CN108339115A - Use the pneumococcus combined vaccine and preparation method thereof of recombinant vector albumen - Google Patents

Abstract

The invention discloses a kind of pneumococcus combined vaccine using recombinant vector albumen, the vaccine includes：Pneumococcal antigens, recombined protein carrier and coupling agent, the recombined protein carrier is obtained by Heat-labile enterotoxin B (LTB) albumen and cholera toxin B albumen (CTB) recombination, the coupling agent is Streptavidin, biotin is modified on the pneumococcal antigens and recombined protein carrier, the pneumococcal antigens and the recombined protein carrier are coupled by the coupling agent.It will be after the recombinant vector albumen of modified biological element and pneumococcal antigens coupling by Streptavidin, the conjugated degree of recombinant vector albumen and pneumococcal antigens greatly improves, and the presence of Streptavidin and biotin does not interfere with the immune effect of combined vaccine, therefore, the immune effect of combined vaccine of the invention is greatly improved.

Description

Use the pneumococcus combined vaccine and preparation method thereof of recombinant vector albumen

Technical field

The present invention relates to biotechnology more particularly to a kind of pneumococcus combined vaccines using recombinant vector albumen And preparation method thereof.

Background technology

One, pathogenic microorganism and vaccine

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

Bacteriogenic toxin can be divided into exotoxin and endotoxin two major classes.Exotoxin is pathogen during growth and breeding It is secreted into a kind of metabolite of ambient environment, is mainly generated 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 with certain chemical substances to heat, is held It is vulnerable to destruction.It is common as：The tetanus toxin, suddenly that diphtherotoxin that corynebacterium diphtheriae generates, clostridium tetani generate The botulinum toxin etc. of enterotoxin, clostridium botulinum generation that random vibrios generates.Most of gramnegative bacteriums can generate endotoxin, Actually it is present in the outer layer of bacteria cell wall, belongs to the component part of cell wall, is not secreted into environment under normal circumstances In, only just released after bacterolysis, thus referred to as endotoxin, 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.Immune refers to just the one of body identification and exclusion antigen foreign matter (such as pathogenic microorganism) Kind aversion response.In general, it is advantageous body, 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 vaccine to be scientist develop so that body is generated specific immunity to resist cause of disease micro- The biological products that biology encroaches on human body, are usually prepared by pathogenic microorganism itself.Bacterium, virus and rickettsia Vaccine is made in the pathogenic microorganisms such as family name's body, after injecting body, body is made to generate specificity or sensitization lymphocyte, secretion is anti- Body reaches specific immunity effect.

And it is therapeutic and two kinds preventative that vaccine, which is divided into, and disease is treated by therapeutic vaccine, and pass through preventative epidemic disease Seedling protects human body not encroached on by invasive organism.By effort for many years, medical field has developed a variety of different epidemic diseases Seedling is to prevent, bacterium, virus and fungi etc., and various diseases caused by infection greatly improve the healthy water of the mankind It is flat.The continuous development of biotechnology promotes the diversification 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 big point of the biology such as useful proteins and polysaccharide for preventing bacterial infectious disease The bacterium class vaccine that sub- purification technique developed, as tetanus toxoid, diphtheria toxoid, pertussis toxoid and its Asia are thin Born of the same parents' component, epidemic meningitis Streptococcus polysaccharides and 23 valence pneumococal polysaccharides etc..More advanced useful half chemical combination technology is opened The bacterial vaccine of the prevention meningitis and pneumonia that issue, such as popular influenzae type polysaccharide-protein conjugate vaccines, 7 valences or 10 Valence pneumococcal polysaccharide-protein conjugate vaccines and 4 valences meningococcal polysacharide-albumen conjugate vaccines.By to biotechnology It continuously improves, more new generation vaccine products can be developed to deal with challenge of the different pathogenic microorganisms to human health.

Two, mucosa-immune

Mucosal immune system is distributed widely under respiratory tract, gastrointestinal tract, urogenital mucous membrane and at some exocrine glands Lymphoid tissue, be the main place for executing local specific immune function.There are about 95% bacterium, virus and parasites for body Infection all originate in mucous membrane surface.Mucosal immune system is first of immunization barrier that body resists pathogen invasion, is had The independent immune system of unique texture and function has positive effect for the field planting and intrusion that prevent pathogen.It is different from biography The immune system of system, mucosal immune system are a large amount of immunocytes and immune molecule the disperse lamina propria under mucosal epithelium or mucous membrane (diffused lymphoid tissue), or the mucosa-associated lymphoid tissue that is gathered by single or multiple lymph follicles, 50% or more body The immunocyte of lymphoid tissue and 80% or more concentrates 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 generates sIgA, this is mucous membrane The main mechanism of immanoprotection action.In addition, mucosa-immune also induces mucous membrane ctl response, and generate the CD4 of secretion IFN-C⁺ T cell, this is very important 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 obtain ideal 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 has 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.Currently, 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；Third class is antigen delivery system.

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. is the bacteriotoxin with mucosal adjuvant function reported recently.LT and CT are Bacteriotoxin, 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 builds removal toxicity or reduces toxicity, is retained simultaneously 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, a large amount of anti-LT can be found in Respiratory Tract of Mice secretion and small intestine contents IgA antibody.It takes small intestinal mucosa to be sliced, a large amount 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 Answering position also has same response.

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

Whether two subunits of LT play a role and play respectively much effects in its adjuvanticity, and there is also very at present Big dispute.People use the 192-GLY LT for lacking GM1 affinity but the complete ADP- ribosylating activities of reservation small as adjuvant immunity Mouse.It was found that with adjuvanticity as wild type LT, therefore, it is considered that the affinity combined with GM1 to the adjuvanticity of LT not It is required.

Three, Escherichia coli and its epidemiology

Escherichia coli are the abbreviations of escherichia coli (Escherichia coli, E.coli), by German bacteriologist Theodor Escherich were most detached earlier than 1885 from infant faeces, were enterobacteriaceae (Entero- Bacteriaceae), the member of Escherichia (Escherichia), original name are Bacterium coli commune, the meaning It refer to enteron aisle common bacteria.In fact, only a small number of a part of E.coli bacterial strains can directly result in host disease in enteron aisle, and Most of E.coli are non-pathogenic in enteron aisle, but if the tissue or organ that are displaced to outside enteron aisle may then cause it is parenteral Infection.Pathogenic E.coli is not conditioned pathogen (such as resistance reduces, normal flora is out of proportion or displacement), and The Escherichia coli itself for being certain serotype groups (type) are pathogen.For the enteropathogenic E. Coli of enteral, according to pathogenic The difference of mechanism, clinical symptoms and epidemic etc. is mainly the following type：Enterotoxigenic escherichia coli (Enterotoxigenic E.coli, ETEC), shiga toxin producing escherichia coli (Shiga toxin-producing E.coli, STEC), enterohemorrhagic large intestine traitor bacterium

(Enterohemorrhagic E.coli, EHEC), enteropathogenic E.Coli (Enteropathogenic E.coli, EPEC) and enteroinvasive E.Coli (Enteroinvasive E.coli, EIEC) etc..In addition, also urethra causes Characteristic of disease Escherichia coli (Uropthogenic E.coli, UPEC) inhale off-type Escherichia coli (Attaching and Effacing E.coli, AEEC), production gangrenosum acne toxin large intestine sweat bacterium (Necrotoxigenic E.coli, NTEC), intestines adhesion large intestine traitor bacterium

(Enteroaggregative E.coli, EAEC) and intestines concentration large intestine traitor bacterium (Enteroaggregative E.coli, EAggEC) etc..

