CN107961371A - Season influenza-RSV combined vaccine and its preparation method and application - Google Patents

Abstract

The invention discloses a kind of season influenza RSV combined vaccines and its preparation method and application, including：Season influenza virus protein and the fusion protein and immunologic adjuvant of Respiratory Syncytial Virus(RSV) (RSV).The fusion protein is selected from the well-conserved protein sequence of season influenza virus and Respiratory Syncytial Virus(RSV), and the fusion protein and immunologic adjuvant of structure are with (0.5~2)：1 ratio mixing, obtains season influenza RSV combined vaccine.The recombination fusion protein that the present invention uses is as novel antigens, during oral nasal administration, causes the IgG in serum and the increase of mucosal system IgA antibody titre.Reacted it is an advantage of the invention that causing for the co-immunization of season influenza and RSV viruses, coordinate mucosal adjuvant, cause mucosa-immune, more enhance immune effect.

Description

Season influenza-RSV combined vaccine and its preparation method and application

Technical field

The present invention relates to a kind of season influenza-RSV combined vaccines and its preparation method and application, belong to biotechnology neck Domain.

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, mucosa-immune

Mucosal immune system is distributed widely under respiratory tract, intestines and stomach, urogenital mucous membrane and at some exocrine glands Lymphoid tissue, be the main place for performing local specific immune function.Body there are about 95% bacterium, virus and parasite Infection all originate in mucous membrane surface.Mucosal immune system is first of immunization barrier of body resistance pathogen invasion, is had The independent immune system of unique texture and function, has positive effect for the field planting and intrusion for preventing pathogen.It is different from biography The immune system of system, mucosal immune system are a large amount of immunocyte and immune molecule disperses lamina proprias under mucosal epithelium or mucous membrane (diffused lymphoid tissue), or the mucosa-associated lymphoid tissue being gathered into by single or multiple lymph follicles, body more than 50% Lymphoid tissue and more than 80% immunocyte concentrate on mucosal immune system.Mucosa-immune can induce local mucous membrane generation point The protection antibodies such as secreting property IgA (sIgA), IgM and IgG, and the mucous membrane that can induce other positions also produces sIgA, this is mucous membrane The main mechanism of immanoprotection action.In addition, mucosa-immune also induces mucous membrane ctl response, and produce the CD4 of secretion IFN-C⁺ T cell, this is very important for the prevention and removing of pathogen intrusion.Therefore mucosa-immune is protection body from cause of disease The important barrier that body is invaded, is of great significance in the design of vaccine.

Based on the vaccine of mucosa-immune since the immune of induction often reacts weaker, the duration is short, it is difficult to obtains preferable Immune protective effect.It is now recognized that such as less immunogenic of recombinant protein, synthesis polypeptide and DNA antigens is important original One of because, it is therefore desirable to try to improve the intensity of immune response, and also have some vaccines to need to change immune response type, with Prominent mucosa-immune etc..The problem of these aspects, makes the use of adjuvant seem particularly urgent and important, therefore for mucosa-immune The research of adjuvant has become a research hotspot of infection immunity and vaccines arts.At present, the Mucosal Adjuvants reported It can be divided mainly into three classes：The first kind is bacterial substances, including albumen (mainly bacteriotoxin) and nucleic acid；Second class is various Cell factor；Three classes are antigen delivery systems.

Most-often used as Mucosal Adjuvants in bacteriotoxin is E.coli LT (LT) and cholera Toxin (CT), people have been carried out it more research.It is close connection toxin (zonula occludens toxin, Zot), thin Cellular toxicity necrosin (cytotoxic necrotizing factor 1, CNF1) and dermotoxin (dermonecrotic toxin, DNT) etc. be report recently there is the bacteriotoxin of mucosal adjuvant.LT and CT are Bacteriotoxin, its nucleotide sequence homology about 80%, structure is also essentially identical.The special CD4 of the main inducing antigens of CT⁺Th2 Type cell, and LT then can induce the CD4 of mixing⁺Th1 and Th2 type cells.CT and LT is strong Mucosal Adjuvants, but by Its application in vaccine for man field is hindered with toxicity in it, therefore is built and is removed toxicity or reduce toxicity, is retained at the same time The mutant of adjuvant attribute is very necessary.

LT is one of certified most effective mucosa-immune original in humans and animals experiment so far.It can effectively be situated between CD4 of the guide pin to LT⁺T cell and B cell reaction.Through alimentary canal approach (oral or stomach in approach) injection LT to mouse, Hypersecretion and systemic antibody response are induced, substantial amounts of anti-LT can be found in Respiratory Tract of Mice secretion and small intestine contents IgA antibody.Take small intestinal mucosa to cut into slices, substantial amounts of thick liquid cell can be seen in mucosa lamina propria aggregate nodules.LT inductions stick Film secretory antibody response it is most strong be antigen deposition site, but be not limited to antigen deposition site, imitated in other mucous membranes Position is answered also to have same response.

LT and LTB has good immunogenicity, and LTB, which contains, causes most of advantage of T specific antibody responses to resist Former epitope, both of which can effectively start the T cell and B cell immune response of body generation locally and systemically, moreover it is possible to make T, B thin Born of the same parents produce long-term anamnestic response.

3rd, season influenza and its epidemiology

Influenza virus is orthomyxoviridae family, is segmented minus-stranded rna virus.According to the nucleoprotein (NP) and matrix egg of virus (M) antigenicity is different in vain, and influenza virus is divided into first/A types, second/Type B, third/c-type.Flu-A has height variation, Su Zhufan The features such as extensive is enclosed, the threat to public health is maximum.Different according to the hemagglutinin (HA) of virus surface, influenza A virus can It is divided into H1-16 hypotypes, according to neuraminidase NA differences, is divided into N1-9 hypotypes.Flu-A popular in crowd disease at present Poison mainly has H1, H2, H3 and N1, N2 hypotypes, and novel influenza popular in crowd has 2009H1N1, H7N9 fowl in recent years Influenza virus, the former causes flu outbreak, and the lethality of the latter is up to 27.2%.

