatctc aagtcaatga aaaaatcaat 1500

caaagtttag cttttattcg tagatctgat gaattactac ataatgtaaa tactggcaaa 1560

tctactacaa atattatgat aactacaatt attatagtaa tcattgtagt attgttatca 1620

ttaatagcta ttggtttgct gttgtattgc aaagccaaaa acacaccagt tacactaagc 1680

aaagaccaac taagtggaat caataatatt gcattcagca aatag 1725

<210> 6

<211> 31

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 6

cccaagcttc agaaaaccgt gacctatcaa g 31

<210> 7

<211> 32

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 7

ccctcgagac atgaagtttt gcctcactag ta 32

<210> 8

<211> 2306

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 8

cttagttatt caaaaactac atcttagcag aaaaccgtga cctatcaagc aagaacgaaa 60

ttaaacctgg ggcaaataac catggagctg ctgatccaca ggttaagtgc aatcttccta 120

actcttgcta ttaatgcatt gtacctcacc tcaagtcaga acataactga ggagttttac 180

caatcgacat gtagtgcagt tagcagaggt tattttagtg ctttaagaac aggttggtat 240

accagtgtca taacaataga attaagtaat ataaaagaaa ccaaatgcaa tggaactgac 300

actaaagtaa aacttataaa acaagaatta gataagtata agaatgcagt gacagaatta 360

cagctactta tgcaaaacac accagctgcc aacaaccggg ccagaagaga agcaccacag 420

tatatgaact atacaatcaa taccactaaa aacctaaatg tatcaataag caagaagagg 480

aaacgaagat ttctgggctt cttgttaggt gtaggatctg caatagcaag tggtatagct 540

gtatccaaag ttctacacct tgaaggagaa gtgaacaaga tcaaaaatgc tttgttatct 600

acaaacaaag ctgtagtcag tctatcaaat ggggtcagtg ttttaaccag caaagtgtta 660

gatctcaaga attacataaa taaccaatta ttacccatag taaatcaaca gagctgtcgc 720

atctccaaca ttgaaacagt tatagaattc cagcagaaga acagcagatt gttggaaatc 780

aacagagaat tcagtgtcaa tgcaggtgta acaacacctt taagcactta catgttaaca 840

aacagtgagt tactatcatt gatcaatgat atgcctataa caaatgatca gaaaaaatta 900

atgtcaagca atgttcagat agtaaggcaa caaagttatt ctatcatgtc tataataaag 960

gaagaagtcc ttgcatatgt tgtacagcta cctatctatg gtgtaataga tacaccttgc 1020

tggaaattac acacatcacc tctatgcacc accaacatca aagaaggatc aaatatttgt 1080

ttaacaagga ctgatagagg atggtattgt gataatgcag gatcagtatc cttctttcca 1140

caggctgaca cttgtaaagt acagtccaat cgagtatttt gtgacactat gaacagtttg 1200

acattaccaa gtgaagtcag cctttgtaac actgacatat tcaattccaa gtatgactgc 1260

aaaattatga catcaaaaac agacataagc agctcagtaa ttacttctct tggagctata 1320

gtgtcatgct atggtaaaac taaatgcact gcatccaaca aaaatcgtgg gattataaag 1380

acattttcta atggttgtga ctatgtgtca aacaaaggag tagatactgt gtcagtgggc 1440

aacactttat actatgtaaa caagctggaa ggcaagaacc tttatgtaaa aggggaacct 1500

ataataaatt actatgaccc tctagtgttt ccttctgatg agtttgatgc atcaatatct 1560

caagtcaatg aaaaaatcaa tcaaagttta gcttttattc gtagatctga tgaattacta 1620

cataatgtaa atactggcaa atctactaca aatattatga taactacaat tattatagta 1680

atcattgtag tattgttatc attaatagct attggtttgc tgttgtattg caaagccaaa 1740

aacacaccag ttacactaag caaagaccaa ctaagtggaa tcaataatat tgcattcagc 1800

aaatagacaa aaaaccacct gatcatgttt caacaacagt ctgctgatca ccaatcccaa 1860

atcaacccat aacaaacact tcaacatcac agtacaggct gaatcatttc ttcacatcat 1920

gctacccaca caactaagct agatccttaa ctcatagtta cataaaaacc tcaagtatca 1980

caatcaaaca ctaaatcaac acatcattca caaaattaac agctggggca aatatgtcgc 2040

gaagaaatcc ttgtaaattt gagattagag gtcattgctt gaatggtaga agatgtcact 2100

acagtcataa ttactttgaa tggcctcctc atgccttact agtgaggcaa aacttcatgt 2160

taaacaagat actcaagtca atggacaaaa gcatagacac tttgtctgaa ataagtggag 2220

ctgctgaact ggacagaaca gaagaatatg ctcttggtat agttggagtg ctagagagtt 2280

acataggatc tataaacaac ataaca 2306

<210> 9

<211> 274

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 9

Met Ala Ala Thr Ala Pro Cys Cys Leu Pro 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 Ala Ile Gly Thr Gly Leu Ala Ala Ala Leu Thr Thr Pro Leu