One of the most common is diarrhea caused by ETEC.ETEC is popular in late period the 1960s earliest, is people and dynamic One of important pathogen body of object infectious diarrhea.Some data show that ETEC is caused in Cholera Epidemic Area original inhabitants adult resident The most commonly encountered diseases of cholera syndrome because；Be developed country or even developing country children, traveler's diarrhea most commonly encountered diseases because；It is also Water source and food faecal contamination and draw play one of key factors such as diarrhea, nausea, low fever, cramp.

ETEC generates 2 kinds of different type toxin, that is, heat-labile toxins (Heat-labile toxin, LT) and resistance to warmheartedness poison Plain (Heat-stable toxin, ST).The physical property of both toxin of LT and ST has larger difference, ST at 100 DEG C, The basic non-inactivations of 30min, but LT is at 65 DEG C, 30min, that is, complete deactivation.ST molecular weight it is relatively low (<9000ku), no antigen, can It is divided into two kinds of ST1 and ST2, wherein ST1 plays an important role on epidemiology, and the two all activates guanylate cyclase, makes thin Intracellular cGMP levels increase, and upset dielectric metabolism, cause diarrhea.LT and ST the difference is that, LT is a kind of immune protein, There is more substantial connection with cholera enterotoxin (Cholrea toxin, CT), forms a toxin family.ETEC can also cause Human body generates choleraic diarrhea, and feeling well, just character and dewatering symptom are similar to cholera.

According to the difference of toxin source, LT can be divided into people source (LT-h/hLT) and pig source (LT-p/pLT) two ethnic group class, it Chemical property and immunological characteristic it is very much like.According to encoding gene difference, LT is divided into as two types：LT1 and LT2.LT1 It is most commonly seen in nature by the Large plasmid coding in endochylema, it is the main component that LT plays biological activity.It is general described LT all refer to LT1 mostly；And LT2 is encoded by chromosomal DNA and is generated.Although their composition is similar with the mode of action, without base Because of homology and cross immunogenicity.Described LT refers both to LT1 below.

The full length gene 1148bp of LT is encoded, is located on the Large plasmid in wild type enterotoxigenic E．Coli cell dress.LT Operon contains 2 structural genes, respectively encodes the toxA genes of the toxB genes and coding A subunits of B subunits, they It is chained together and 1 mRNA chain can be transcribed into.Wherein toxB genes are located at the end of toxA genes 3', its ribosomes knot It closes site (GGGA) to be located in toxA genes, and 2, the ends toxA3' codon (TTATGA) and 2, the ends toxB 5' codon (ATGAAT) there are 4 nucleotide overlapped.B subunits coding region gene overall length 372bp, coding are made of 124 amino acid Polypeptide；A subunits coding region gene overall length 777bp encodes the polypeptide of 259 amino acid.ToxB before initiation codon 3 Have SD sequences at a nucleotide, 5 nucleotide therein can with 5 complementations in the nucleotide of 12, the ends 16S rRNA 3', And the nearly ends ATG base is A.These structures are conducive to improve ribosomal joint efficiency, and the secondary structure of toxB mRNA also has Conducive to the translation of toxB genes, so as to cause in the same promoter downstream, although toxB is located at distal end, expression quantity is more than As many as 5 times of toxA.This more special structure 2 subunits of toxin promoter pair that may be more convenient for play preferable regulation and control and make With adjusting their expression quantity, make it easy to reach the best condition of toxin synthesis.

A, B subunit in endochylema is all to carry the presence of signal propeptide.It can be just assembled into after passing through cell membrane Whole LT.The effect of B subunits is specifically bound with the GM-1 ganglioside receptors of eukaryocyte film surface, with convenient A subunits enter target cell.A subunits have the ADP- ribosylation transferase actives of GTP dependences, are mediated by G-protein ADP- ribosylation reaction upset degradation and the balance of intracellular cAMP, stimulation cAMP contents increase, to cause toxicity effect It answers.LT-h and LT-p acts on different G-proteins, the former is Gs albumen, and the latter is Gi albumen, and final effect is all to lead to egg White kinases A inactivations, cause a series of biochemical reaction, cAMP contents are caused to increase, stimulate the mistake of intestinal mucosa water and electrolyte Degree secretion, leads to diarrhea.

LTB has been found as the function of immunologic adjuvant, and has higher and higher attention rate, but LTB is separately as load The research of body protein is fewer and fewer, and individual LTB albumen is only used as the carrier of LTA at present, and which greatly limits LTB's Use scope.LTB due to its have with the protein carrier built after good architecture basics, with other protein fusion expressions it is wide General prospect of the application.

Four, comma bacillus is summarized

CT is a kind of heat-labile toxin of comma bacillus secretion, identical with five by a toxicity A subunit (CTA) B subunits (CTB) composition, formed AB5 structures.A subunits are one single-stranded, are often cracked between 194,195 residues after synthesis CTA1 and CTA2, is connected with disulfide bond.Wherein CTA1 has ADP- ribosyl-transferase activities, and the adjuvant effect with CT has closely Relationship, CTA2 then connect CTA1 and CTB.5 subunits of CTB are combined into pentamer with non-covalent bond each other, then pass through CTA2 rings AB5 structures are formed around CTA.Binding force between the subunit of CTB pentamers is very strong, is more than the binding force of they and A subunits.

CTB is widely paid attention to as the application of immune carrier in recent years, and CTB has as nontoxic unit and combines GM1 Function, be that toxicity subunit A is combined closely in intestinal mucosa cell surface, make fusion protein be more easy to and alimentary canal mucous membrane make With, and then cause a series of biochemical reactions, stronger immune effect is generated, CTB is also used as certain allogenic polypeptides Stable carrier can evoke fusion epitope antigen after it enters in vivo simultaneously with the antigen of chemical coupling or Gene Fusion Immunogenicity makes body generate strong immune response, reaches immanoprotection action.