Influenza A and Type B influenza virus are all containing 8 segmented negative sense single stranded RNAs.Influenza A gene Group at least encodes 11 polypeptides.Genetic fragment 1-3 encodes 3 polypeptides, the RNA polymerase that composition viral RNA relies on.Fragment 1 is compiled Code polymerase complex protein PB2.Other polymerase protein PB1 and PA is encoded by fragment 2 and fragment 3 respectively.It is in addition, some The fragment 1 of influenza A strain also encodes a little albumen PB1-F2, is produced by another reading frame in PB1 code areas Raw.Fragment 4 encodes hemagglutinin (HA) surface glycoprotein, participates in cell adherence and virus is entered cell in infection period. Fragment 5 encodes nucleocapsid nucleoprotein (NP) polypeptide, this is a kind of major structural protein being connected with viral RNA.Fragment 6 encodes god Through amidase (NA) envelope glycoprotein.Fragment 7 encodes two kinds of stromatins, is referred to as M1 and M2, is by through different modes montage MRNA translation.Fragment 8 encodes NS1 and NS2 (NEP), this is two kinds of non-structural proteins, by the mRNA through other modes montage Translation.

HA, NA glycoprotein play an important role in virus infection, reproduction process, and important antigen, can stimulate body Produce neutrality protection antibody.HA is present in virus envelope surface in the form of homotrimer, is identified by antibody in host Major antigen, it influenza virus invade before identification host cell receptor and it is in connection in favor of virus absorption and wear film, HA precursors just have infectivity by host protein enzymatic lysis, influenza virus.Anti- HA antibody is that have currently used for assessment influenza vaccines The important indicator of effect property, although anti-NA antibody can mitigate clinical symptoms, the previously color producing reaction based on β acetonformaldehydes acid, by In, suitable high-volume examination poisonous, cumbersome using reagent, thus anti-NA antibody tests are not widely popularized.Nineteen ninety, Enzyme chain immunodetection of the Claude R.Lambr é reports based on the detection of arachidonic acid combination galactolipin group, is subsequently found Anti- HA antibody as and Virus Interaction, which can also influence the combination with NA in conformation, so that it is false positive anti-NA antibody occur Property.2009, SandbulteMR research teams were used for anti-NA antibody using the virus-like particle (VLP) for containing only influenza virus NA Detection, this method patent applied for, because the VLP yield of preparation is limited, may also have baculoviral, VLP technologies are lacked in system The laboratory of weary grasp or vaccine producer are difficult to carry out.

Vaccine inoculation is the most effective mode that flu-prevention occurs and propagates.The influenza vaccines of existing market application mainly have Three kinds：Inactivated virus vaccine, split vaccine, subunit vaccine.The influenza vaccines applied at present it is efficient 60%~ Between 85%, its major antigen composition is influenza virus memebrane protein HA and NA.These vaccines infect same subtype influenza virus pre- It is anti-effective, but the protecting effect between different subtype virus is weaker.In addition, the speed of mutation of influenza virus is very fast, influenza Vaccine will be replaced every year, and prepared by the production to vaccine causes inconvenience.The World Health Organization is according to the whole world then Next year influenza vaccines production component is predicted and recommended to scope influenza virus situation of change, and the accuracy of prediction will be direct The protective efficacy of vaccine is influenced, as prediction of failure will cause the potential threat of flu outbreak prevalence.Therefore, developing one kind has The universal vaccine of extensive protective effect establishes rapidly community immunity screen for the unexpected outburst after reply influenza virus mutant Barrier, blocks flu outbreak sprawling, has extremely important practical significance in terms of reducing its harmfulness.

FLUMist is a kind of attenuated live vaccine, and children and adult can be protected not to suffer from influenza.FLUMist vaccine strains, which contain, to be come Come from HA the and NA genetic fragments of wild-type strain currently popular and from common main donor virus (common Masterdonor virus) (MDV) six gene segments：PB1, PB2, PA, NP, M and NS.The A types influenza disease of FLUMist The MDV (MDV-A) of strain be continuously reduce temperature under conditions of in primary chicken nephridial tissue culture by wild type A/Ann Obtained from the serial passage of Arbor/6/60 Strain (A/AA/6/60).MDV-A can effectively be replicated at 25 DEG C (ca, it is cold suitable Should), but its growth is suppressed (ts, temperature sensitive) in 38 DEG C and 39 DEG C.In addition, this virus is in infected ferret intrapulmonary Not reproducible (att, attenuation).This temperature sensitive phenotype, which is believed to be, to be limited its position in human respiratory outside most cold-zone domain and answers The reason for making and causing its toxicity to weaken.Animal model test and clinical test show that this characteristic is quite stable.With leading to This ts phenotypes difference of the strains of influenza viruses of mutagenesis preparation is crossed, the ts characteristics of MDV-A in the hamster of infection by passing This characteristic of generation or the separation strains through passage isolated from children will not reduce.

After influenza virus is normally due to touch the nasal cavity and respiratory mucosa of host, into host cell, virus is completed The duplication and transcription of genome.Therefore the first line of defence as human immunity, the timely activation of mucosa-immune are flowed for prevention The diseases such as sense have important practical significance.

4th, Respiratory Syncytial Virus(RSV) and its epidemiology

Respiratory infectious disease is still to cause one of main causes of 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.At present, the existing safely and effectively different type vaccine of influenza, guarantee is provided for the prevention and control of influenza. And RSV there is no effective vaccine, challenge proposed for its prevention and control due to autoimmune properties.RSV is to cause infant's lower respiratory tract Most important cause of disease is infected, and causes the elderly and immune deficiency to be grown up and is in hospital and the major reason because of pneumonia death.According to system Count, the infant within 6 months causes to be even as high as up to the childhood infection rate within 70%, 2 one full year of life in hospital because of rsv infection 99%.For RSV because its scope of causing a disease is wide, the state of an illness is occurred frequently, and can cause serious complication etc., is made safely to mankind's health and lives Into serious threat.RSV vaccines are set to one of 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 formed, wherein fusion protein F (Fusion protein, F) and attachment Protein G (Attchment protein, G) is that RSV excitating organisms produce the most important virus protein of protection antibody.G-protein is high Difference is spent, two hypotypes of A and B can be divided into according to G-protein antigenic specificity RSV；The F protein of RSV is highly conserved, mediate retroviral bag The fusion of film and host cell membrane so that virus successfully invades host cell and can also cause the fusion between flanking cell plasma membrane 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 The virulence that F and G-protein have been acknowledged as RSV is caused a disease molecule and protective antigens.

