CN107007829A

CN107007829A - A kind of pneumonia multivalence combined vaccine and preparation method thereof

Info

Publication number: CN107007829A
Application number: CN201710277403.4A
Authority: CN
Inventors: 艾智武; 刘昊智; 史晋; 吴克
Original assignee: BRAVOVAX Co Ltd
Current assignee: Bravovax Co ltd; SHANGHAI BOWO BIOTECHNOLOGY CO Ltd
Priority date: 2014-07-25
Filing date: 2014-09-23
Publication date: 2017-08-04
Anticipated expiration: 2034-09-23
Also published as: CN104208671A; CN107007829B; CN104208671B

Abstract

The invention discloses a kind of pneumonia multivalence combined vaccine, the combined vaccine is multivalent pneumococcal polysaccharide and combined vaccine of two or more the carrier protein through connector, wherein, connector is magnetic Nano microsphere.The preparation method of the pneumonia multivalence combined vaccine forms multivalent pneumococcal polysaccharide with two kinds or more of carrier protein with nanoparticle through coupling respectively.The pneumonia multivalence combined vaccine preparation technology that the present invention is provided is simple, nanoparticle is used to strengthen mouse Th1 type immune responses for the pneumonia multivalence combined vaccine of attachment, and immune lasting effect, specificity and the compatibility of polysaccharide specificity antibody, it additionally can induce mouse and produce rotavirus antibody；Possesses the preventive effect of two kinds of vaccines；Therefore with very wide application prospect.

Description

A kind of pneumonia multivalence combined vaccine and preparation method thereof

Technical field

It is to be related to a kind of pneumonia multivalence combined vaccine vaccine and preparation method thereof specifically the present invention relates to a kind of vaccine, Belong to biological technical field.

Background technology

First, harm and counter-measure of the microorganism to human body

Microorganism typically refers to the biocenose that those body volume diameters are generally less than 1mm, and they are simple in construction, mostly It is unicellular, also some even eucaryotic cell structures do not have yet, and microorganism is not present in we all the time at one's side, it will usually by Microscope or electron microscope can just see their form and structure clearly；Wherein, pathogenic microorganisms be refer to cause the mankind, The disease of animal and plant, with pathogenic microorganism.A kind of pathogenic attack to host for depending on it of pathogen and Host resistance is bred and resisted in vivo without the ability eliminated by it.Microorganism is pathogenic generic character, and pathogenecity is strong Weak degree is referred to as virulence.The establishment of infectious diseases not by the virulence unilateral decision of microorganism, will also regard the strong of host Health situation and immune functional state.In general, the body that the strong microorganism infection of virulence be not immunized, can cause pathology to damage There is apparent infection etc. in evil, and normal body can resist the infringement of many low toxicity microorganisms (such as conditioned pathogen), but works as place Then can be susceptible and pathogenic to these microorganisms during main resistance reduction.The virulence of pathogen and host resistance between the two compared with Amount, draws the generation of infectious diseases, develops, lapses to and prognosis, because adaptedness is different between pathogen and host, both sides The final result contended with is different, produces a variety of patterns of infection, the i.e. different manifestations of course of infection.Pathogenic is to specific host Speech, what is had only has pathogenic to the mankind, have only to some animals, and the then category infecting both domestic animals and human microorganism having.And now place Master is typically only possible by the infection that the immune system of itself carrys out combating microorganisms, and the death rate is very high, causes each researcher to grind Study carefully vaccine and resist infringement of the invasive organism to human body.