CTB has very strong immunogenicity, can enhance bacterial virus and the immunogenicity of other antigens, especially pass through Mucosal immune can not only enhance the specific IgG antibodies response of serum, also can induce stronger specific performance mucous membrane IgA immune responses have the characteristics that the antigenic of epitope can be reinforced.CTB can be with dendron shape in stimulator antigen presenting cell Cell is to the transport capacity of antigen, and Dendritic Cells is for transporting antigen to lymphoid organ, and activation antigen CD4+ and CD8+T Cell.CTB can activate the maturation of Dendritic Cells, keep the dendron of Dendritic Cells longer, volume bigger.CTB cells may be used also To adjust the generation of t cell responses and internal antibody in system.

Five, pneumococcus 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, have in worldwide higher incidence and Case fatality rate, especially to 2 years old children and old man below.The S. pneumoniae capsular saccharide vaccine and capsular glycoprotein listed at present Matter combined vaccine, design are all based on S. pneumoniae capsular saccharide, cover the most common serotype for leading to pneumococcal disease. But S. pneumoniae capsular saccharide is thymus independent antigen (Thymus independent antigen, TI-Ag), antibody response Depend on its recurring unit composition linear epitope, in the case where no T lymphocytes assist directly with bone-marrow-derived lymphocyte table The IgM receptors in face are crosslinked, and the antibody induced is mainly IgM and IgG2, lacks preferable complement activation ability, antibody level is not The sufficiently long time can be maintained, and is unable to inducing immunological memory, immunoprotection can not be generated in child at 2 years old or less.Capsular saccharides Complicated structure causes the immunogenicity of each serotype different, can not generate effective immune response.Pneumococcus combines Vaccine includes that Serotypes are more, and specificity structure of each type for combination is different, leads to the combined method phase of each of which type It is different.Modification to capsular saccharides and and carrier protein combination not lost ensureing the special group of capsular saccharides, antigenicity and immune It is carried out under the premise of originality is unaffected, while the requirement of the excessive crosslinking and conjugate aseptic filtration in order to avoid sugar chain, it is right Capsular saccharides and conjugate bulk of molecule should have certain control.7 valence vaccines were approved to use in 2 months 2000 in the U.S..Due to Pneumococcus type is more, needs binding protein ingredient in the manufacturing process of combined vaccine, because protein ingredient can cause part anti- It answers, so producing comprising combined vaccines more than 12 types with regard to highly difficult.Combined vaccine antibody living after initial immunity is dense Degree is only capable of maintaining some months, will then drop to pre-immune levels；And it needs to add in the entire technical process of combined vaccine Enter a variety of chemical reagent and participate in reaction, and capsular glycoprotein combined vaccine serotype coverage rate is low and nonvaccine serotype lung The increase of scorching coccus infectious diseases is so that more researchers begin to focus on the Pnu-Imune 23 exploitation in other directions.

The research and development mainstream of pneumovax nearly ten years is had become using the pneumococcus combined vaccine of recombinant vector albumen, is had There is the vaccine based on species specificity antigen by the attention of research staff, has attracted a large amount of sight.But due to albumen sheet The limitation of the formation and physic-chemical property of body, many pneumoproteins cannot be used directly for human immunity, need to spend a large amount of Man power and material studies and clinical verification.Protein vaccine utilize biotechnology synthetic proteins antigen, proteantigen by What it is in its selection is highly conserved pneumococcus specific proteins, to eliminate the immune difference between different serotypes It is different, and due to not needing other kinds of common protein carrier, eliminating existing Pnu-Imune 23 cannot be with general load The obstacle that the vaccine of body protein same type is used in combination simultaneously；Its proteantigen itself can also be used as carrier protein and pneumonia ball Bacterium capsular saccharides carry out spontaneity and are conjugated, therefore immune effect is more preferable, realize specific immunity and combine realization with what is be immunized extensively.

Pneumococcus causes the big stability of the antigenic structure of its antigen itself poor, it is difficult to other since its serotype Vaccine, which is combined, coexists use, and existing S. pneumoniae capsular saccharide protein conjugate vaccines use diphtheria or tetanus toxoid to make For protein carrier, the main component of this vaccine is the capsular saccharides of serotype specificity, to other blood being not included in vaccine Clear type pneumococcal infection is invalid, that is, lacks Cross immunogenicity effect；And will with used in children's routine immunization Diphtheria and tetanus vaccine generate interference, destroy existing immune effect.Therefore the enough good energy of a autoantigenic is studied Enough be stabilized becomes the task of top priority that vaccine researches and develops field using the pneumococcus combined vaccine of recombinant vector albumen.

Invention content

In view of the above-mentioned problems existing in the prior art, the object of the present invention is to provide a kind of use of good immune effect recombinations The pneumococcus combined vaccine of carrier protein.

For achieving the above object, the pneumococcus combined vaccine provided by the invention using recombinant vector albumen uses Technical solution it is as follows：

Vaccine includes：The recombined protein carrier and strepto- for being modified with the pneumococcal antigens of biotin, being modified with biotin Avidin, recombined protein carrier are sub- by Heat-labile enterotoxin B (LTB) albumen and comma bacillus enterotoxin B Unit-protein (CTB) recombination obtains, the pneumococcal antigens for being decorated with biotin and the recombination egg for being modified with biotin Bai Zaiti is coupled to by biotin on the Streptavidin, the pneumococcal antigens on the Streptavidin and recombination egg The conjugated connections of Bai Zaiti.

LTB nucleic acid sequences are bridged with CTB nucleic acid sequences by primer sequence, the LTB-CTB nucleic acid sequences such as sequence of structure Table SEQ ID NO：Shown in 5, the carrier protein sequence such as sequence table SEQ ID NO of coding：Shown in 6.

Encode the nucleotide sequence such as sequence table SEQ ID NO of LTB albumen：Shown in 1, the amino acid sequence such as sequence of coding List SEQ ID NO：Shown in 2.

Encode the nucleotide sequence such as sequence table SEQ ID NO of CTB albumen：Shown in 3, the amino acid sequence such as sequence of coding List SEQ ID NO：Shown in 4.

Pneumococcal antigens include pneumococcal protein antigen, pneumococcal polysaccharide antigen and/or pneumococal polysaccharide-egg White conjugated antigen.

Pneumococcal protein antigen includes：Pneumococcus dissolved blood protein and its modification derivant, pneumococcal surface protein And its modification derivant, pneumonia ball surface adhesion albumen and its modification derivant, three histidine protein family of pneumococcus and Its modification derivant and/or pneumococcus stick virulence factor.