For RSV, in the 1960s, the formalin-inactivated vaccine (FI-RSV) of the development such as Fulginiti VA Too drastic cause 2 death of child due to inducing Th2 types and being immunized and 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 can use so far without granted RSV vaccines.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 produce Give birth to effective mucous membrane and cell immune response and immune protective effect is limited, there are potential security and total length F, G for DNA vaccination There are the bottleneck problems such as potential Th1/Th2 loss of equilibrium are urgently to be resolved hurrily for protein vaccine.In recent years, with influenza virus reverse genetics Technology day is also ripe and the multiple protein carrier such as influenza vectors it is increasingly ripe, using carrier protein as delivery system successfully The RSV vaccine candidates strain of research and development can produce Double immune protecting effect, and safe, easily operated, have wide hair Exhibition prospect, is expected to provide new thinking for RSV vaccine researches.The beautiful pearl of pa of MedImmune companies of U.S. development and production (palivizumab) mainly for 263-275 amino acids in RSVA type F protein epitopes II, as global first spy The opposite sex is directed to the Humanized monoclonal antibodies of RSV F protein neutralizing epitopes, and the beautiful pearl of pa confirms that inoculation crowd can be significantly reduced Natural infection rate, plays a significant role during the prevention of RSV and early intervention.Ratified by FDA at present, successfully on City, takes the lead in finishing in the world RSV without Miao Kefang, the situation to past medical help.

Influenza virus since its antigenic (referring to HA and NA) continuously morphs, at this two classes antigen it is hand-made Although standby influenza vaccines technology of preparing is more mature, high using degree, it prepares speed far away from the viral change friction speed of itself Degree so that the immune range of existing influenza all-virus inactivated vaccine in use is very limited, it is difficult to which the new variation of covering produces Influenza virus.Nucleocapsid protein (NP) and stromatin (M) are conservative structural proteins in influenza virus, in virus evolution mistake Aberration rate is very low in journey, therefore has the feasibility of the research and development permanent vaccine of influenza in theory.

Applicant further study show that, the coating and capsid protein of the virus surface of Respiratory Syncytial Virus(RSV) (RSV) (M2) also there is higher conservative, vaccine is prepared based on highly conserved albumen, can solve to exempt from existing RSV natural immunities Epidemic disease memory time section, antigen are easy to be denatured, and cause the technical problem of patient's superinfection.

Easily trigger respiratory disease after influenza infection, clinically RSV takes place frequently with influenza mixed infection at present.Therefore Research and develop it is a kind of can at the same time flu immunization virus and Respiratory Syncytial Virus(RSV) vaccine, can not only reduce inoculator economy and Body burden, and can prevent in time because of influenza virus infection and caused by respiratory disease.

The content of the invention

In view of the above-mentioned problems existing in the prior art, the object of the present invention is to provide a kind of season influenza-RSV combined vaccines And its preparation method and application.

For achieving the above object, the technical solution that season influenza-RSV combined vaccine of the invention uses is as follows：

A kind of season influenza-RSV combined vaccines, including：Season influenza virus protein and Respiratory Syncytial Virus(RSV) (RSV) Fusion protein and immunologic adjuvant.The fusion protein is selected from the well-conserved albumen of season influenza virus and Respiratory Syncytial Virus(RSV) Sequence, the fusion protein and immunologic adjuvant of structure are with (0.5~2)：1 ratio mixing, obtains season influenza-RSV combined vaccine.

Preferably, season influenza virus protein is selected from for Flu-A structural proteins：PBI、PBZ、PA、HA、NA、NP、ML And/or M.

Preferably, Respiratory Syncytial Virus(RSV) albumen is selected from least one of following albumen：Transmembrane protein G, F, SH；Matrix Albumen M, M2；Nucleocapsid protein N, P, L.

It is highly preferred that fusion protein combines albumen for M2-1-NP, the nucleotide sequence such as sequence of M2-1-NP fusion proteins is encoded List SEQ ID NO：Shown in 9, its amino acid sequence such as sequence table SEQ ID NO encoded：Shown in 10.

Further, the nucleotide sequence for encoding M2-1-NP fusion proteins is NP nucleotide sequences and M2-1 nucleotide sequences, NP cores Acid sequence such as sequence table SEQ ID NO：Shown in 1, its amino acid sequence such as sequence table SEQ ID NO encoded：Shown in 2；M2-1 Nucleotide sequence such as sequence table SEQ ID NO：Shown in 3, its amino acid sequence such as sequence table SEQ ID NO encoded：Shown in 4.

Two pairs of primer sequences, the wherein primer sequence of NP nucleic acid are separately designed according to NP nucleotide sequences and M2-1 nucleotide sequences Such as sequence table SEQ ID NO：Shown in 5 and 6, the primer sequence table such as sequence table SEQ ID NO of M2-1 nucleic acid：Shown in 7 and 8.

Preferably, adjuvant includes sero-immunity adjuvant and Mucosal Adjuvants.

It is highly preferred that sero-immunity adjuvant includes freund adjuvant；Mucosal Adjuvants include CT adjuvants.It is any can be with egg White vaccine combination is considered as falling under the scope of the present invention using the adjuvant for playing enhancing immune effect.

The mixed proportion of joint albumen and adjuvant is 1：1.

The present invention another goal of the invention be to provide a kind of preparation method of season influenza-RSV combined vaccines, its In：

s1：Kit is rapidly purified using viral RNA/DNA and extracts RSV and Influenza Virus RNA respectively, by what is extracted RNA carries out reverse transcription (RT), NP and M2 gene magnifications are carried out by template of the cDNA products of reverse transcription；

s2：Design primer expands to obtain M2-1 genetic fragments, NP genetic fragments respectively, then using this two sections of genetic fragments as mould Plate carries out the M2-1-NP full length fragments that PCR amplification is recombinated, and recycles M2-1-NP genetic fragments and pGEX-2T carriers, prepares Recombinant plasmid pGEX-2T-M2-1-NP；

s3：Recombinant plasmid pGEX-2T-M2-1-NP translation tables are reached into bacterial strain, obtain recombination fusion protein M2-1-NP.

