CN107080841A

CN107080841A - Cholera rotavirus combined vaccine based on recombinant vector albumen and preparation method thereof

Info

Publication number: CN107080841A
Application number: CN201710258940.4A
Authority: CN
Inventors: 史晋; 马涛; 王文灏; 李津
Original assignee: BRAVOVAX Co Ltd
Current assignee: Bravovax Co ltd; SHANGHAI BOWO BIOTECHNOLOGY CO Ltd
Priority date: 2017-03-30
Filing date: 2017-04-19
Publication date: 2017-08-22
Anticipated expiration: 2037-04-19
Also published as: CN107098976A; CN107080841B; CN107050443B; CN107050443A; CN107098976B

Abstract

The invention discloses a kind of cholera rotavirus combined vaccine based on recombinant vector albumen and preparation method thereof, combined vaccine includes：A. cholera vaccine, including：Heat-labile enterotoxin B (LTB) albumen and cholera toxin B albumen (CTB) recombinant protein, and cholera antigen；B. Rotavirus Vaccine；Wherein, Rotavirus Vaccine faces with suspension with cholera vaccine.The carrier protein that cholera rotavirus combined vaccine based on recombinant vector albumen that the present invention is provided and preparation method thereof is used not only has good immunogenicity, with more good immunologic adjuvant effect, good mucosa-immune effect can be caused, can as a variety of antigens excellent carrier, be with a wide range of applications.

Description

Cholera based on recombinant vector albumen-rotavirus combined vaccine and preparation method thereof

Technical field

The present invention relates to a kind of cholera based on recombinant vector albumen-rotavirus combined vaccine and preparation method thereof, category In biomedicine field.

Background technology

First, pathogenic microorganism and vaccine

Human body or animal body can be caused to occur the microorganism of infectious disease, referred to as pathogenic microorganism or pathogenic microorganisms.Infect Refer to after 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 mutually, therefore It can produce communicable diseases, be on the one hand pathogenic or virulence depending on the pathogenecity of pathogenic microorganism, another aspect is additionally depended on The resistance of body is immunity.Pathogenic bacteria causes the capacity of water of infection, is exactly the virulence or pathogenic of bacterium.Carefully The power of the presence or absence of bacterium power and virulence depends primarily on its invasiveness, toxigenicity and the ability for causing hypersensitivity.

Bacteriogenic toxin can be divided into exotoxin and the major class of endotoxin two.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 be produced 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 is produced, clostridium tetani produce Enterotoxin, botulinum toxin of clostridium botulinum generation that random vibrios produces etc..Most of gramnegative bacteriums can produce endogenous toxic material Element, actually it be present in the outer layer of bacteria cell wall, belong to the part of cell membrane, be not secreted into generally In environment, only just discharged after bacterolysis, thus referred to as endotoxin, its toxicity is lower than exotoxin, antigenicity It is weak.

The Different Individual of same organism, after they are contacted with pathogen, some illness, what is had then safe and sound, reason It is that the immunity of Different Individual is different.It is immunized just referring to body identification and excluding antigen foreign matter (such as pathogenic microorganism etc.) A kind of aversion response.In general, it be to body it is favourable, in exception conditions, it is also possible to damage body.Human body It is immune to be divided into nospecific immunity and specific immunity.Wherein specific immunity refers to body for a certain or a certain micro- life of class Special resistance produced by thing or product.And to be scientist develop vaccine body is produced specific immunity resistance disease The biological products that pathogenic microorganism is encroached on human body, are generally prepared from itself by pathogenic microorganism.Bacterium, virus and The pathogenic microorganisms such as rickettsia are made after vaccine, injection body, body is produced specificity or sensitization lymphocyte, Secretory antibody, reaches specific immunity effect.

And vaccine is divided into therapeutic and preventative two kinds, disease is treated by therapeutic vaccine, and passes 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 epidemic diseases Seedling is to prevent, bacterium, virus and fungi etc., infects the various diseases caused, drastically increases the healthy water of the mankind It is flat.Continuing to develop for biotechnology, promotes the variation of vaccine kind.There is inactivation to infectious disease caused by pre- anti-virus The vaccine that virus technology is developed, such as Vaccinum Encephalitidis Epidemicae, polio vaccine, influenza vaccines；Attenuated virus technological development Attenuated live vaccine out, such as Rotavirus Vaccine, oral polio virus vaccine, measles virus vaccines, parotitis disease Malicious vaccine, rubella virus vaccine and chicken pox vaccine etc..Useful proteins and polysaccharide to prevent bacterial infectious disease etc. are biological big The bacterium class vaccine that molecular purifications technological development comes out, such as tetanus toxoid, diphtheria toxoid, DT-Pa and its Subcellular components, epidemic meningitis Streptococcus polysaccharides and 23 valency pneumococal polysaccharides etc..More advanced useful half chemical combination skill Prevention meningitis and the bacterial vaccine of pneumonia that art is developed, 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 right Updating for biotechnology, can develop more new generation vaccine products and the mankind are good for deal with different pathogenic microorganisms The challenge of health.

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 parasitism The infection of worm all originates in mucous membrane surface.Mucosal immune system is first of immunization barrier of body resistance pathogen invasion, tool There is the independent immune system of unique texture and function, have positive effect for the field planting and intrusion for preventing and treating pathogen.It is different from Traditional immune system, mucosal immune system is that a large amount of immunocytes and immune molecule disperse are solid under mucosal epithelium or mucous membrane Have layer (diffused lymphoid tissue), or a mucosa-associated lymphoid tissue being gathered into by single or multiple lymph follicles, body 50% with On lymphoid tissue and more than 80% immunocyte concentrate on mucosal immune system.Mucosa-immune can induce local mucous membrane The protection antibodies such as secretory IgA (sIgA), IgM and IgG are produced, and can induce the mucous membrane at other positions and also produce sIgA, this It is the main mechanism of mucosa-immune protective effect.In addition, mucosa-immune also induces mucous membrane ctl response, and produce secretion IFN- C CD4⁺T cell, this prevention invaded for pathogen and removing are very important.Therefore mucosa-immune is protection body The important barrier invaded from pathogen, it is significant in the design of vaccine.