Pneumococcal protein antigen includes：Pneumococcus dissolved blood protein Ply, modified pneumococcus dissolved blood protein △ A146Ply, pneumococcus dissolved blood protein derivative PlyD1, pneumococcus dissolved blood protein derivative PlyD B, pneumococcus haemolysis Protein derivatives PlyD T, pneumococcal surface protein C, Pneumococcal Surface adhesion protein A, Pneumococcal Surface adhesion protein C, three histidine protein D of pneumococcus and/or pneumococcus stick virulence factor A.

The pneumococcal polysaccharide antigen is the capsular polysaccharide on separating-purifying Pneumococcal serotype pod membrane, the pneumonia The serotype of coccus include 1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and/or 33F.

It is further preferred that the serotype of pneumococcal polysaccharide antigen includes 4,6B, 9V, 14,18C, 19F and 23F.

Preferably, the mass ratio between each of S. pneumoniae capsular saccharide and pneumoprotein serotype and albumen is 1.5~4.5:1.

The inventive concept total as one, the present invention also provides a kind of above-mentioned pneumococcus using recombinant vector albumen The preparation method of combined vaccine, includes the following steps：

1) pneumococcal antigens and biotin are added in the first phosphate buffered saline solution, it is anti-that first is carried out under the conditions of being protected from light It answers, modified biological element on pneumococcal antigens, reaction product is made to remove extra biotin through dialysis, be repaiied after freeze-drying It is decorated with the pneumococcal antigens of biotin；Recombined protein carrier and biotin are added in the second phosphate buffered saline solution, are protected from light Under the conditions of carry out the second reaction, so that modified biological element of dialysing on recombined protein carrier, reaction product is removed extra life through dialysis Object element, obtains the recombined protein carrier for being modified with biotin after freeze-drying；

(2) phosphate buffered saline solution containing Streptavidin is prepared, the pneumococcal antigens for being modified with biotin are added With the recombined protein carrier for being modified with biotin, third reaction is carried out under the conditions of being protected from light, obtains the first reaction solution；

(3) it is added in the first reaction solution obtained by step (2) and agent progress conjugation reaction is conjugated, sediment is collected by centrifugation, The sediment obtains the pneumococcus combined vaccine using recombinant vector albumen through gel column purification after freeze-drying.

In the step (1), in first phosphate buffered saline solution, the quality of the pneumococcal antigens and biotin Than being 1: 0.05~0.15, a concentration of 0.1~0.2mol/L of the phosphate-buffered salt；It is described first reaction time be 30~ 60min；In second phosphate buffered saline solution, the recombined protein carrier, the phosphate-buffered salt a concentration of 0.1~ 0.2mol/L；The time of second reaction is 30~60min.

In the step (2), in the phosphate buffered saline solution containing Streptavidin, Streptavidin is modified with life The pneumococcal antigens of object element, be modified with biotin recombined protein carrier mass ratio be 1: 1.5~4.5: 1；The third The time of reaction is 30~60min.

In the step (3), the conjugated agent is carbodiimide, and the conjugation reaction reacts 4 under room temperature acidic environment ~6d.

Compared with prior art, the present invention is using a variety of capsular saccharides of recombinant vector albumen connection pneumococcus, the carrier Also there is albumen the adjuvant effect of activation mucosa-immune can not only cause the blood of high titre after being conjugated with pneumococcal antigens Clear antibody can also quickly cause mucous membrane serum antibody for a long time.The optional pneumoprotein of pneumococcus antibody and pneumonia Coccus capsular polysaccharide, when the valence state of vaccine is higher, the protein carrier that recombination can be used combines with pneumococcus homologous protein and makes With the immune response that existing Pneumococcus serotypes cause can not only be enhanced, and can cause the mucosa-immune of lung.

The pneumococcus combined vaccine using recombinant vector albumen in the present invention has certain structural elasticity, has more The conjugated combination of kind polysaccharide protein, carrier protein promotes body to generate specific antigen, but itself is not pathogenic, will not generate friendship The Immunological Effect of fork property, therefore the vaccine has very extensive use value.

Applicant had found by subsequent application study, Heat-labile enterotoxin B (LTB) albumen and suddenly The recombinant vector albumen that random vibrios enterotoxin subunit B albumen (CTB) recombination obtains and the conjugated degree of pneumococcal antigens be not high, leads Cause the immune effect of combined vaccine bad.To enhance the conjugated degree of recombinant vector albumen and pneumococcal antigens, applicant does It is a large amount of to attempt, it finally finds, biotin is easy to be combined with recombinant vector albumen and pneumococcal antigens, since Streptavidin has The ability for having excellent combination biotin is resisted the recombinant vector albumen of modified biological element and pneumococcus by Streptavidin After original coupling, the conjugated degree of recombinant vector albumen and pneumococcal antigens greatly improves, and Streptavidin and biotin In the presence of the immune effect for not interfering with combined vaccine, therefore, the immune effect of combined vaccine of the invention is greatly improved.

Description of the drawings

Fig. 1 is the structure schematic diagram of structure pET28a-LTB-CTB plasmids provided by the invention.

Fig. 2 is the plasmid schematic diagram of structure pET28a-LTB-CTB plasmids provided by the invention.

Fig. 3 is recombination LTB-CTB carrier proteins induction purification result schematic diagram provided by the invention.

Fig. 4 is specific antibody titres in Respiratory Tract of Mice mucus provided by the invention.

Fig. 5 is specific antibody titres in mice serum provided by the invention.

Specific implementation mode

The present invention is made further in detail, completely to illustrate with reference to embodiment.Experimental method in following embodiments, Unless otherwise specified, it is conventional method.Experiment material as used in the following examples is market purchase unless otherwise specified It obtains.

Embodiment 1：

One, the clone of recombination LTB and CTB albumen and prokaryotic expression

LT and CT etc., six glycoprotein polyprotein precursor of AB5 types that LT is made of A, B Liang Zhong subunits (LT-A and LT-B).A、B Liang Ge subunits are combined by non-covalent bond, and individual subunit does not have bioactivity, and being only combined together just has full poison The biology and chemical characteristic of element.A subunits are the virulence centers of toxin, and molecular weight is about 28KD, have ADP- ribosylation Enzymatic activity.A subunits can be cut into two segments of A1 (LT-A1) and A2 (LT-A2), A1 segments and A2 segments point with reduction reaction Not Cheng foldable structure and helical structure, the two by disulfide bond be connected.A1 is the active part of LT toxin, and A2 is and B subunits Connected part.When the disulfide bond for connecting A1 and A2 is reduced, the A1 subunits with enzymatic activity are released.B is sub- Unit is made of two α spirals and six β lamellar structures, a cysteine residues at the ends N- and the one and half Guang ammonia at the ends C- Sour residue forms disulfide bond, and two ends of molecule are linked together.The molecules align that five molecular weight are about 11.5KD exists Structure annular in shape together can be combined with specific receptor nervon GM1 on whip cell.A subunits are inserted into the Asias B by A2 segments Unit pentamer center, forms a complete LT molecule.