Preferably, the restriction enzyme site of M2-1 is I restriction enzyme sites of EcoR, and the restriction enzyme site of NP bases is I restriction enzyme sites of Xho.

Preferably, the bacterial strain that recombinant plasmid pGEX-2T-M2-1-NP translation tables reach is e. coli strain bl21.

The 3rd goal of the invention of the present invention is to protect a kind of season influenza-RSV combined vaccines preventing or treating season Application in the medicine for the disease that throttling sense and Respiratory Syncytial Virus(RSV) induce.

Compared with prior art, the present invention uses the fusion protein of restructuring as novel antigens, while causes inoculator to produce Life is directed to two kinds of viral immune responses.Coordinate mucosal adjuvant, during oral nasal administration, this vaccine can cause mucosa-immune, enhancing Immune effect；And trigger the raising of the IgG and schneiderian membrance system IgA antibody titre in serum at the same time.And this vaccine construct is steady Fixed, adverse reaction is few, and one time inoculating two kinds are immunized, vaccination ways simple and effective, and body and the economy for significantly reducing patient are negative Load.

It is of the invention main by the use of comma bacillus (CT) as a kind of effective Mucosal Adjuvants, CT by with cell surface GM1 combine, the immune response effect of body fight original can not only be strengthened, additionally it is possible to change the type of mucosal immune response, bag Include the expression for the cell factor for promoting Th1 and Th2 type cell effects, CD8⁺The apoptosis and CD4 of T cell⁺The propagation of T cell；Make B MHC II of cell activation, the increase of ICAM-1, CD25, CD40 developed by molecule, influence B cell and macrophage antigen processing and Presentation pathway.

The fusion protein that the present invention uses, can not only cause for two kinds of viral immune responses at the same time, due to selection Albumen be the highly conserved albumen of virus, therefore easily cause the immune response of full hypotype for a long time after being inoculated with, carry significantly High immune effect, for the virus of the height variation such as influenza, causes the permanent vaccine of full hypotype immune response, really does Sick preceding prevention has been arrived, inoculation crowd and inoculation scope has not only been expanded, extends inoculation time, there is good popularization and economy Value, also provides a new research direction for vaccine research staff.

Brief description of the drawings

Fig. 1 is M2-1-NP fusion proteins gel electrophoresis figure provided by the invention.

Fig. 2 is the M2-1-NP fusion protein Ss DS-PAGE figures of the offer of the present invention.

Fig. 3 is M2-1-NP fusion proteins ELISA antiserum titre figures provided by the invention.

Embodiment

The present invention is made with reference to embodiment further in detail, intactly to illustrate.The embodiments described below is example Property, it is only used for explaining the present invention, and be 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.

RSVM2 genes are between F and L genes, containing two open reading frames, i.e. M2-1 and M2-2.M2-1 genes are complete 826 nucleotide are about, encode 195 amino acid, positioned at 5 ' ends of M2 genes, its 3 ' end is overlapping with M2-2 gene head ends, M2-l is RSV peploses and capsid composition, and viral capsid is formed together with N, P albumen.

First, the clone of M2-1-NP fusion proteins and prokaryotic expression

NP nucleotide sequences (the Gene of A/California/07/2009 (H1N1) influenza virus recorded in Genebank ID:23308125) CDS areas complete sequence, sequence such as sequence table SEQ ID NO：Shown in 1, its amino acid sequence such as SEQ encoded ID NO：Shown in 2.Nucleotide sequence (the Gene ID of M2-1 albumen are encoded in RSV:1489826) such as sequence table SEQ ID NO：3 institutes Show, its amino acid sequence such as SEQ ID NO encoded：Shown in 4.

According to M2CDS sequences (Gene ID in the full nucleotide sequences of the RSV recorded in Genebank:1489826), design is drawn Thing is as follows：

M2-1-Fwd is followed successively by by 5 ' ends to 3 ' ends：I restriction enzyme sites of protection base EcoR -- M2-1 albumen n end sequences.Draw Thing (such as sequence table SEQ ID NO：Shown in 5) it is specific as follows：

M2-1-Fwd：5’--TAAGAATTC--atgtcgcgaagaaatcctt-3’

M2-1-Rev is followed successively by by 5 ' ends to 3 ' ends：Protection base Eco311 restriction enzyme sites -- NP albumen n end sequences -- M2- 1 PROTEIN C terminal sequence.Primer (such as sequence table SEQ ID NO：Shown in 6) it is specific as follows：

M2-1-Rev：5’--CTAGGGTCTC--gtcttatgacacta-3’

The nucleocapsid protein (NP) of influenza virus is the major structural protein encoded by viral RNA fragment 5.Share 1497 Nucleotide, from the code area that 46~1542 are NP, encodes 498 amino acid.NP albumen and virus group RNA and varial polymerases 3 subunits PB1, PB2, PA be connected, form ribonucleocapsids albumen (RNP) complex, participate in the duplication and transcription of RNA, Work in the assembling of virus and the building-up process of RNA.

According to the NP nucleotide sequences of A/California/07/2009 (H1N1) influenza virus recorded in Genebank (Gene ID:23308125) CDS areas complete sequence, design primer are as follows：

NP-Fwd is followed successively by by 5 ' ends to 3 ' ends：Protection base -- Eco311 restriction enzyme sites -- M2-1 PROTEIN C terminal sequences -- NP albumen n end sequences.Primer (such as sequence table SEQ ID NO：Shown in 7) it is specific as follows：

NP-Fwd：5’-CTAGGGTCTC-taaatggcgtctcaaggcaccaaacgat-3’

NP-Rev is followed successively by by 5 ' ends to 3 ' ends：Protection base -- I restriction enzyme sites of Xho -- NP PROTEIN C terminal sequences.Primer (such as sequence table SEQ ID NO：Shown in 8) it is specific as follows：

NP-Rev：5’-CGCTCGAG-tcaactgtcatactcct-3’

Kit is rapidly purified using viral RNA/DNA and extracts the RSV and RNA of influenza virus, the bacterium that will be extracted respectively Body RNA carries out reverse transcription (RT), NP and M2 gene magnifications are carried out by template of the cDNA products of reverse transcription.