Vaccine based on mucosa-immune often reacts weaker due to the immune of induction, and the duration is short, it is difficult to obtain preferable Immune protective effect.It is now recognized that the less immunogenic of such as recombinant protein, synthesis polypeptide and DNA antigens is important original Therefore one, 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, To protrude mucosa-immune etc..The problem of these aspects, makes the use of adjuvant seem particularly urgent and important, therefore exempts from for mucous membrane The research of epidemic disease adjuvant has become a study hotspot of infection immunity and vaccines arts.At present, the mucosa-immune assistant reported Agent can be divided mainly into three classes：The first kind is bacterial substances, including albumen (mainly bacteriotoxin) and nucleic acid；Equations of The Second Kind is Various cell factors；3rd class is antigen delivery system.

In bacteriotoxin as Mucosal Adjuvants it is most-often used be E.coli LT (LT) and cholera Toxin (CT), people have been carried out more research to it.Close connection toxin (zonula occludens toxin, Zot), Cytotoxicity 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, 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 can then induce the CD4 of mixing⁺Th1 and Th2 type cells.CT and LT are strong Mucosal Adjuvants, but Because there is toxicity to hinder its application in vaccine for man field for it, therefore removal toxicity or reduction toxicity are built, protected simultaneously The mutant for staying adjuvant attribute is very necessary.

LT is one of certified maximally 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 (approach in oral or stomach) injection LT to mouse, It can be found in induction hypersecretion and systemic antibody response, Respiratory Tract of Mice secretion and respiratory tract content substantial amounts of Anti- LT IgA antibody.Take respiratory mucosa to cut into slices, substantial amounts of thick liquid cell can be seen in mucosa lamina propria aggregate nodules.LT is lured It is antigen deposition site that the mucosal secretory antibodies response led is most strong, but is not limited to antigen deposition site, at other Mucous membrane effective-site also has same response.

LT and LTB have 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 Cell produces long-term anamnestic response.

3rd, Escherichia coli and its epidemiology

Escherichia coli are ETEC (Escherichia coli, E.coli) abbreviations, by German bacteriologist Theodor Escherich are separated most earlier than 1885 from infant faeces, are enterobacteriaceae (Entero- Bacteriaceae), the member of Escherichia (Escherichia), original name is Bacterium coli commune, meaning Think of refers to enteron aisle common bacteria.In fact, only a small number of a part of E.coli bacterial strains can directly result in host's disease in enteron aisle Disease, 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 draw Play parenteral infection.Not for conditioned pathogen, (such as resistance is reduced pathogenic E.coli, normal flora is out of proportion or is moved Position etc.), but the Escherichia coli of some serotypes group (type) are pathogen in itself.For the enteropathogenic E. Coli of enteral, Bu Tong mainly there are following several types with epidemic etc. according to pathogenesis, clinical symptoms：Produce enterotoxin large intestine bar Bacterium (Enterotoxigenic E.coli, ETEC), shiga toxin producing escherichia coli (Shiga toxin-producing E.coli, STEC), the wicked bacterium (Enterohemorrhagic E.coli, EHEC) of enterohemorrhagic large intestine, the pathogenic large intestine bar of intestines Bacterium (Enteropathogenic E.coli, EPEC) and enteroinvasive E.Coli (Enteroinvasive E.coli, EIEC) etc..In addition, also avian pathogenic 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), the wicked bacterium (Enteroaggregative E.coli, EAEC) of intestines adhesion large intestine and intestines concentration large intestine traitor Bacterium (Enteroaggregative E.coli, EAggEC) etc..

Most common of which is diarrhoea caused by ETEC.ETEC is popular in late period 1960s earliest, is people and dynamic One of important pathogen body of thing infectious diarrhea.Some data show that ETEC is that indigenous be grown up in resident in Cholera Epidemic Area causes 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 water source and food faecal contamination and draw play one of key factors such as diarrhoea, nausea, low fever, cramp.

It is heat-labile toxin (Heat-labile toxin, LT) and resistance to warmheartedness poison that ETEC, which produces 2 kinds of different type toxin, 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, at 65 DEG C, 30min 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 are raised, and are upset dielectric metabolism, are caused diarrhoea.LT and ST differences are that LT is a kind of immune egg In vain, there is more substantial connection with cholera enterotoxin (Cholrea toxin, CT), constitute a toxin family.ETEC also may be used Human body is caused to produce choleraic diarrhea, 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 people's species, it Chemical property and immunological characteristic it is very much like.Different according to encoding gene, LT is divided into two types again：LT1 and LT2. LT1 is encoded by the Large plasmid in endochylema, most commonly seen in nature, is the main component that LT plays biological activity.Typically Described LT refers to LT1 mostly；And LT2 is encoded by chromosomal DNA and produced.Although their composition is similar with the mode of action, Without genetic homology and cross immunogenicity.Described LT refers both to LT1 below.

LT full length gene 1148bp is encoded, on the Large plasmid in wild type enterotoxigenic E．Coli cell dress.LT Operator contains 2 structural genes, respectively encodes the toxB genes of B subunits and the toxA genes of coding A subunits, it Be chained together and can be transcribed into 1 mRNA chain.Wherein toxB genes are located at toxA genes 3' end, its ribose Body binding site (GGGA) is located in toxA genes, and 2, toxA3' ends codon (TTATGA) and 2, toxB 5' ends are close Numeral (ATGAAT) has 4 nucleotides overlapped.B subunits coding region gene total length 372bp, is encoded by 124 amino acid The polypeptide of composition；A subunits coding region gene total length 777bp, encodes the polypeptide of 259 amino acid.ToxB is in initiation codon There are SD sequences at 3 preceding nucleotides, 5 nucleotides therein can be with 5 in the nucleotides of 12, ends of 16S rRNA 3' Complementation, and nearly ATG ends base is A.These structures are conducive to improving ribosomal joint efficiency, and toxB mRNA two grades of knots Structure is also beneficial to the translation of toxB genes, so as to result in same promoter downstream, although toxB is located at distal end, expression Amount but exceedes as many as 5 times of toxA.This more special structure may be more convenient for toxin promoter 2 subunits are played compared with Good regulating and controlling effect, adjusts their expression quantity, makes it easy to reach the best condition of toxin synthesis.

A, B subunit in endochylema all exists to carry signal propeptide.It can be just assembled into after cell membrane is passed through Whole LT.The effect of B subunits is the GM-1 ganglioside receptors specific binding with eukaryotic cell membrane surface, with facility Enter target cell in A subunits.A subunits have the ADP- ribosylation transferase actives of GTP dependences, are situated between by G-protein Intracellular cAMP degraded and balance are upset in the ADP- ribosylation reaction led, and stimulate cAMP contents to increase, so as to trigger toxicity Effect.LT-h and LT-p act on different G-proteins, and the former is Gs albumen, and the latter is Gi albumen, and final effect is all led Protein kinase A inactivation is caused, causes a series of biochemical reaction, causes cAMP contents to increase, intestinal mucosa water and electrolyte is stimulated Excessive secretion, cause diarrhoea.