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.Come prevention disease it is the mankind in generation more than one by inoculating against property vaccine In the clinical practice of discipline, it was demonstrated that be effective means.By effort for many years, medical field has been developed a variety of Vaccine is to prevent, bacterium, virus and fungi etc., infects the various diseases caused, drastically increases the health of the mankind Level.Continuing to develop for biotechnology, promotes the variation of vaccine kind.Today, to infectious disease caused by pre- anti-virus There are the vaccine that inactivation of viruses technological development comes out, such as Vaccinum Encephalitidis Epidemicae, polio vaccine, influenza vaccines；Use attenuated virus The attenuated live vaccine that technological development comes out, such as Rotavirus Vaccine, oral polio virus vaccine, measles virus vaccines, Mumps virus vaccine, rubella virus vaccine and chicken pox vaccine etc..Prevent useful proteins and polysaccharide of bacterial infectious disease etc. raw The bacterium class vaccine that thing macromolecule purifying 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..Use gene recombinant protein technological development Vaccine out, such as hepatitis B surface antigen (prevention hepatitis B), human nipple shape viruslike particle virus (prevention cervix Cancer) etc..The prevention meningitis and the bacterial vaccine of pneumonia developed with half chemical combination technology, such as popular haemophilus b Type polysaccharide-protein combined vaccine, 7 valencys or 10 valency pneumococcal polysaccharide-protein combined vaccines and 4 valency meningococcal polysacharides- Protein conjugate vaccines.As can be seen here, the development of medical biotechnology, is the motive power that vaccine product is continued to develop, by life Updating for thing technology, can develop more new generation vaccine products and human health is chosen to deal with different infectious diseases War.

2nd, combined vaccine is summarized

Polysaccharide is a kind of important immune active ingredient in pathogen, have mycelial polysaccharides (OPS) and capsular polysaccharide (CPS) it Point.After pathogen invades body, they can stimulate body to produce protective immune response as immunogene.But, many sugars Son belongs to the not dependent antigen of T cell (Ti-Ag), less immunogenic, and immune effect is especially undesirable after inoculation infant, and It is many at present to endanger serious disease meningitis, E.Coli O 157 as caused by haemophilus influenzae (Hib):It is small caused by H7 Occurred frequently in the infant and clinical nothings such as youngster's hemorrhagic diarrhea dehydration effectively treat method, case fatality rate height (Wang Yan etc., with reference to epidemic disease Seedling summarizes [J], microbiology immunology progress, 2000,28 (1):60-63).In order to improve the immunogenicity of polysaccharide vaccine, state The outer chemical bond vaccine that polysaccharide and albumen have been risen at the beginning of 1920's, i.e. conjugate vaccines, with reference to (using albumen as carrier Bacterial polysaccharides class) vaccine using chemical method by polysaccharide covalent combine polysaccharide-protein combination epidemic disease is prepared on protein carrier Seedling, such as immunogenicity for improving bacterial vaccine polysaccharide antigen, b types haemophilus influenzae combined vaccine, meningococcus knot Vaccine and pneumococcal conjugated vaccine etc. are closed, short decades, its development was quite rapid, had obtained notable achievement.

3rd, the harm and its epidemiological study of pneumococcus

It is the main cause of whole world morbidity and mortality as the infection caused by pneumococcus (lung chain).Pneumonia, hair Hot bacteremia and meningitis are the most common forms of expression of invasive pneumococcal disease, and the bacterium diffusion in respiratory tract Middle ear infection, sinusitis or recurrent bronchitis can be caused.Compared with invasive disease, the form of expression of Noninvasive is generally not It is so serious but more common.Due to the diffusion of antibiotics resistance sexually transmitted disease, and pneumococcal pneumonia is often in influenza infection Occur afterwards, the possibility that pneumococcal disease breaks out during influenza further increases.

The disease triggered by streptococcus pneumonia has turned into a global important public health problem.Pneumococcus has turned into The number one killer of global children.The case fatality rate of China's pneumonia is 16.4%, wherein more than 50 years old the elderly and less than 1 years old baby children Youngster is up to 28.6% and 22.0% respectively.Carrying rate of China pneumococcus in healthy children is higher, and statistics are shown, Carrying rate in northern area healthy children is 24.2%, and southern area is 31.3%.And the disease is to cause less than 5 years old children Dead major reason.Main reason is that the development of infant immunisation system is not perfect, immunity is weaker.And the age gets over Small baby, immunity is weaker.About 90 kinds serotypes (bacterial strain) of pneumococcus, the statistics of China shows pneumococcus Several serotypes are followed successively by before infection strain：5th, 6,19,23,14,2,4 type.860 plants of pneumococcus separation strains are have collected according to one Studies have shown that there are 109 plants (12.7%) to show as sero-group 6, reached in the erythromycin resistance of Chinese Pneumococcus serotypes 6 100%, 6A, 6B and 6C type therein is respectively 62 plants (56.9%), 38 plants (34.9%) and 9 plants (8.2%).