According to above-mentioned nucleotide sequence and primer sequence, NP nucleotide sequences and M2-1 nucleotide sequences are expanded respectively, expanded After increasing, amplified production is examined in gel electrophoresis.As shown in Figure 1, band 1 is DNA Marker, band 2 is blank control, and band 3 is NP amplified productions, size are about 1500bp, and band 4 is M2-1 amplified productions, and size is about 800bp, and band is clear, meets expection Theoretical value.

According to above-mentioned primer, pGEX-2T is selected to build pGEX-2T-M2-1-NP plasmids as carrier.After being put up a bridge with primer Amplification, amplified production is through double digestion, gel recycling M2-1-NP fragments and linear carrier pGEX-2T.Obtained after being inserted into successfully M2-1-NP nucleotide sequences such as sequence table SEQ ID NO：Shown in 9, its amino acid sequence such as sequence table SEQ ID NO encoded：10 It is shown.Censorship after amplified production glue reclaim is sequenced, sequencing result is compared in NCBI websites, it is as a result completely correct.

Glue reclaim recycles PCR purpose fragments using freeze-thaw method, and a hole is pricked with EP bottom of the tube of No. 18 syringe needles in 0.5mL, A hole is similarly pricked on lid.EP pipes with holes are put in a new 1.5mLEP pipe.Spread in gel imager Clean preservative film, the gel after electrophoresis is placed on preservative film, observes the position of purpose band.It is careful with blade in the UV lamp Purpose band is cut, is put into EP pipes with holes.The EP pipes of overlapping placement are centrifuged into 5min together.After centrifugation, film becomes broken, from band The EP pipes in hole are dropped in following pipe, if still having glue not change in pipe above, then are centrifuged, until all glue all drops to down In the pipe in face.Pipe above is taken away, the neutral phenol of the balance of equivalent is added in following EP pipes, is mixed.Put to -80 DEG C of refrigerators Middle jelly 30min, then room temperature melt.Upper strata aqueous phase is drawn in centrifugation, is put into new EP pipes.The PCI of equivalent is added, is vortexed and mixes, Centrifuge 5min.Supernatant is put into new centrifuge tube, adds the 3M sodium acetates of 1/10 volume, the absolute ethyl alcohol of 2.5 times of volumes, is mixed Even, -20 DEG C of refrigerators place lh.12000rpm centrifuges 20min, then abandons supernatant.1mL70% ethanol, washing are added in EP pipes Precipitation, centrifuges 5min, abandons supernatant.After drying to be precipitated, it is dissolved in spare in 10 μ L TE.

2nd, M2-1-NP combines clone and the prokaryotic expression of albumen

Glue is returned into product DNA ligase, 16 DEG C of conversion DH5a competent cells, monoclonal expands, after small upgrading grain, I double digestion of EcoR I and Xho, the screening positive clone under amicillin resistance, then converts BL21 competent cells.IPTG Induction, adds the X-gal of the IPTG and 15 μ L of 40 μ L 0.1mol/l, 37 DEG C of 12~16h of culture, and centrifuge collects thalline, Carrying out ultrasonic bacteria breaking, collect albumen after through GST affinity chromatography column purifications, obtain recombination fusion protein.SDS-PAGE the result is shown in Occur a band at 64kD, it is close with expected results.The results are shown in Figure 2 by SDS-PAGE, and band 1 is albumen Marker；Band 2 Combine albumen for the M2-1-NP after lyophilized.

Since M2-1-NP fusion proteins are excessive, generally it is present in inclusion bodies in induction bacterium, it is therefore desirable to will be a large amount of The protein product of expression is suspended in 3% beta -mercaptoethanol of 4mL 8M urea (Tris0.01M), and piping and druming is uniform, stirs 2h, 10000rpm centrifuges 15min, takes supernatant.Chromatography is freezed after collecting and preserved.

3rd, M2-1-NP combines the immunological effect experiment of albumen

1st, fusion protein prepares

Freund adjuvant is added in M2-1-NP fusion protein systems, fusion protein presses 1 with freund adjuvant：1 volume ratio is mixed Added after conjunction in beaker, 4 DEG C are emulsified completely with stirrer stirring at low speed to freund adjuvant.

By fusion protein and CT adjuvants (1mg/mL) by volume 1：1 mixing, gently shaking is uniformly mixed it.

Peritoneal immunity and mucosa-immune are carried out using fusion protein.Healthy BALB/c mouse is taken to be tested, peritoneal immunity Combine each four groups of mucosa-immune group, be respectively joint protein immunization group, commercially available influenza vaccines immune group, RSV vaccine immunities group and Negative control group.

Commercially available influenza vaccines selection inspires confidence in Luo Ke (influenza virus subunit vaccine, lot number 20110801) and is given birth to by day scholar's power Jenner Thing technology (Tianjin) Co., Ltd produces, the F-G eggs of RSV vaccines selection liberation army microorganism epidemic research institute laboratory restructuring White subunit vaccine, negative control compare for PBS with the mixing of adjuvant.

2nd, peritoneal immunity

2.1st, 20 BALB/C mices are randomly divided into 4 groups, and first to fourth group is joint protein immunization group, commercially available stream respectively Influenza vaccine immune group, RSV vaccine immunities group and negative control group；

2.2nd, during first immunisation, by recombinant protein and Freund's complete adjuvant mixture through experimental mice is injected intraperitoneally, often Only 200 μ L (containing 15 μ g recombinant proteins)；At the same time with PBS and Freund's complete adjuvant mixture through control group mice is injected intraperitoneally, often Only 200 μ L；

2.3rd, second week and the 3rd week after first immunisation, it is respectively that recombinant protein and freund 's incomplete adjuvant mixture is trans-abdominal Chamber injects experimental mice, every 200 μ L (containing 15 μ g recombinant proteins)；Passed through at the same time with PBS and freund 's incomplete adjuvant mixture Control group mice, every 200 μ L are injected intraperitoneally.