It is as follows according to the GeneBank LTB and CTB nucleic acid sequences logged in and amino acid sequence, particular sequence：

Nucleic acid, LTB nucleic acid sequences (375bp) such as sequence table SEQ ID NO：Shown in 1, ammonia that thus nucleic acid sequence encoding goes out Base acid sequence (124a.a, MW=14133.74) such as sequence table SEQ ID NO：Shown in 2.

CTB nucleic acid sequences (375bp) such as sequence table SEQ ID NO：Shown in 3, CTB amino that thus nucleic acid sequence encoding goes out Acid sequence (124a.a.MW=13896.46) such as sequence table SEQ ID NO：Shown in 4.

Select pET28a as carrier, according to the overall length CDS sequences of the GeneBank LTB and CTB logged in, design primer is as follows：

LTB-Fwd is followed successively by by 5 ' ends to 3 ' ends：Protection base -- I restriction enzyme sites of BamH -- LTB albumen n end sequences.Draw Object (such as sequence table SEQ ID NO：Shown in 7) it is specific as follows：

5’-CG--GGATCC--atgaataaagtaaaatgttatgttttatttacggcgtta-3’

LTB-Rev is followed successively by by 5 ' ends to 3 ' ends：Protection base -- Eco311 restriction enzyme sites -- CTB albumen n end sequences -- LTB PROTEIN C terminal sequences.Primer (such as sequence table SEQ ID NO：Shown in 8) it is specific as follows：

5’-CTAG--GGTCTC--cat--gtttttcatactgattgccgcaa-3

CTB-Fwd is followed successively by by 5 ' ends to 3 ' ends：Protection base -- Eco311 restriction enzyme sites -- LTB PROTEIN C terminal sequences -- CTB albumen n end sequences.Primer (such as sequence table SEQ ID NO：Shown in 9) it is specific as follows：

5’-CTAG--GGTCTC--AAC--atgattaaattaaaatttggtgttttttttacagtttta-3’

CTB-Rev is followed successively by by 5 ' ends to 3 ' ends：Protection base -- I restriction enzyme sites of Xho -- CTB PROTEIN C terminal sequences.Primer (such as sequence table SEQ ID NO：Shown in 10) it is specific as follows：

5’-CC--CTCGAG--ttaatttgccatactaattgcggcaa-3’

As shown in Fig. 1~2, pET28a-LTB-CTB plasmids are built.There is higher homology due to LTB and CTB, because This is inserted into the segment of CTB in LTB segments, is connected with primer, puts up a bridge and carries out PCR, amplifiable to obtain recombination LTB-CTB carrier proteins Full length fragment.The complete sequence of the pET28a plasmids of structure such as sequence table SEQ ID NO：Shown in 5, the amino acid sequence such as sequence of coding Table SEQ ID NO：Shown in 6.

After carrier and PCR product are by I double digestion of BamH I and Xho, gel recycles LTB and CTB segments.After glue recycling, use DNA ligase, overnight, connection conversion bacillus coli DH 5 alpha, monoclonal expands for 16 DEG C of connections, after small upgrading grain, BamH I and Xho The about 800bp bands of I double digestion identification, the screening positive clone under amicillin resistance are sequenced after positive-selecting, will be sequenced As a result it is compared in the websites NCBI, it is as a result completely correct.

Two, the expression and purity of LTB-CTB carrier proteins is recombinated

Recombination LTB-CTB carrier proteins induce 30h at 20 DEG C, a concentration of 0.3mmol/L of IPTG, and inductive condition is best.It lures It goes the supernatant of appropriate ultrasonic treatment product to precipitate after leading and carries out SDS-PAGE, while setting the albumen before induction as control. After broken bacterium, it is found that expression protein exists with inclusion bodies.After inclusion body is washed, dissolved with the urea of 8mol/L, through multiple Property liquid renaturation obtain the recombination fusion egg that purity is about 93% then through Glutathione S transferase (GST) affinity chromatography column purification In vain.SDS-PAGE results show in recombinant bacterium cracking precipitation the additional band of a treaty 30kD occur, close with expected results.Due to Albumen is mainly expressed in the form of inclusion body, therefore selects the long-time induction of lower temperature, to ensure expressing quantity.Induce table The purification result reached is as shown in figure 3, band 1 is albumen Marker；2 be the preceding total albumen of induction；3 be total albumen after induction；4 be thin Bacterium is crushed supernatant after liquid centrifugation；5 be bacteria breaking liquid centrifuged deposit；Band 6 is the recombination LTB-CTB carrier proteins after freeze-drying.

Three, pneumoprotein-capsular polysaccharide combination is prepared

3.1.1 the preparation of pneumococal polysaccharide (7 valence)

Choose 7 kinds of serotypes (4,6B, 9V, 14,18C, 19F, 23F) pneumococcus fermented and cultured after, using sand culture from Scheming, disc centrifuge or other large capacity centrifuges are separately cultured liquid, collect centrifugation supernatant；Supernatant will be centrifuged with 100KD Film packet is concentrated by ultrafiltration, and carries out fractional precipitation using 25~80% ethyl alcohol, collects and precipitate and washed respectively with absolute ethyl alcohol and acetone It washs, obtains crude polysaccharide；Sterilized water for injection dissolves polysaccharide and after NaTDC is handled, using ion-exchange packing, with The method of series connection chromatography or step chromatography carries out raw sugar and refines, and collection flows through peak, adopts GE Sephadex G25Coarse to flowing through Peak carries out desalination, ethanol precipitation or freeze-drying recycling polysaccharide, is placed in -20 DEG C and saves backup.

3.1.2 LTB-CTB carrier protein pneumococal polysaccharide conjugates are recombinated

It takes each serotype polysaccharide to be dissolved in the 0.02mol/LPBS of pH7.2, for preparing 10mL vaccinogen liquids, pod is added Film polysaccharide 4,9V, 14,19F and 23F 20 μ g of each 40 μ g, oligosaccharides 18C and 80 μ g of polysaccharide 6B, add 30 μ g biotins, are protected from light React 60min.Reaction product is dialysed 72h in the phosphate buffered saline solution of 150mmol/L, removes free biotin；Dialysis After be freeze-dried, obtain the S. pneumoniae capsular saccharide for being modified with biotin.