LTB has been found to as the function of immunologic adjuvant, and with higher and higher attention rate, but LTB is separately as load The research of body protein is few, and single LTB albumen is only used as LTA carrier at present, and which greatly limits LTB Use scope.LTB has good architecture basics due to it, with the protein carrier tool built after other protein fusion expressions There is extensive prospect of the application.

4th, comma bacillus and its epidemiology

Cholera is a kind of severe intestinal infectious disease of threat human survival, is one of China's category A infectious disease.Own history Since record, the whole world has seven cholera and is very popular, and is very popular results in people's infection hundreds thousand of or even up to a million every time, thousands of People up to ten thousand are dead.Even to this day, cholera is still wreaked havoc in global multiple countries.

Cholera is the severe intestinal infectious disease caused by comma bacillus, clinically rushed down with violent Silent Neuritis tell, water in which rice has been washed sample Stool, serious dehydration, muscle cramp and peripheral circulatory failure etc. are characterized.In the 1960s, once developing in the world A variety of parenterally cholera vaccines, but due to adverse reaction weight, protection is only within 50%, and the protection period is short, WHO biological standards Change from the Committee of Experts is decided by 1999 and stop using.

Oral vaccine can induce enteron aisle to produce antibody, and enteron aisle is the front for resisting cholera.Moreover, oral vaccine Medical treatment cost can also be reduced, it is to avoid unnecessary medical injury, therefore, oral cholera vaccine achieves large development in recent years.

Currently available oral cholera vaccine mainly has：Inactivate whole cell cholera vaccine (WC vaccines), inactivation whole cell Plus B subunits cholera vaccine (WC/rBs vaccines) and attenuation cholera live vaccine (CVD103-HgR attenuated live vaccines).

Killed whole-cell vaccine (WC) is initially, for O1 groups of cholera, to be achieved in the field test of Vietnam good Protecting effect (Trach DD, Clemens JD, Ke NT, et al.Field tial ofa locally produced, Killed, oral cholera vaccine in Viet Nam.Lancet, 1997,349 (9047)：231-235.), each year Effective percentage of the age group after 8 months is 66%, but only in a few countries production such as Vietnam and Indonesia.Then, according to WHO The suggestion for disease vaccine steering committee of suffering from diarrhoea, is developed into the full cell divalence vaccine of 2nd generation inactivation, this vaccine is by O1 Group and the full cellularity of O139 comma bacillus, wherein containing the O139 of 5 × 1010cfu Formalin inactivations.Hanoi's Research is oral 2 doses, every dose of 1.5ml, is spaced 2 weeks, but immune effect is undesirable.

Choleratoxin B subunit-inactivation comma bacillus whole cell vaccine (BS-WC) adds cholera on the basis of WC vaccines Toxin B subunit (BS).BS is an advantage over cholera toxoid but is slightly inferior to the good immunogene of cholera toxin.With BS and inactivation suddenly The combination-vaccine (BS-WC) that random vibrios whole cell vaccine (WC) is constituted entered in 1985~1989 years in Bangladesh Cholera Epidemic Area Field test is gone.After 63498 people's vaccine inoculations the immune protective rate of initial 6 months be 85%, 3 years after be down to 51%, it was demonstrated that BS-WC vaccines have good protective effect to cholera.It is this sub- single by the cholera toxin B recombinated in terms of O1 groups of cholera are prevented The oral vaccine of the full cell composition of O1 group's cholera of position and inactivation, has been achieved for good effect, therefore, world health group Knit to give this vaccine and recommend.In the nineties in last century, the vaccine is in Sweden's approval registration production.But this vaccine is needed Hydrochloric acid in gastric juice is neutralized using antiacid when oral, therefore adverse reaction is than larger.And in cholera explosively, inherently lack Weary clean water source, in addition it is also necessary to which substantial amounts of water dissolves antiacid, virtually improves the cost of medication.

Entering human trial in BS-WC vaccines proves that it has good security, immunogenicity and higher protective rate On the basis of, the development work of oral cholera vaccine replace natural again to the deeper step of step by step advance one using restructuring BS (rBS) Cholera BS, constructs rBS-WC cholera Vaccines.Clear an ancient unit of weight of Bioengineering Research Institute of Chinese military medicine academy of sciences horse etc. is studied Member, have developed first class national new drug rBS-WC oral cholera vaccines.This is the oral cholera epidemic disease of China's scientist's independent development One of seedling, oral cholera vaccine as World Health Organization's formal recommendation.

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 into after synthesis between 194,195 residues CTA1 and CTA2, with disulfide bond.Wherein CTA1 has ADP- ribosyl-transferase activities, has close with CT adjuvant effect Relation is cut, CTA2 then connects CTA1 and CTB.CTB 5 subunits are combined into pentamer with non-covalent bond each other, then pass through CTA2 Around CTA formation AB5 structures.Adhesion between the subunit of CTB pentamers is very strong, more than their adhesions with A subunits.

CTB is widely paid attention to as the application of immune carrier in recent years, and CTB is as nontoxic unit, with combining 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 produced, CTB is also used as some allogenic polypeptides Stable carrier, after the antigen of it and chemical coupling or Gene Fusion enters in vivo simultaneously, can evoke fusion epitope antigen Immunogenicity, makes body produce strong immune response, reaches immanoprotection action.

CTB has very strong immunogenicity, can strengthen bacterial virus and the immunogenicity of other antigens, particularly pass through Mucosal immune, can not only strengthen the specific IgG antibodies response of serum, also can induce stronger specific performance mucous membrane IgA immune responses, with antigenic feature of epitope can be strengthened.CTB can be with dendron shape in stimulator antigen presenting cell Cell is to the transport capacity of antigen, and BMDC is used to transporting antigen to lymphoid organ, and activation antigen CD4+ and CD8+ T cells.CTB can activate the maturation of BMDC, make that the dendron of BMDC is longer, and volume is bigger.CTB cells are also Can adjust the t cell responses in being, and internal antibody generation.