3rd, mucosal immunity

3.1st, 20 BALB/C mices are randomly divided into 4 groups, the 5th group to the 8th group be joint protein immunization group respectively, it is commercially available Influenza vaccines immune group, RSV vaccine immunities group and negative control group；

3.2nd, with 1.5% yellow Jackets intraperitoneal injection of anesthesia mouse, to mouse no longer activity, by recombinant protein and CT Adjuvant mixture instills experimental mice nasal cavity, every 30 μ L (containing 15 μ g recombinant proteins) dropwise with sterile pipette tips, while uses PBS With CT adjuvant mixture instillation control group mice nasal cavities, every 30 μ L；It is immunized once weekly, continuous immunity surrounding.

4th, sampling evaluation

4.1st, the collection of mice serum

Blood is taken through mouse orbit after a week in final immunization, separation serum (adopt by peritoneal immunity group and mucosal immunity group Collection)；With PBS by each group mice serum from 1：10000 start doubling dilution to 1：5120000, indirect ELISA detection antibody effect Valency.Peritoneal immunity detection serum IgG titers (geometrical mean).

4.2nd, the collection final immunization of mouse salivary after a week, by carbachol through making its generation in Intraperitoneal injection Mice Body Saliva, every mouse 30ul, saliva (collection of mucosal immunity group) is collected with sterile pipette tips；With PBS by each group mouse salivary from 1： 200 start doubling dilution to 1：25600, indirect ELISA detection antibody titer.Mucosa-immune detection saliva IgA titres (put down by geometry Average).

Testing result is shown in Table 1, wherein first group to the 4th group detection serum IgG titers (1：), the 5th group to the 8th group inspection Survey saliva IgA titres (1：).

Mice antibody titer table after 1 peritoneal immunity of table and mucosa-immune

	Parallel 1	Parallel 2	Parallel 3	Parallel 4	Parallel 5
						First group	22449	20378	27241	22337	23097
Second group	27755	28476	28830	26532	26770
						3rd group	26803	26577	28719	25761	26879
4th group	0	0	0	0	0
						5th group	12842	12569	11430	12907	13243
6th group	4813	3906	5083	5611	6507
						7th group	3851	4044	3984	4077	3577
8th group	0	0	0	0	0

As can be seen from Table 1, artificial synthesized M2-1-NP combines albumen compared with commercial available vaccines, can cause equal drop The immune response of degree, and immune effect is good.

After the saliva of mucosa-immune is pressed gradient dilution, influence of the analysis antigen concentration to ELISA testing results, the 5th group The results are shown in Figure 3, and antigen concentration and specific antibody IgA titres are in a linear relationship, illustrate that mucosa-immune stimulates successfully.

Finally be necessary described herein be：Above example is served only for making technical scheme further detailed Ground explanation, it is impossible to be interpreted 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 belong to protection scope of the present invention.

SEQUENCE LISTING

<110>Wuhan Bo Wo bio tech ltd

<120>Season influenza-RSV combined vaccine and its preparation method and application