It takes 160 μ g recombined protein carriers and 16 μ g biotins to be added in 150mmol/L phosphate buffered saline solutions, is protected from light anti- Answer 60min.Reaction product is dialysed 72h in the phosphate buffered saline solution of 150mmol/L, removes free biotin, dialysis knot It is freeze-dried after beam, obtains the recombined protein carrier for being modified with biotin.

The phosphate buffered saline solution for preparing the 150mmol/L containing 16 μ g Streptavidins adds and above-mentioned is modified with biology The pneumococcal antigens of element and the recombined protein carrier for being modified with biotin, react 60min under the conditions of being protected from light, obtain the first reaction solution.

The pH value of the first reaction solution is adjusted to 10.8 or so, 1- cyano -4-dimethylaminopyridine-tetrafluoro of 30 μ g is added Change boron (CDAP), at room temperature activated polysaccharide 10min.Add the adipoyl of 0.5mol/L identical with the first reaction solution volume Hydrazine reacts at room temperature 60min.50KD film packet ultrafiltration removes CDAP and adipoyl hydrazine.It is eventually adding the carbodiimide of 30 μ g, room temperature, 5d is reacted under pH5.6.Granular precipitates are collected by centrifugation after reaction, by sediment through gel column (Thermo:43230) purifying is gone Except extra Streptavidin, the conjugate of the pneumococcus and recombination LTB-CTB that are purified.

It recombinates LTB-CTB carrier protein pneumococal polysaccharide conjugates and freeze-dried formulation may be selected, convenient for storage and transport.Freeze Dry dosage form sucrose is not more than 20% as freeze-drying skeleton, wherein initial mass percentage composition of the sucrose in lyophilized stoste, freeze-drying After obtain S. pneumoniae capsular saccharide-protein vaccine lyophilized preparation.

Comparative example 1：

The pneumococcus combined vaccine using recombinant vector albumen of this comparative example, pneumococcal antigens and recombinant protein carry Body is same as Example 1, the difference lies in that pneumococcal antigens are conjugated connection with recombinant vector albumen.

Preparation method is substantially the same manner as Example 1, the difference lies in that step 3.1.2 recombinates LTB-CTB carrier protein pneumonia The preparation process of Streptococcus polysaccharides conjugate is as follows：

It takes each serotype polysaccharide to be dissolved in the 150mmol/LPBS of pH7.2, for preparing 10mL vaccinogen liquids, pod is added Film polysaccharide 4,9V, 14,19F and 23F 20 μ g of each 40 μ g, oligosaccharides 18C and 80 μ g of polysaccharide 6B adjust pH value to 10.8 or so, add Enter the 1- cyano -4-dimethylaminopyridine-tetrafluoride boron (CDAP) of 30 μ g, at room temperature activated polysaccharide 10min.It adds and the The adipoyl hydrazine of the identical 0.5mol/L of one reaction solution volume reacts at room temperature 60min.50KD film packet ultrafiltration removes CDAP and two Hydrazides obtains polysaccharide derivates.150 μ g recombination LTB-CTB carrier proteins are added, the carbon two for being stirring evenly and then adding into 30 μ g is sub- Amine reacts 5d under room temperature, pH5.6, graininess conjugate occurs, and 10min is centrifuged at 4 DEG C and is collected.It is pure through gel chromatography column chromatography Change, the conjugate of the pneumococcus purified and recombination LTB-CTB.

Test alive

The conjugate of the embodiment 1 of successful connection and comparative example 1 is subjected to mouse live infection respectively, takes successful connection Conjugate carries out intraperitoneal injection and intranasal administration to mouse, and 6~8 weeks healthy BALB/c mouses is selected to be tested, every group 5.The One group of administration recombinates LTB-CTB carrier protein pneumococal polysaccharide conjugates, the commercially available 7 valence Pnu-Imune 23 (quotient of second group of administration The name of an article：Abundant youngster), PBS is administered as negative blank control in third group.Peritoneal immunity is administered three times, and is immunized once weekly, dosage is equal For every 0.2ml (containing 15 μ g recombinant vector albumen pneumococal polysaccharides conjugates), every 0.2ml of PBS is injected intraperitoneally in third group.

When nasal administration, incited somebody to action until after mouse no longer activity with 1.5% yellow Jackets intraperitoneal injection of anesthesia mouse Drug instills experimental mice nasal cavity dropwise with sterile pipette tips, and every 30 μ l (contain 15 μ g recombinant vector albumen pneumococal polysaccharides Conjugate), primary, continuous immunity three weeks is immunized weekly.Third group each mouse nasal cavity instillation PBS30 μ l, continuous three weeks.Last Immune latter Zhou Jinhang samplings.

Serum and respiratory tract mucus are collected when sampling.It takes Virus monitory each group mice serum IgG antibody horizontal, takes mouse nose Detection IgA antibody is horizontal after the dilution of chamber irrigating solution.

The preparation of serum：Mouse orbit blood is acquired to centrifuge with 3000r/min after blood collection is placed on 37 DEG C of standing 2h 10min draws serum Cord blood, standby inspection.

It is prepared by respiratory mucus：It pours water and rinses mouse nasal cavity.Flushing liquor is collected, is then diluted with 4 times of PBS, low temperature is protected It deposits, standby inspection.

IgA antibody in IgG antibody in serum and mucous membrane is detected using indirect elisa method, the specific method is as follows：It adopts With 7 kinds after purification each serotype polysaccharide, respectively with optimal dose coated elisa plate, 37 DEG C are incubated overnight, and series is added after board-washing The colour developing of horseradish peroxidase-labeled sheep anti-mouse igg secondary antibody, enzyme mark is added in diluted mice serum to be measured, board-washing after 37 DEG C of incubations Instrument measures, and reads 492nm (630nm is reference wavelength) absorbance value (A values).The standard deviation of+3 times of negative control group serum A mean values Difference is Cutoff values, and 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 The geometric mean titer of each type mouse, as mice serum IgG antibody potency (as shown in table 1).The coupling vaccine egg of embodiment 1 White stoste, the conjugate vaccines albumen stoste of comparative example 1 and commercially available 7 valence positive control polysaccharide vaccine produce height after mouse is immunized The serum antibody of degree, and the titre higher that vaccine protein stoste generates is coupled, illustrate that coupling vaccine has superior immune effect.