5th, rotavirus and its epidemiology

Rotavirus is to cause one of main pathogens of infant's severe diarrhea.The whole world there are about 1 every year.1100000000 are less than 5 The infant in year suffers from RV diarrhoea, and 350,000 to 590,000 infants are there are about every year and die from RV diarrhoea, wherein 82% occurs in development China Family.In terms of epidemiology, in developed country and developing country, the incidence of disease of rotavirus is not significantly different, and this shows to change Kind living-hygienic environment does not have remarkable result to control rotavirus diarrhea.Specific drug is there is no for diarrhoea caused by RV at present, Vaccine inoculation turns into the means of effective and unique prevention and control RV diarrhoea.Widely used three kinds of RV vaccines are at this stage Attenuated live vaccine, although can effectively lower the morbidity and mortality of rotavirus infection, but still suffer from potential danger of causing a disease Danger, vaccine is possible to be mutated because occurring to drive in the wrong direction and recovers virulence in human body.

Rotavirus Reoviridae, rotavirus.Diameter is about 65~75nm under Electronic Speculum, no coating, there is bilayer Shell, the double-stranded RNA genome containing 11 sections.Mrna length is from 680~3300bp, and 11 genes encode 6 knots altogether Structure albumen (VP1, VP2, VP3, VP4, VP6 and VP7) and 6 non-structural proteins (NSP1~NSP6).VP4 and VP7 have type Specific epitope, can produce the specific neutralizing antibody of inducing protective immunity response, and VP6 has group specificity Antigenic determinant, NSP4 albumen then there is enterotoxin to act on, and be the key factor for causing diarrhoea.

Human rotavirus is greatly.Up to the present, due to the independent assortment of G and P albumen and separation cause to Few 42 kinds different P-G serotypes combinations are determined, cause the generation of different bacterial strains.Fortunately, it is only a small number of special Rotavirus strain have the whole world propagate infection the mankind ability.General RV genotypings are compiled with albumen in the world Code frame (ORF) nucleotide sequence homology is relatively determined.Conventional rotavirus serum (gene) parting is mainly by two Structural proteins VP4 and VP7 are determined.According to VP7 genes, rotavirus is divided into 19 kinds of G types (serotype)；According to VP4 genes, Rotavirus is divided into 28 kinds of p-types (genotype).VP4 serotypes, have had now been found that two kinds of types：G1G3 and G4 are that same type is P1A.G2 is P1B.

The colyliform vaccine used at present on the market has the Rotarix (G1P types) that GlaxoSmithKline PLC company is produced, Merck ＆ Co., Inc. The Rotateq (G1~G4P types) of production and rood prestige (G10P) type of Lanzhou Institute of Biological Products of China production.Wherein Rotavirus Vaccine Rotarix and Rotateq has found do not have in the multinational large-scale third stage evaluation studies more than 60,000 people Cause the risk of any unnecessary entembole.But the price of both vaccines is slightly higher, numerous developing countries can not undertake big face Long-pending immunoprophylaxis.And rood prestige, only in CHINESE REGION production and sales, its limitation is also foreseeable.Various countries' health care Tissue is actively researching and developing new Rotavirus Vaccine, but does not have generally acknowledged breakthrough also at present.

Because rotavirus and cholera virus are to cause diarrhoea to be reacted, and onset environment similarity is higher, in order to mitigate The financial burden of vaccine recipient and inoculation times, being badly in need of that research and development are a kind of can be with combined immunization rotavirus and cholera virus Combined vaccine.

The content of the invention

In view of the above-mentioned problems existing in the prior art, it is an object of the invention to provide it is a kind of based on recombinant vector albumen suddenly Unrest-rotavirus combined vaccine and preparation method thereof.

For achieving the above object, the cholera based on recombinant vector albumen that the present invention is used-rotavirus joint epidemic disease Seedling and preparation method thereof technical scheme is as follows：

First purpose of the present invention is to provide a kind of cholera based on recombinant vector albumen-rotavirus joint epidemic disease Seedling, including：

A. cholera vaccine, including：Cholera antigen and antigen vectors, wherein cholera antigen are coupled with antigen vectors, and antigen is carried Body is recombined protein carrier, sub- by Heat-labile enterotoxin B (LTB) albumen and comma bacillus enterotoxin B Unit-protein (CTB) genetic recombination obtains recombinant vector albumen；

B. Rotavirus Vaccine；

Rotavirus Vaccine faces with suspension with cholera vaccine.

LTB nucleotide sequences are bridged with CTB nucleotide sequences by primer sequence, the LTB-CTB nucleotide 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.

Preferably, CTB nucleotide sequences are selected from the CDS areas of O139 groups of cholera complete genome sequences.

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

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

Preferably, cholera antigen is proteantigen and/or polysaccharide antigen.

It is highly preferred that cholera antigen is cholera capsular polysaccharide.

As a preferred embodiment, cholera antigen is vibrio cholerae O 139 group cholera capsular polysaccharide.

It is highly preferred that comma bacillus polysaccharide and carrier protein in mass ratio 0.4~0.8：1 is coupled.

Cholera vaccine is any one of spray-type, liquid dosage form, capsule formulation, freeze dried powder, tablet and pill.

Cholera based on recombinant vector albumen-rotavirus combined vaccine and preparation method thereof also includes sucrose, for making For the freeze drying protectant of the lyophilized formulations of cholera vaccine.

It is any to think to fall under the scope of the present invention as the component of vaccine freeze-drying protective agent, the present invention In freeze drying protectant be not limited to sucrose.

Preferably, Rotavirus Vaccine includes inactivated rotavirus vaccine or rotavirus attenuated live vaccine.

Second object of the present invention is to provide a kind of cholera based on recombinant vector albumen-rotavirus combined vaccine And preparation method thereof, step includes：

S1. according to LTB and CTB CDS areas nucleotide sequence, two pairs of primers are designed, pET28a-LTB-CTB plasmids are built, After PCR amplifications, by BamH I and the double digestions of Xho I, gel reclaims LTB-CTB fragments and expression vector pET28a；

S2. connection converts and monoclonal amplification is carried out after e.colistraindh5α, screening positive clone, is reflected after IPTG inductions It is fixed；

S3. cholera antigen is coupled with recombinant vector albumen, detection combines product after coupling.

When selecting comma bacillus polysaccharide as cholera antigen, S3 is specially：

Cholera polysaccharide is coupled with recombinant vector albumen, polysaccharide is 0.5~4 in mass ratio with carrier protein：1, it is even Detection combines product after connection.