<130> 2017

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 1497

<212> DNA

<213> Unknown

<220>

<223>NP DNA sequence dnas

<400> 1

atggcgtctc aaggcaccaa acgatcatat gaacaaatgg agactggtgg ggagcgccag 60

gatgccacag aaatcagagc atctgtcgga agaatgattg gtggaatcgg gagattctac 120

atccaaatgt gcactgaact caaactcagt gattatgatg gacgactaat ccagaatagc 180

ataacaatag agaggatggt gctttctgct tttgatgaga gaagaaataa atacctagaa 240

gagcatccca gtgctgggaa ggaccctaag aaaacaggag gacccatata tagaagagta 300

gacggaaagt ggatgagaga actcatcctt tatgacaaag ragaaataag gagagtttgg 360

cgcctagcaa acaatggcga agatgcaaca gcaggtctta ctcatatcat gatttggcat 420

tccaacctga atgatgccac atatcagaga acaagagcgc ttgttcgcac cggaatggat 480

cccagaatgt gctctctaat gcaaggttca acacttccca gaaggtctgg tgccgcaggt 540

gctgcggtga aaggagttgg aacaatagca atggagttaa tcagaatgat caaacgtgga 600

atcaatgacc gaaatttctg gaggggtgaa aatggacgaa ggacaagggt tgcttatgaa 660

agaatgtgca atatcctcaa aggaaaattt caaacagctg cccagagggc aatgatggat 720

caagtaagag aaagtcgaaa cccaggaaac gctgagattg aagacctcat tttcctggca 780

cggtcagcac tcattctgag gggatcagtt gcacataaat cctgcctgcc tgcttgtgtg 840

tatgggcttg cagtagcaag tgggcatgac tttgaaaggg aagggtactc actggtcggg 900

atagacccat tcaaattact ccaaaacagc caagtggtca gcctgatgag accaaatgaa 960

aacccagctc acaagagtca attggtgtgg atggcatgcc actctgctgc atttgaagat 1020

ttaagagtat caagtttcat aagaggaaag aaagtgattc caagaggaaa gctttccaca 1080

agaggggtcc agattgcttc aaatgagaat gtggaaacca tggactccaa taccctggaa 1140

ctgagaagca gatactgggc cataaggacc aggagtggag gaaataccaa tcaacaaaag 1200

gcatccgcag gccagatcag tgtgcagcct acattctcag tgcagcggaa tctccctttt 1260

gaaagagcaa ccgttatggc agcattcagc gggaacaatg aaggacggac atccgacatg 1320

cgaacagaag ttataagaat gatggaaagt gcaaagccag aagatttgtc cttccagggg 1380

cggggagtct tcgagctctc ggacgaaaag gcaacgaacc cgatcgtgcc ttcctttgac 1440

atgagtaatg aagggtctta tttcttcgga gacaatgcag aggagtatga cagttga 1497

<210> 2

<211> 498

<212> Protein

<213> Unknown

<220>

<223>NP amino acid sequences

<400> 2

masqgtkrsy eqmetggerq dateirasvg rmiggigrfy iqmctelkls dydgrliqns 60

itiermvlsa fderrnkyle ehpsagkdpk ktggpiyrrv dgkwmrelil ydkxeirrvw 120

rlanngedat aglthimiwh snlndatyqr tralvrtgmd prmcslmqgs tlprrsgaag 180

aavkgvgtia melirmikrg indrnfwrge ngrrtrvaye rmcnilkgkf qtaaqrammd 240

qvresrnpgn aeiedlifla rsalilrgsv ahksclpacv yglavasghd feregyslvg 300

idpfkllqns qvvslmrpne npahksqlvw machsaafed lrvssfirgk kviprgklst 360

rgvqiasnen vetmdsntle lrsrywairt rsggntnqqk asagqisvqp tfsvqrnlpf 420

eratvmaafs gnnegrtsdm rtevirmmes akpedlsfqg rgvfelsdek atnpivpsfd 480

msnegsyffg dnaeeyds 498

<210> 3

<211> 780

<212> DNA

<213> Unknown

<220>

<223>M2-1 DNA sequence dnas

<400> 3

atgtcgcgaa gaaatccttg taaatttgag attagaggtc attgcttgaa tggtagaaga 60

tgtcactaca gtcataatta ctttgaatgg cctcctcatg ccttactagt gaggcaaaac 120

ttcatgttaa acaagatact caagtcaatg gacaaaagca tagacacttt gtctgaaata 180

agtggagctg ctgaactgga cagaacagaa gaatatgctc ttggtatagt tggagtgcta 240

gagagttaca taggatctat aaacaacata acaaaacaat cagcatgtgt tgctatgagt 300

aaacttctta ttgagatcaa tagtgatgac attaaaaagc tgagagataa tgaagaaccc 360

aattcaccta agataagagt gtacaatact gttatatcat acattgagag caatagaaaa 420

aacaacaagc aaacaatcca tctgctcaaa agactaccag cagacgtgct gaagaagaca 480

ataaaaaaca cattagatat ccacaaaagc ataatcataa gcaacccaaa agagtcaacc 540

gtgaatgatc aaaatgacca aaccaaaaat aatgatatta ccggataaat atccttgtag 600

tatatcatcc atattgattt caagtgaaag catgattgct acattcaatc ataaaaacat 660

attacaattt aaccataacc atttggataa ccaccagcgt ttattaaata atatatttga 720

tgaaattcat tggacaccta aaaacttatt agatgccact caacaatttc tccaacatct 780

<210> 4

<211> 90

<212> Protein

<213> Unknown

<220>

<223>M2-1 amino acid sequences

<400> 4

mtkpkimilp dkypcsissi lissesmiat fnhknilqfn hnhldnhqrl lnnifdeihw 60

tpknlldatq qflqhlnipe diytiyilvs 90

<210> 5

<211> 28

<212> DNA

<213> Unknown

<220>

<223>M2-1-Fwd primer sequences

<400> 5

taagaattca tgtcgcgaag aaatcctt 28

<210> 6

<211> 25

<212> DNA

<213> Unknown

<220>

<223>M2-1-Rev primer sequences

<400> 6

cgctcgagtc aactgtcata ctcct 25

<210> 7

<211> 38

<212> DNA

<213> Unknown

<220>

<223>NP-Fwd primer sequences

<400> 7

ctagggtctc taaatggcgt ctcaaggcac caaacgat 38

<210> 8

<211> 25

<212> DNA

<213> Unknown

<220>

<223>NP-Rev primer sequences

<400> 8

cgctcgagtc aactgtcata ctcct 25

<210> 9

<211> 2274

<212> DNA

<213> Unknown

<220>

<223> M2-1-NP DNA

<400> 9

atggcgtctc aaggcaccaa acgatcatat gaacaaatgg agactggtgg ggagcgccag 60

gatgccacag aaatcagagc atctgtcgga agaatgattg gtggaatcgg gagattctac 120

atccaaatgt gcactgaact caaactcagt gattatgatg gacgactaat ccagaatagc 180

ataacaatag agaggatggt gctttctgct tttgatgaga gaagaaataa atacctagaa 240

gagcatccca gtgctgggaa ggaccctaag aaaacaggag gacccatata tagaagagta 300

gacggaaagt ggatgagaga actcatcctt tatgacaaag ragaaataag gagagtttgg 360

cgcctagcaa acaatggcga agatgcaaca gcaggtctta ctcatatcat gatttggcat 420

tccaacctga atgatgccac atatcagaga acaagagcgc ttgttcgcac cggaatggat 480

cccagaatgt gctctctaat gcaaggttca acacttccca gaaggtctgg tgccgcaggt 540

gctgcggtga aaggagttgg aacaatagca atggagttaa tcagaatgat caaacgtgga 600

atcaatgacc gaaatttctg gaggggtgaa aatggacgaa ggacaagggt tgcttatgaa 660

agaatgtgca atatcctcaa aggaaaattt caaacagctg cccagagggc aatgatggat 720

caagtaagag aaagtcgaaa cccaggaaac gctgagattg aagacctcat tttcctggca 780

cggtcagcac tcattctgag gggatcagtt gcacataaat cctgcctgcc tgcttgtgtg 840

tatgggcttg cagtagcaag tgggcatgac tttgaaaggg aagggtactc actggtcggg 900

atagacccat tcaaattact ccaaaacagc caagtggtca gcctgatgag accaaatgaa 960