Each serotype antibody titer of 17 valence pneumococcus of table detects (geometrical mean) (1：)

	Couple product IgG	Conjugation product IgG	Commercial available vaccines IgG	Negative control IgG
					4 type polysaccharide	18725	15832	17864	0
6B type polysaccharide	19810	16029	18754	0
					9V type polysaccharide	20936	18957	19801	0
14 type polysaccharide	16759	12003	15642	0
					18C type polysaccharide	17450	14421	16788	0
19F type polysaccharide	18021	15788	16835	0
					23F type polysaccharide	17811	14358	16523	0

Detection of Stability

The pneumococcus combined vaccine of embodiment 1 preserves 4 weeks for 12 months or 37 DEG C at 4 DEG C, visual examination and discrimination test No abnormality seen, pH value and antigenic content keep relative stability, and carry out mouse test and also do not find that notable difference occurs for antibody level. Illustrate that pneumococcus combined vaccine using recombinant vector albumen preserves 4 weeks for 12 months or 37 DEG C at 4 DEG C to have good stability.

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>Use the pneumococcus combined vaccine of recombinant vector albumen

<130> 2017

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 375

<212> DNA

<213> Unknown

<220>

<221> gene

<222> (1)..(375)

<223>LTB DNA sequence dnas

<400> 1

atgaataaag taaaatgtta tgttttattt acggcgttac tatcctctct atatgcacac 60

ggagctcccc agactattac agaactatgt tcggaatatc gcaacacaca aatatatacg 120

ataaatgaca agatactatc atatacggaa tcgatggcag gcaaaagaga aatggttatc 180

attacattta agagcggcga aacatttcag gtcgaagtcc cgggcagtca acatatagac 240

tcccagaaaa aagccattga aaggatgaag gacacattaa gaatcacata tctgaccgag 300

accaaaattg ataaattatg tgtatggaat aataaaaccc ccaattcaat tgcggcaatc 360

agtatgaaaa actag 375

<210> 2

<211> 124

<212> PRT

<213> Unknown

<220>

<221> UNSURE

<222> (1)..(124)

<223>LTB amino acid sequences

<400> 2

Met Asn Lys Val Lys Cys Tyr Val Leu Phe Thr Ala Leu Leu Ser Ser

1 5 10 15

Leu Tyr Ala His Gly Ala Pro Gln Thr Ile Thr Glu Leu Cys Ser Glu

20 25 30

Tyr Arg Asn Thr Gln Ile Tyr Thr Ile Asn Asp Lys Ile Leu Ser Tyr

35 40 45

Thr Glu Ser Met Ala Gly Lys Arg Glu Met Val Ile Ile Thr Phe Lys

50 55 60

Ser Gly Glu Thr Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp

65 70 75 80

Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Thr

85 90 95

Tyr Leu Thr Glu Thr Lys Ile Asp Lys Leu Cys Val Trp Asn Asn Lys

100 105 110

Thr Pro Asn Ser Ile Ala Ala Ile Ser Met Lys Asn

115 120

<210> 3

<211> 375

<212> DNA

<213> Unknown

<220>

<221> gene

<222> (1)..(375)

<223>CTB DNA sequence dnas

<400> 3

atgattaaat taaaatttgg tgtttttttt acagttttac tatcttcagc atatgcaaat 60

ggaacacctc aaaatattac tgatttgtgt gcagaatacc acaacacaca aatacatacg 120

ctaaatgata agatattttc gtatacagaa tctctagctg gaaaaagaga gatggctatc 180

attactttta agaatggtgc aacttttcaa gtagaagtac caggtagtca acatatagat 240

tcacaaaaaa aagcgattga aaggatgaag gataccctga ggattgcata tcttactgaa 300

gctaaagtcg aaaagttatg tgtatggaat aataaaacgc ctcatgcgat tgccgcaatt 360

agtatggcaa attaa 375

<210> 4

<211> 124

<212> PRT

<213> Unknown

<220>

<221> UNSURE

<222> (1)..(124)

<223>CTB amino acid sequences

<400> 4

Met Ile Lys Leu Lys Phe Gly Val Phe Phe Thr Val Leu Leu Ser Ser

1 5 10 15

Ala Tyr Ala Asn Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu

20 25 30

Tyr His Asn Thr Gln Ile His Thr Leu Asn Asp Lys Ile Phe Ser Tyr

35 40 45

Thr Glu Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys

50 55 60

Asn Gly Ala Thr Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp

65 70 75 80

Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala

85 90 95

Tyr Leu Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys

100 105 110

Thr Pro His Ala Ile Ala Ala Ile Ser Met Ala Asn

115 120

<210> 5

<211> 747

<212> DNA

<213> Unknown

<220>

<221> unsure

<222> (1)..(747)

<223> LTB-CTB DNA

<400> 5

atgaataaag taaaatgtta tgttttattt acggcgttac tatcctctct atatgcacac 60

ggagctcccc agactattac agaactatgt tcggaatatc gcaacacaca aatatatacg 120

ataaatgaca agatactatc atatacggaa tcgatggcag gcaaaagaga aatggttatc 180

attacattta agagcggcga aacatttcag gtcgaagtcc cgggcagtca acatatagac 240

tcccagaaaa aagccattga aaggatgaag gacacattaa gaatcacata tctgaccgag 300

accaaaattg ataaattatg tgtatggaat aataaaaccc ccaattcaat tgcggcaatc 360

agtatgaaaa acatgattaa attaaaattt ggtgtttttt ttacagtttt actatcttca 420

gcatatgcaa atggaacacc tcaaaatatt actgatttgt gtgcagaata ccacaacaca 480

caaatacata cgctaaatga taagatattt tcgtatacag aatctctagc tggaaaaaga 540

gagatggcta tcattacttt taagaatggt gcaacttttc aagtagaagt accaggtagt 600

caacatatag attcacaaaa aaaagcgatt gaaaggatga aggataccct gaggattgca 660

tatcttactg aagctaaagt cgaaaagtta tgtgtatgga ataataaaac gcctcatgcg 720

attgccgcaa ttagtatggc aaattaa 747

<210> 6

<211> 248

<212> PRT

<213> Unknown

<220>

<221> UNSURE

<222> (1)..(248)

<223>LTB-CTB amino acid sequences

<400> 6

Met Asn Lys Val Lys Cys Tyr Val Leu Phe Thr Ala Leu Leu Ser Ser

1 5 10 15

Leu Tyr Ala His Gly Ala Pro Gln Thr Ile Thr Glu Leu Cys Ser Glu

20 25 30

Tyr Arg Asn Thr Gln Ile Tyr Thr Ile Asn Asp Lys Ile Leu Ser Tyr

35 40 45

Thr Glu Ser Met Ala Gly Lys Arg Glu Met Val Ile Ile Thr Phe Lys

50 55 60

Ser Gly Glu Thr Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp

65 70 75 80

Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Thr

85 90 95

Tyr Leu Thr Glu Thr Lys Ile Asp Lys Leu Cys Val Trp Asn Asn Lys

100 105 110

Thr Pro Asn Ser Ile Ala Ala Ile Ser Met Lys Asn Met Ile Lys Leu

115 120 125

Lys Phe Gly Val Phe Phe Thr Val Leu Leu Ser Ser Ala Tyr Ala Asn

130 135 140

Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Tyr His Asn Thr

145 150 155 160

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

165 170 175

Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Thr

180 185 190

Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys

195 200 205

Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu

210 215 220

Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala

225 230 235 240

Ile Ala Ala Ile Ser Met Ala Asn

245

<210> 7

<211> 47

<212> DNA

<213> Unknown

<220>

<221> unsure

<222> (1)..(47)