When selecting cholera albumen as cholera antigen, S3 is specially：

Cholera albumen is coupled with recombinant vector albumen, cholera albumen is 0.4~1 in mass ratio with monomeric protein： 1, detection combines product after coupling.

Compared with prior art, the present invention is using the LTB-CTB protein carriers of restructuring and the capsular polysaccharide of O139 plants of cholera Coupling, faces with being suspended with colyliform vaccine, can not only cause the sero-immunity of vaccinee, can more cause glutinous in enteron aisle Film is immunized.The combined vaccine toxin expelling rate that the present invention is provided is low, and the influence of environment is smaller to external world, and reduce horizontal transmission can Can property.Because the carrier protein in the present invention and cholera antigen have homology, GL-PP conjugate can be spontaneously formed, The antigen can not only cause the more comprehensive immune response of body, and under synergy, with more preferable immune range and Persistence, with wide market prospects.

Brief description of the drawings

Fig. 1 is the structure schematic diagram for the structure pET28a-LTB-CTB plasmids that the present invention is provided.

Fig. 2 is the plasmid schematic diagram for the structure pET28a-LTB-CTB plasmids that the present invention is provided.

Fig. 3 is the restructuring LTB-CTB carrier proteins induction purification result schematic diagram that the present invention is provided.

Embodiment

The present invention is made with reference to embodiment further in detail, intactly to illustrate.Experimental method in following embodiments, Unless otherwise specified, it is conventional method.Experiment material used is market purchase unless otherwise specified in following embodiments It can buy.

First, the clone of restructuring LTB and CTB albumen and prokaryotic expression

LT and CT etc., the glycoprotein polyprotein precursor of AB5 types six that LT is made up of A, B Liang Zhong subunits (LT-A and LT-B).A、 B Liang Ge subunits are combined by non-covalent bond, and single subunit does not have bioactivity, and being only combined together just has entirely The biology and chemical characteristic of toxin.A subunits are the virulence centers of toxin, and molecular weight is about 28KD, with ADP- ribosyls Change enzymatic activity.A subunits can be cut into two fragments of A1 (LT-A1) and A2 (LT-A2), A1 fragments and A2 pieces with reduction reaction Section is in foldable structure and helical structure respectively, and the two is connected by disulfide bond.A1 is the active part of LT toxin, and A2 is sub- with B The connected part of unit.When the disulfide bond for connecting A1 and A2 is reduced, the A1 subunits with enzymatic activity are to be released Come.B subunits are made up of two α spirals and six β lamellar structures, a cysteine residues at its N- end and the one of C- ends Individual cysteine residues formation disulfide bond, two ends of molecule are linked together.Point that five molecular weight are about 11.5KD Son structure annular in shape arranged together, can be combined with specific receptor nervon GM1 on whip cell.A subunits pass through A2 pieces Section insertion B subunit's pentamers center, constitutes a complete LT molecule.

According to the GeneBank LTB and CTB nucleotide sequences logged in and amino acid sequence, particular sequence is as follows：

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

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

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

LTB-Fwd is followed successively by by 5 ' ends to 3 ' ends：Protection base -- restriction enzyme sites of BamH I -- LTB albumen n end sequences.Draw Thing (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 -- restriction enzyme sites of Xho I -- 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, Therefore CTB fragment is inserted in LTB fragments, is connected, is put up a bridge into performing PCR with primer, it is amplifiable to obtain recombinating LTB-CTB carriers Full length protein fragment.The complete sequence of the pET28a plasmids of structure such as sequence table SEQ ID NO：Shown in 5, the amino acid sequence of coding Such as sequence table SEQ ID NO：Shown in 6.

After carrier and PCR primer are by BamH I and the double digestions of Xho I, gel reclaims LTB and CTB fragments.After glue reclaim, With DNA ligase, 16 DEG C of connections are stayed overnight, connection conversion bacillus coli DH 5 alpha, monoclonal amplification, after small upgrading grain, BamH I and It is sequenced after the about 800bp bands of the double digestions of Xho I identification, the screening positive clone under amicillin resistance, positive-selecting, will Sequencing result is compared in NCBI websites, as a result completely correct.

2nd, the expression and purity of LTB-CTB carrier proteins is recombinated

Recombinate LTB-CTB carrier proteins and 30h is induced at 20 DEG C, IPTG concentration is 0.3mmol/L, and inductive condition is optimal.Lure Go the supernatant of appropriate ultrasonic treatment product to precipitate after leading and carry out SDS-PAGE, while setting the albumen before induction as control. After broken bacterium, it is found that marking protein exists with inclusion bodies.After inclusion body is washed, dissolved with 8mol/L urea, through multiple Property liquid renaturation, then purified through Glutathione S transferase (GST) affinity column, obtain restructuring that purity is about 93% fusion egg In vain.SDS-PAGE results show in recombinant bacterium cracking precipitation the additional band of a treaty 30kD occur, close with expected results. Expressed because albumen is main in the form of inclusion body, therefore the long-time induction of selection lower temperature, to ensure protein expression Amount.The purification result of induced expression is as shown in figure 3, band 1 is albumen Marker；2 be albumen total before induction；After 3 is inductions Total albumen；4 be supernatant after the centrifugation of bacteria breaking liquid；5 be bacteria breaking liquid centrifuged deposit；Band 6 is the restructuring after freezing LTB-CTB carrier proteins.

3rd, the result experiment of restructuring LTB-CTB carrier protein connection cholera lipopolysaccharides (LPS)

The extraction of 3.1 cholera lipopolysaccharides

Lipopolysaccharide molecule is made up of O- side chains, core oligosaccharide and lipid A.O- side chains constitute oligosaccharides by 2~4 glycosyls The long-chain of repetitive structure；The branch oligonucleotide chain that core oligosaccharide is made up of 9~10 glycosyls；Lipid A moiety is the bone of lipopolysaccharides Frame, basic composition unit is the D-Glucose amine disaccharide being connected with β -1,6- glycosidic bonds.Lipopolysaccharides on comma bacillus cell membrane Both somatic antigen (O antigens), was both virulence factor and important protective antigens.