aacccagctc acaagagtca attggtgtgg atggcatgcc actctgctgc atttgaagat 1020

ttaagagtat caagtttcat aagaggaaag aaagtgattc caagaggaaa gctttccaca 1080

agaggggtcc agattgcttc aaatgagaat gtggaaacca tggactccaa taccctggaa 1140

ctgagaagca gatactgggc cataaggacc aggagtggag gaaataccaa tcaacaaaag 1200

gcatccgcag gccagatcag tgtgcagcct acattctcag tgcagcggaa tctccctttt 1260

gaaagagcaa ccgttatggc agcattcagc gggaacaatg aaggacggac atccgacatg 1320

cgaacagaag ttataagaat gatggaaagt gcaaagccag aagatttgtc cttccagggg 1380

cggggagtct tcgagctctc ggacgaaaag gcaacgaacc cgatcgtgcc ttcctttgac 1440

atgagtaatg aagggtctta tttcttcgga gacaatgcag aggagtatga cagtatgtcg 1500

cgaagaaatc cttgtaaatt tgagattaga ggtcattgct tgaatggtag aagatgtcac 1560

tacagtcata attactttga atggcctcct catgccttac tagtgaggca aaacttcatg 1620

ttaaacaaga tactcaagtc aatggacaaa agcatagaca ctttgtctga aataagtgga 1680

gctgctgaac tggacagaac agaagaatat gctcttggta tagttggagt gctagagagt 1740

tacataggat ctataaacaa cataacaaaa caatcagcat gtgttgctat gagtaaactt 1800

cttattgaga tcaatagtga tgacattaaa aagctgagag ataatgaaga acccaattca 1860

cctaagataa gagtgtacaa tactgttata tcatacattg agagcaatag aaaaaacaac 1920

aagcaaacaa tccatctgct caaaagacta ccagcagacg tgctgaagaa gacaataaaa 1980

aacacattag atatccacaa aagcataatc ataagcaacc caaaagagtc aaccgtgaat 2040

gatcaaaatg accaaaccaa aaataatgat attaccggat aaatatcctt gtagtatatc 2100

atccatattg atttcaagtg aaagcatgat tgctacattc aatcataaaa acatattaca 2160

atttaaccat aaccatttgg ataaccacca gcgtttatta aataatatat ttgatgaaat 2220

tcattggaca cctaaaaact tattagatgc cactcaacaa tttctccaac atct 2274

<210> 10

<211> 588

<212> Protein

<213> Unknown

<220>

<223>M2-1 amino acid sequences

<400> 10

masqgtkrsy eqmetggerq dateirasvg rmiggigrfy iqmctelkls dydgrliqns 60

itiermvlsa fderrnkyle ehpsagkdpk ktggpiyrrv dgkwmrelil ydkxeirrvw 120

rlanngedat aglthimiwh snlndatyqr tralvrtgmd prmcslmqgs tlprrsgaag 180

aavkgvgtia melirmikrg indrnfwrge ngrrtrvaye rmcnilkgkf qtaaqrammd 240

qvresrnpgn aeiedlifla rsalilrgsv ahksclpacv yglavasghd feregyslvg 300

idpfkllqns qvvslmrpne npahksqlvw machsaafed lrvssfirgk kviprgklst 360

rgvqiasnen vetmdsntle lrsrywairt rsggntnqqk asagqisvqp tfsvqrnlpf 420

eratvmaafs gnnegrtsdm rtevirmmes akpedlsfqg rgvfelsdek atnpivpsfd 480

msnegsyffg dnaeeydsmt kpkimilpdk ypcsissili ssesmiatfn hknilqfnhn 540

hldnhqrlln nifdeihwtp knlldatqqf lqhlnipedi ytiyilvs 588

Claims (10)

1. A kind of 1. season influenza-RSV combined vaccines, it is characterised in that including：Season influenza virus protein and respiratory syncystial disease The fusion protein and immunologic adjuvant of malicious (RSV), the fusion protein are selected from the height of season influenza virus and Respiratory Syncytial Virus(RSV) Conservative protein sequence, builds the albumen of fusion with immunologic adjuvant with (0.5~2)：1 ratio mixing, obtain season influenza- RSV combined vaccines.
2. 2. season influenza-RSV combined vaccines according to claim 1, it is characterised in that season influenza virus protein is first Type influenza structural proteins are selected from：PBI, PBZ, PA, HA, NA, NP, ML and/or M.
3. 3. season influenza-RSV combined vaccines according to claim 1, it is characterised in that Respiratory Syncytial Virus(RSV) albumen selects From at least one of following albumen：Transmembrane protein G, F, SH；Matrix protein, M2；Nucleocapsid protein N, P, L.
4. 4. season influenza-RSV combined vaccines according to claim 1, it is characterised in that：Joint albumen melts for M2-1-NP Hop protein.
5. 5. season influenza-RSV combined vaccines according to claim 4, it is characterised in that：Encode M2-1-NP fusion proteins Nucleotide sequence such as sequence table SEQ ID NO：Shown in 9, its amino acid sequence such as sequence table SEQ ID NO encoded：Shown in 10.
6. 6. season influenza-RSV combined vaccines according to claim 4, it is characterised in that：According to NP nucleotide sequences and M2-1 Nucleotide sequence separately designs two pairs of primer sequences, the wherein primer sequence of NP nucleic acid such as sequence table SEQ ID NO：Shown in 5 and 6, The primer sequence table of M2-1 nucleic acid such as sequence table SEQ ID NO：Shown in 7 and 8.
7. 7. season influenza-RSV combined vaccines according to claim 1, it is characterised in that：Adjuvant is selected from sero-immunity adjuvant And Mucosal Adjuvants.
8. 8. season influenza-RSV combined vaccines according to claim 1, it is characterised in that：The mixing of fusion protein and adjuvant Ratio is 1：1.
9. 9. the preparation method of season influenza-RSV combined vaccines according to claim 1, it is characterised in that：

   s1：Kit is rapidly purified using viral RNA/DNA and extracts RSV and Influenza Virus RNA respectively, by the RNA extracted into Row reverse transcription (RT), NP and M2 gene magnifications are carried out by template of the cDNA products of reverse transcription；

   s2：Design primer expands to obtain respectively M2-1 genetic fragments, NP genetic fragments, then using this two sections of genetic fragments as template into The M2-1-NP full length fragments that row PCR amplification is recombinated, recycle M2-1-NP genetic fragments and pGEX-2T carriers, Prepare restructuring Plasmid pGEX-2T-M2-1-NP；

   s3：Recombinant plasmid pGEX-2T-M2-1-NP translation tables are reached into bacterial strain, obtain recombination fusion protein M2-1-NP.
10. 10. season influenza-RSV combined vaccines according to claim 1 are in prevention or treat season influenza and respiratory tract conjunction Application in the medicine for the disease that cellular virus induces.