<223>LTB-Fwd primer sequences

<400> 7

cgggatccat gaataaagta aaatgttatg ttttatttac ggcgtta 47

<210> 8

<211> 36

<212> DNA

<213> Unknown

<220>

<221> unsure

<222> (1)..(36)

<223>LTB-Rev primer sequences

<400> 8

ctagggtctc catgtttttc atactgattg ccgcaa 36

<210> 9

<211> 52

<212> DNA

<213> Unknown

<220>

<221> unsure

<222> (1)..(52)

<223>CTB-Fwd primer sequences

<400> 9

ctagggtctc aacatgatta aattaaaatt tggtgttttt tttacagttt ta 52

<210> 10

<211> 34

<212> DNA

<213> Unknown

<220>

<221> unsure

<222> (1)..(34)

<223>CTB-Rev primer sequences

<400> 10

ccctcgagtt aatttgccat actaattgcg gcaa 34

Claims (10)

1. a kind of pneumococcus combined vaccine using recombinant vector albumen, which is characterized in that the vaccine includes：It is modified with life The pneumococcal antigens of object element, the recombined protein carrier and Streptavidin for being modified with biotin, recombined protein carrier is by large intestine Bacillus heat labile enterotoxin B subunit (LTB) albumen and cholera toxin B albumen (CTB) recombination obtain, described Be decorated with the pneumococcal antigens of biotin and the recombined protein carrier for being modified with biotin be coupled to by biotin it is described On Streptavidin, the pneumococcal antigens on the Streptavidin and the conjugated connection of recombined protein carrier.

2. the pneumococcus combined vaccine according to claim 1 using recombinant vector albumen, which is characterized in that LTB cores Acid sequence is bridged with CTB nucleic acid sequences by primer sequence, the LTB-CTB nucleic acid sequences such as sequence table SEQ ID NO of structure：5 It is shown, the carrier protein sequence such as sequence table SEQ ID NO of coding：Shown in 6.

3. the pneumococcus combined vaccine according to claim 2 using recombinant vector albumen, it is characterised in that：Coding The nucleotide sequence of LTB albumen such as sequence table SEQ ID NO：Shown in 1, the amino acid sequence such as sequence table SEQ ID of coding NO：Shown in 2.

4. the pneumococcus combined vaccine according to claim 3 using recombinant vector albumen, it is characterised in that：Coding The nucleotide sequence of CTB albumen such as sequence table SEQ ID NO：Shown in 3, the amino acid sequence such as sequence table SEQ ID of coding NO：Shown in 4.

5. using the pneumococcus combined vaccine of recombinant vector albumen according to Claims 1 to 4 any one of them, feature exists In：Pneumococcal antigens include that pneumococcal protein antigen, pneumococcal polysaccharide antigen and/or pneumococcal polysaccharide-protein are sewed Close antigen.

6. the pneumococcus combined vaccine according to claim 5 using recombinant vector albumen, it is characterised in that：Pneumonia ball Mycoprotein antigen includes：Pneumococcus dissolved blood protein Ply, modified pneumococcus dissolved blood protein △ A146Ply, pneumococcus haemolysis Protein derivatives PlyD1, pneumococcus dissolved blood protein derivative PlyD B, pneumococcus dissolved blood protein derivative PlyDT, pneumonia Coccus surface protein C, Pneumococcal Surface adhesion protein A, Pneumococcal Surface adhesion protein C, three histidine egg of pneumococcus White D and/or pneumococcus stick virulence factor A.

7. the pneumococcus combined vaccine according to claim 5 using recombinant vector albumen, which is characterized in that the lung Scorching Streptococcus polysaccharides antigen is the capsular polysaccharide on separating-purifying Pneumococcal serotype pod membrane, the serotype packet of the pneumococcus Include 1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and/or 33F。

8. a kind of as claim 1~7 any one of them uses the preparation of the pneumococcus combined vaccine of recombinant vector albumen Method includes the following steps：

(1) pneumococcal antigens and biotin are added in the first phosphate buffered saline solution, the first reaction are carried out under the conditions of being protected from light, Modified biological element on pneumococcal antigens, reaction product is set to remove extra biotin through dialysis, be modified after freeze-drying There are the pneumococcal antigens of biotin；Recombined protein carrier and biotin are added in the second phosphate buffered saline solution, item is protected from light The second reaction is carried out under part, and modified biological element of dialysing on recombined protein carrier, reaction product is made to remove extra biology through dialysis Element obtains the recombined protein carrier for being modified with biotin after freeze-drying；

(2) phosphate buffered saline solution containing Streptavidin is prepared, the pneumococcal antigens for being modified with biotin are added and is repaiied It is decorated with the recombined protein carrier of biotin, third reaction is carried out under the conditions of being protected from light, obtains the first reaction solution；

(3) it is added in the first reaction solution obtained by step (2) and agent progress conjugation reaction is conjugated, sediment is collected by centrifugation, it is described Sediment obtains the pneumococcus combined vaccine using recombinant vector albumen through gel column purification after freeze-drying.

9. the preparation method of the pneumococcus combined vaccine according to claim 8 using recombinant vector albumen, feature It is：In the step (1), in first phosphate buffered saline solution, the mass ratio of the pneumococcal antigens and biotin It is 1: 0.05~0.15, a concentration of 0.1~0.2mol/L of the phosphate-buffered salt；It is described first reaction time be 30~ 60min；In second phosphate buffered saline solution, the recombined protein carrier, the phosphate-buffered salt a concentration of 0.1~ 0.2mol/L；The time of second reaction is 30~60min；In the step (2), the phosphoric acid containing Streptavidin is slow It rushes in salting liquid, Streptavidin, the pneumococcal antigens that are modified with biotin, the recombined protein carrier for being modified with biotin Mass ratio is 0.1~0.2: 1.5~4.5: 1；The time of the third reaction is 30~60min.

10. the preparation method of the pneumococcus combined vaccine using recombinant vector albumen according to claim 8 or claim 9, It is characterized in that：In the step (3), the conjugated agent is carbodiimide, and the conjugation reaction reacts 4 under room temperature acidic environment ~6d.