O139 serotypes comma bacillus is selected to extract lipopolysaccharides in the present embodiment.Thalline adds 95% benzene after expanding culture Phenol, ultracentrifugation collects supernatant, and it is 70% to collect and absolute ethyl alcohol to concentration of alcohol is added after supernatant, 4 DEG C overnight after centrifuge and abandon Clearly, transparent gel-form precipitation is taken to be dissolved in 2mmol/LMgSO₄In -50mmol/L Tris (pH7.6) buffer solution, add in the solution Enter DNase and RNase, put in incubator overnight；15mg Proteinase Ks are added, solution is transferred in bag filter, room temperature dialysis 24h, Dialyzate is above-mentioned buffer solution；Treat that enzyme hydrolysis is complete, bag filter is put into water, put 4 DEG C of dialysis 2d, a water is changed per 12h；Thoroughly Solution centrifugal after analysis, precipitation is dissolved in a small amount of water and freezes preservation, the LPS as purified.

The LPS of the purification after testing, confirms that its molecular size range is errorless.Total nucleic acid content≤5% in LPS samples, it is remaining Protein content≤2%.

3.2 cholera lipopolysaccharides are combined with restructuring LTB-CTB carrier proteins

The freeze-dried powder of restructuring LTB-CTB carrier proteins, which adds to be dissolved to after water for injection, needs concentration, standby after dissolving.

Lyophilized lipopolysaccharides is diluted with water for injection, 60 μ l CDAP (100mg/ are added in 1mlLPS (10mg/ml) Ml acetonitrile solution), pH5.8-6 is controlled, 30s is reacted；Then the 140 μ l TEA aqueous solution are added to pH7.0,2min is reacted； 1ml 5mg/ml restructuring LTB-CTB carrier protein solutions are added, pH8.0 are adjusted with 0.1M NaOH, 4 DEG C of rotations are stayed overnight, most After add 100 μ l ethanolamine solutions terminating reactions.

3.3 detections combine product

It will be purified with reference to product by gel permeation chromatography post, eluent is 0.2M NaCl, detects ultraviolet 280nm The light absorption value at place, collects the material in an eluting peak.

Confirm to combine containing restructuring LTB-CTB components in product using SDS-PAGE methods, and detect protein content.Using benzene Phenol-sulfuric acid method detects polyoses content, confirms that polyoses content is about 95 μ g/ml in product.

Whether combined by GM1-ELISA detections with reference to product with the GM1 of intestinal tract surface, stimulate mucosal system to produce mucous membrane IgA.Specific assay method is as follows：

1) it is coated with：Buffer solution is coated with carbonate, and GM1 is diluted to 10 μ g/ml.Add in each reacting hole of 96 orifice plates The 100 above-mentioned envelope antigens of μ l, 4 DEG C stand overnight.Next day, solution in hole is removed, washed with lavation buffer solution PBST 3 times；

2) close：The μ l of BSA solution 200 for plus 4% are in above-mentioned coated reacting hole, and 37 DEG C are placed 2 hours.Discard Confining liquid, is washed 3 times；

3) it is loaded：The CTB solution and conjugate sample solution of a series of dilution gradients are separately added into, per the μ l of hole 100, is placed in 37 DEG C are incubated 1.5 hours, are washed out 3 times；

4) primary antibody is added：1 is added per hole：The μ l of rabbit-anti CTB IgG antibodies 100 of 4000 dilutions, 37 DEG C are incubated 2 hours；

5) enzyme-added labeling antibody：In each reacting hole, the μ l of enzyme mark donkey anti-rabbit IgG antibody 100 of diluted fresh are added, 37 DEG C incubate Educate 1 hour, wash 3 times；

6) substrate solution colour developing is added：The μ l of tmb substrate solution 100 are added in each reacting hole, room temperature is placed 10~15 minutes；

7) terminating reaction：The μ l of 2M sulfuric acid 50 are added in each reacting hole；

8) reading：On ELIASA, the light absorption value in each hole, print record are detected at ultraviolet 450nm.

Testing result shows that the OD values of CTB pure proteins are 0.30 or so, and stable with GM1 binding ability.Restructuring The OD values of LTB-CTB albumen are fluctuated between 0.25~0.48, illustrate with reference to the restructuring LTB-CTB in product still maintain with The ability that GM1 is combined, and the ability is stable, and good mucosa-immune can be caused to react.

3.4 test alive

The combination product of successful connection is subjected to mouse live infection, takes the conjugate of successful connection to carry out mouse peritoneal note Administration is penetrated, selects 6~8 weeks healthy BALB/c mouses to be tested, every group 10, dosage is 1mg.First group of administration knot Close product, the unloaded restructuring LTB-CTB carrier proteins of second group of administration, the 3rd group of administration CT albumen, the 4th group of administration LT egg In vain, the 5th group of administration physiological saline is as negative blank control, Continuous Observation mouse systemic in 30 DEG C of successive administrations 3d, 3~7d First time sampling is carried out after reaction, 7d, 2 mouse samplings are put to death every 7 days each groups.Serum and small intestine mucus are collected during sampling, Jejunitas 24h before sampling, does not cut off the water supply, to reduce intestinal contents every time.Virus monitory each group mice serum IgG antibody level is taken, IgA antibody level in 1 detection small intestine mucus of every group of execution mouse.

The preparation of serum：Gather and be placed in after mouse orbit blood, blood collection after 37 DEG C of standing 2h, centrifuged with 3000r/min L0min, draws serum Cord blood, standby inspection.

The mouse put to death after blood sampling, dissects immediately, separation about 10cm jejunums stage casing.Blank pipe weight is weighed in advance, scrapes intestines Mucus is contained in Guan Zhonghou and claims its gross weight, and it is again scraped mucus weight that gross weight, which subtracts blank pipe, and then the PBS with 4 times is dilute Release, Cord blood, standby inspection.

Using IgA contents in IgG content in indirect ELISA detection blood and enteron aisle.IgG content testing result is shown in blood IgA content detection results are shown in such as table 2 below in such as table 1 below, enteron aisle：

IgG content testing result in the blood of table 1

	7d	14d	21d	28d	35d
						First group	0.413	0.608	1.003	1.011	0.907
Second group	0.132	0.159	0.274	0.351	0.237
						3rd group	0.375	0.559	0.868	0.694	0.501
4th group	0.486	0.604	0.815	0.733	0.472
						5th group	0.015	0.006	0.027	0.014	0.013

IgA content detection results in the enteron aisle of table 2

	7d	14d	21d	28d	35d
						First group	0.231	0.328	0.698	0.814	0.788
Second group	0.112	0.149	0.195	0.188	0.191
						3rd group	0.203	0.298	0.385	0.317	0.337
4th group	0.215	0.307	0.362	0.345	0.271
						5th group	0.006	0.011	0.030	0.051	0.004

Note：Each group of data is obtained through experiment is repeated several times, and data result statistical analysis is without significantly between each parallel group Difference.