35 40 45

Ala His Leu Ala Leu Gly Leu Leu Ser Leu Thr Leu Gly Ala Ser Ile

50 55 60

Pro Gly Ala Gly Thr Leu Thr Leu Ser Ala Gly Gly Ala Gly Leu Thr

65 70 75 80

Pro Leu Ala Gly Ala Leu Ala Ala Ser Leu Ala Thr Gly Leu Leu Leu

85 90 95

Ala Ala Leu Ile Ser Ala Pro Ala Pro Val Gly Leu Ile Gly Val Ala

100 105 110

Gly Gly Thr Ile Thr Leu Ala Ser Gly Gly Pro Gly Ile Thr Leu Gly

115 120 125

Ala His Ser Ala Val Val Ala Leu Gly Ile Gly Leu Ile Ala Ala Pro

130 135 140

Ala Leu Ile Ala Ser Leu Ile Ala Gly Ala Ser Pro Leu Val Ser Gly

145 150 155 160

Leu Gly Gly Gly His Thr Ala Pro Ala Gly Leu Pro Gly Thr Ala Pro

165 170 175

Gly Ser Ala Ala Ala Ser Gly Leu Leu Thr Thr Thr Ile Ala Pro Ala

180 185 190

Ala Leu Gly Gly His Gly Leu Ile Gly His Leu Leu Ser Pro Gly Leu

195 200 205

Ala Val Ala Leu Ala Ala Ser Ala Ile Leu Pro Ala Leu Leu Ala His

210 215 220

Ala Val Ile Ser Gly Ser Val Leu Thr Ala Gly Ala Gly Leu Gly Ser

225 230 235 240

Thr Ser Leu Gly Ile Pro Gly Gly Gly Ala Gly Gly Val Ala Gly Ser

245 250 255

Ala Gly Val Gly Thr Ala Ala Gly Ile Ala His Ile Gly Leu Ala Ala

260 265 270

Leu Gly

<210> 10

<211> 825

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 10

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

Claims (10)

1. a kind of anti-Hib-RSV- meningococcus-pneumococcus combined vaccine, which is characterized in that the combined vaccine includes：

A) intermediate is immunized in Hib- pneumococcus, and it is conservative that the height that intermediate is expressed with pneumococcus is immunized in the Hib- pneumococcus Property and the albumen with immunogenicity be carrier protein, Hib capsular polysaccharides are conjugated；

B) intermediate is immunized in meningococcus, and it includes Δ fHbp that intermediate, which is immunized, in the meningococcus, the recombination Δ fHbp Including fHbp can variant V1 VA, VB structural domain and fHbp can variant V3 VC, VD, VE structural domain；

C) intermediate is immunized in Respiratory Syncytial Virus(RSV), and the antigen that intermediate is immunized in the Respiratory Syncytial Virus(RSV) is that can cause body The RSV film surfaces fusion protein F and/or attachment protein G of immune response, wherein the nucleic acid sequence of expression proteantigen is binned in core On acid vectors, the nucleic acid sequence of recombinant protein is to be attenuated intracellular bacteria as bacteria carrier；

The immune intermediate of said components is prepared as freeze dried powder respectively, faces with melting mixing again.

2. anti-Hib-RSV- meningococcus-pneumococcus combined vaccine according to claim 1, it is characterised in that：Institute It further includes the pneumococcal capsular polysaccharide being conjugated with carrier protein to state Hib- pneumococcus and intermediate is immunized.

3. anti-Hib-RSV- meningococcus-pneumococcus combined vaccine according to claim 1, it is characterised in that：Lung The one kind or more of scorching coccus capsular polysaccharide in following serotype：1、2、3、4、5、6B、7F、8、9N、9V、10A、11A、 12F, 14,15B, 17F, 18C, 19F, 19A, 20,22F, 23F and/or 33F.

4. anti-Hib-RSV- meningococcus-pneumococcus combined vaccine according to claim 1, which is characterized in that lung Scorching coccus carrier protein includes：Pneumococcus dissolved blood protein and its modification derivant, pneumococcal surface protein and its modification are spread out Biology, three histidine protein family fusion protein of pneumonia ball surface adhesion albumen and its modification derivant or pneumococcus.

5. anti-Hib-RSV- meningococcus-pneumococcus combined vaccine according to claim 1, which is characterized in that lung Scorching coccus carrier protein is specially：Modified pneumococcus dissolved blood protein △ A146Ply, pneumococcus dissolved blood protein derivative DPly, three histidine protein PhtA, PhtB of Pneumococal surface protein A, Pneumococcal Surface adhesion protein A or pneumococcus, PhtD、PhtE、PhtDE。

6. anti-Hib-RSV- meningococcus-pneumococcus combined vaccine according to claim 1, it is characterised in that：RSV Albumen is F protein, and specific primer, primer sequence such as sequence table SEQ ID NO are designed according to the nucleic acid sequence of expression albumen：6 With SEQ ID NO：Shown in 7.

7. anti-Hib-RSV- meningococcus-pneumococcus combined vaccine according to claim 1, it is characterised in that：Weight The rsv protein antigen of group is pcDNA3.1-F.

8. anti-Hib-RSV- meningococcus-pneumococcus combined vaccine according to claim 1, it is characterised in that：Subtract Bacteria carrier is selected from toxicyst：L3261, SL7207 or ty21a.

9. anti-Hib-RSV- meningococcus-pneumococcus combined vaccine according to claim 1, it is characterised in that：Turn Dye to the RSV antigens in bacteria carrier are SL7207/pcDNA3.1-F.

10. anti-Hib-RSV- meningococcus-pneumococcus combined vaccine according to claim 1, it is characterised in that：Institute State the amino acid sequence such as sequence table SEQ ID NO of recombination Δ fHbp：Shown in 9, nucleotide coding sequence such as sequence table SEQ ID NO：Shown in 10.