From table 1 and table 2, restructuring LTB-CTB carrier proteins do not have the toxicity of comma bacillus or Escherichia coli in itself, Activation specific immune response can not be oriented, the recombinant antigen protein and simple CT albumen and LT albumen of lipopolysaccharides is connected to Compare, with stronger antibody titer, and the initiation time of immune response is short, good immune effect, immune response it is lasting when Between it is long.Mucous membrane IgA content detection result illustrates that the albumen after restructuring serves not only as good antigen vectors and played to enter live body After trigger immune response, can more trigger mucosa-immune in mucosal system, orientation enhances the immune effect of regional area.

3.5 quantizations for combining product are produced

Lipopolysaccharides and restructuring LTB-CTB carrier proteins after purification is taken, using mass ratio as 0.5~4：1 ratio carries out 3.2 Described in step combination, obtain combine product it is purified, examine after, add sucrose as freeze drying protectant, wherein sucrose exists Initial mass percentage composition in lyophilized stoste is not more than 20%, obtains recombinating cholera vaccine lyophilized formulations after freezing.

Wherein step of freeze drying is specially：

1. compound concentration is 70%, the sucrose mother liquor through 121 DEG C of sterilization treatment 15min；

2. the Pnu-Imune 23 that the gained of embodiment 1 is isolated and purified is placed in sterile chamber, add what step 1 was configured to Sucrose mother liquor, makes sucrose concentration to 10%, obtains mixing and is prepared into containing the protectant virus stock solution used semi-finished product of sucrose, then distinguishes With 1.0ml/ bottles of specification it is filling in cillin bottle to carry out follow-up lyophilized technique；

3. step 2 gained semi-finished product are carried out into pre-freeze, fast cooling maintains 15h~20h to -55 DEG C during pre-freeze；

4. semi-finished product after step 4 gained pre-freeze are subjected to first stage drying：- 45 DEG C~-40 DEG C maintenance 50h are warming up to, It is warming up to -40 DEG C~-33 DEG C maintenance 22h；

5. semi-finished product carry out second stage drying after the step 4 gained first stage is dried：Set in different time and heat up To 0 DEG C~30 DEG C, the different temperature rise periods continue to 28h；The restructuring cholera that sucrose concentration in lyophilized stoste is 10% is prepared again Vaccine, the preparation method of freeze dried vaccine refers to prior art preparation, will not be described here.

4th, the immunological evaluation of rotavirus cholera combined vaccine

4.1 prepare rotavirus cholera combined vaccine

Due to the combined vaccine in the present invention to improve the combined immunization ability of rotavirus and comma bacillus, by causing Strong mucosa-immune is main improvement direction for the resistivity of enteron aisle venereal disease poison so as to improve inoculator, therefore this reality Colyliform vaccine can be prepared using prior art by applying in example, colyliform vaccine includes inactivation colyliform vaccine and deactivation colyliform vaccine.

G3 plant of rotavirus P [2] of Vero Cell Culture Vaccines is selected in the present embodiment, purifying after virus multiplication is carried out, There are P [2] G3 virions of complete infectious after purification, and viral genome does not change after testing, it is remaining Vero cell DNA contents are no more than 500pg.

The redissolution liquid of cholera combination product lyophilized protein is used as using P [2] G3 virus liquids after purification, that is to say, that facing P [2] G3 virus liquids are added in the lyophilized formulations of the cholera vaccine combination product based on recombinant vector albumen, will combined with preceding Product is redissolved, and intramuscular injection can be carried out after being well mixed.

The immunological test of 4.2 rotavirus cholera combined vaccines

4.2.1 the antigen detection of rotavirus

Because the rotavirus strain of selection is A rotavirus, for detecting wild A rotavirus antigenic reagent box .Whether contain wheel virus antigen in ELISA (EIA) detection excrement sample.Sample melts first, then samples And virus preservation liquid dissolving is added, finally operated according to EIA kit specifications in reacting hole.

Three groups of mouse point is tested, and every group 3 parallel, and each parallel 10 mouse, testing result takes each group parallel flat Average.First group of inoculation or rotavirus cholera combined vaccine, second group of G3 plants of inoculation rotavirus P [2], the 3rd group is negative Control.After vaccine inoculation in 14d, stool in mice is gathered, every part takes 5~10g or 5~10ml or so, carried out in kit EIA is detected.There is the mouse quantity suffered from diarrhoea in statistics each group, and calculates average diarrhea rate.Testing result is as shown in table 3.

The EIA testing results of table 3

As shown in Table 3, the rotavirus cholera combined vaccine in the present embodiment is caused more compared with commercially available colyliform vaccine Antagonism or competitive relation between strong immune response, two kinds of vaccines is very small.Inoculation will not trigger inoculator strong simultaneously It is strong discomfort (except immune deficiency person), and this combined vaccine diarrhea rate compared with commercially available vaccine, adverse reaction is similar, but production The time of raw adverse reaction is short, that is to say, that the toxin expelling phase is shorter.

4.2.2 the antibody titer detection of combined vaccine

Detecting step is with 3.4 test alive steps, and that examines twice differs only in, and administration species is different.Contrast administration When cholera vaccine selection can only fit capsulae enterosolubilis (Oravacs).It is administered orally.

Using IgA contents in IgG content in indirect ELISA detection blood and enteron aisle.IgG antibody titre detection knot in blood Fruit sees that such as table 4 below IgA antibody titre testing result is shown in such as table 5 below in enteron aisle：

IgG antibody titre testing result in the blood of table 4

IgA content detection results in the enteron aisle of table 5

From testing result, commercially available traditional vaccine in terms of serum antibody titer with the combined vaccine water in the present invention It is flat similar, but be less able to cause mucosa-immune, and cause the specific antibody of time short generation few, therefore in the present invention Rotavirus-cholera live vaccine causes enough immune responses in mucous layer, stimulates body to produce enough and response time Long specific antibody.With better immune effect.

4.2.3 the Detection of Stability of combined vaccine

Combined vaccine is preserved 4 weeks in 4 DEG C of 12 months or 37 DEG C, visual examination and discrimination test no abnormality seen, pH value and is resisted Former content keeps relative stability, and carries out mouse test and does not also find that notable difference occurs for antibody level.Illustrate to use recombinant vector The cholera of albumen-rotavirus combined vaccine is preserved 4 weeks in 4 DEG C of 12 months or 37 DEG C and had good stability.

Finally be necessary described herein be：Above example is served only for making further detailed to technical scheme Ground explanation, it is impossible to be interpreted as limiting the scope of the invention, those skilled in the art is according to the above of the invention Some the nonessential modifications and adaptations made belong to protection scope of the present invention.

Sequence table

<110>Wuhan Bo Wo bio tech ltd

<120>Cholera based on recombinant vector albumen-rotavirus combined vaccine and preparation method thereof

<130> 2017

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 375

<212> DNA

<213> Unknown

<220>

<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> Protein

<213> Unknown

<220>

<223>LTB amino acid sequences

<400> 2

MNKVKCYVLF TALLSSLYAH GAPQTITELC SEYRNTQIYT INDKILSYTE SMAGKREMVI 60

ITFKSGETFQ VEVPGSQHID SQKKAIERMK DTLRITYLTE TKIDKLCVWN NKTPNSIAAI 120

SMKN 124

<210> 3

<211> 375

<212> DNA

<213> Unknown

<220>

<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> Protein

<213> Unknown

<220>

<223>CTB amino acid sequences

<400> 4

MIKLKFGVFF TVLLSSAYAN GTPQNITDLC AEYHNTQIHT LNDKIFSYTE SLAGKREMAI 60

ITFKNGATFQ VEVPGSQHID SQKKAIERMK DTLRIAYLTE AKVEKLCVWN NKTPHAIAAI 120

SMAN 124

<210> 5

<211> 747

<212> DNA

<213> Unknown

<220>

<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> Protein

<213> Unknown

<220>

<223>LTB-CTB amino acid sequences

<400> 6

MNKVKCYVLF TALLSSLYAH GAPQTITELC SEYRNTQIYT INDKILSYTE SMAGKREMVI 60

ITFKSGETFQ VEVPGSQHID SQKKAIERMK DTLRITYLTE TKIDKLCVWN NKTPNSIAAI 120

SMKNMIKLKF GVFFTVLLSS AYANGTPQNI TDLCAEYHNT QIHTLNDKIF SYTESLAGKR 180

EMAIITFKNG ATFQVEVPGS QHIDSQKKAI ERMKDTLRIA YLTEAKVEKL CVWNNKTPHA 240

IAAISMAN 248

<210> 7

<211> 47

<212> DNA

<213> Unknown

<220>

<223>LTB-Fwd primer sequences

<400> 7

cgggatccat gaataaagta aaatgttatg ttttatttac ggcgtta 47

<210> 8

<211> 36

<212> DNA

<213> Unknown

<220>

<223>LTB-Rev primer sequences

<400> 8

ctagggtctc catgtttttc atactgattg ccgcaa 36

<210> 9

<211> 52

<212> DNA

<213> Unknown

<220>

<223>CTB-Fwd primer sequences

<400> 9

ctagggtctc aacatgatta aattaaaatt tggtgttttt tttacagttt ta 52

<210> 10

<211> 34

<212> DNA

<213> Unknown

<220>

<223>CTB-Rev primer sequences

<400> 10

ccctcgagtt aatttgccat actaattgcg gcaa 34

Claims

1. a kind of cholera based on recombinant vector albumen-rotavirus combined vaccine, it is characterised in that the combined vaccine bag Include：

A. cholera vaccine, including：Cholera antigen and antigen vectors, wherein cholera antigen are coupled with antigen vectors, and antigen vectors are Recombined protein carrier, by Heat-labile enterotoxin B (LTB) albumen and cholera toxin B egg (CTB) genetic recombination obtains recombinant vector albumen in vain；

B. Rotavirus Vaccine；

Rotavirus Vaccine faces with suspension with cholera vaccine.

2. the cholera based on recombinant vector albumen-rotavirus combined vaccine according to claim 1, it is characterised in that LTB nucleotide sequences are bridged with CTB nucleotide sequences by primer sequence, the LTB-CTB nucleotide sequences such as sequence table SEQ ID of structure NO：Shown in 5, the carrier protein sequence such as sequence table SEQ ID NO of coding：Shown in 6.

3. the cholera based on recombinant vector albumen-rotavirus combined vaccine according to claim 1, it is characterised in that： CTB nucleotide sequences are selected from the CDS areas of O139 groups of cholera complete genome sequences.

4. the cholera based on recombinant vector albumen-rotavirus combined vaccine according to claim 1, it is characterised in that： Encode the nucleotide sequence such as sequence table SEQ ID NO of LTB albumen：Shown in 1, its amino acid sequence such as sequence table SEQ encoded ID NO：Shown in 2.

5. the cholera based on recombinant vector albumen-rotavirus combined vaccine according to claim 4, it is characterised in that： Encode the nucleotide sequence such as sequence table SEQ ID NO of CTB albumen：Shown in 3, its amino acid sequence such as sequence table SEQ encoded ID NO：Shown in 4.

6. the cholera based on recombinant vector albumen-rotavirus combined vaccine according to claim 1, it is characterised in that： Cholera antigen is proteantigen and/or polysaccharide antigen.

7. the cholera based on recombinant vector albumen-rotavirus combined vaccine according to claim 6, it is characterised in that： Cholera antigen is vibrio cholerae O 139 group cholera capsular polysaccharide.

8. the cholera based on recombinant vector albumen-rotavirus combined vaccine according to claim 1, it is characterised in that： Rotavirus Vaccine includes inactivated rotavirus vaccine or rotavirus attenuated live vaccine.

9. the cholera based on recombinant vector albumen-rotavirus combined vaccine according to claim 1 and preparation method thereof, Characterized in that, step includes：

S1. according to LTB and CTB CDS areas nucleotide sequence, two pairs of primers are designed, pET28a-LTB-CTB plasmids are built, PCR expands After increasing, by BamH I and the double digestions of Xho I, gel reclaims LTB-CTB fragments and expression vector pET28a；

S2. connection converts and monoclonal amplification is carried out after e.colistraindh5α, screening positive clone, is identified after IPTG inductions；

10. the cholera based on recombinant vector albumen-rotavirus combined vaccine according to claim 9 and its preparation side Method, it is characterised in that S3 is specially：

Cholera polysaccharide is coupled with recombinant vector albumen, polysaccharide is 0.5~4 in mass ratio with carrier protein：1, after coupling Detection combines product.