Chemically in structure from the point of view of, above pathogen possesses a cell surface capsular polysaccharide (Capsular Polysaccharide, CPS) or lipopolysaccharides (Lipopolysaccharide, LPS) shell, or both have concurrently, its function is side Help pathogenic infection host.Capsular polysaccharide can shield bacterial cell surface functional component from being recognized by host immune system, Prevent complement system from being swallowed by bacterial surface protein activation and immunocyte, if bacterium is swallowed, capsular polysaccharide can be prevented Bacterium is killed.In most of pathogens, different bacterial strains expresses the capsular polysaccharide and lipopolysaccharides of different structure, and generation is a variety of not Same serological type strain.Pneumonia and meningitis caused by pneumococcus are by a big chunk bacterium in known 90 kinds of serotype Caused by strain infection.

As can be seen here, most of bacterial polysaccharides class vaccines must be pathogenic to improve containing a variety of different types of bacterial polysaccharideses Bacterial strain coverage rate, optimization and selection are an extremely complex epidemic disease in vaccine comprising which kind of bacterium or serotype polysaccharide Knowledge is inscribed.Once specifying which kind of polysaccharide antibody has protective effect, then as immunogene vaccine can be produced with this polysaccharide.

23 valency Pnu-Imune 23s of Chengdu Inst. of Biological Products of Chinese biological technology group production are to have chosen 23 kinds Most common pathogenic bacteria (1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F), distinguish the various polysaccharide on fermented and cultured and separating-purifying pneumococcal capsule, be mixed by equal proportion Vaccine.The polysaccharide of bacterium is a kind of thymus independent antigen, and the main distinction of this antigen and thymus dependent antigen is The former does not need the auxiliary of T lymphocytes to produce antibody.Clinical practice proves that the vaccine that capsular polysaccharide makes is clearly effective, And widely used in multiple countries.But there is problems with this kind of polysaccharide vaccine：(1) in brood or infants only Faint immune response can be produced, immune response is not produced even, immune response strengthens with the growth at age；(2) produce low The antibody of affinity；(3) of short duration immune response is only produced, does not possess immunological memory and Immune-enhancing effect effect during inoculation repeatedly Should；(4) immune tolerance is easily produced；(5) common adjuvant is difficult to play a part of Immune-enhancing effect, 23 valency polysaccharide to this antigen The protective rate that vaccine is infected for intrusion type lung chain is 50-70%, and is only used for the crowd of more than 2 years old and is inoculated with, and pneumonia Peak age of onset be the 6-12 monthly ages；(6) polysaccharide with repetitive structure is the immunogene of the class of T cell self 2, without T The participation of cell, they be can not inducing immunological memory effect, stimulate body produce antibody be mainly IgM and IgG2, it is impossible to Enough effectively activating complement systems.

The method of half chemical combination technology (also referred to as combination technology) vaccine development is the exploitation occurred from 1980s The technology of bacterial vaccine.John Robbins are by by popular influenzae type (Haemophilusinfluenzaetype B, Hib) capsular polysaccharide (Polyribosylribitolphosphate, PRP), it is connected to protein carrier with the form of covalent bond (tetanus toxoid) has synthesized popular influenzae type polysaccharide PRP- tetanus toxoid conjugates (PRP-TT), After immune animal, the protection antibody of sterilization can be produced, so as to start bacterial vaccine development technique of new generation.Especially have Meaning, for the age is less than the infant of 2 years old, due to developing immune system imperfection, polysaccharide is for infant Belong to a kind of T-independent antigen, it is impossible to stimulate body to be produced adult and long-acting be directed to the polysaccharide The bacterium specificity protection IgG antibody in source.Therefore, polysaccharide is a kind of haptens for the infant less than 2 years old, it is impossible to as Vaccine is inoculated with the children less than 2 years old.When bacterial polysaccharides is connected into protein carrier in the form of covalent bond, such as tetanus are malicious Plain (Tetanus Toxoid, TT), because protein is a kind of T cell dependence antigen, the polysaccharide that can connect covalent bond It is transformed into T cell dependence antigen, so as to stimulate body to produce the specific IgG antibodies for being directed to the polysaccharide, protects body Do not infected by bacterium.The success of popular influenzae type polysaccharide-tetanus toxoid (PRP-TT) combined vaccine exploitation, The technology platform of an exploitation bacterial vaccine, i.e., by bacterial polysaccharides, such as capsular polysaccharide, O- specific polysaccharides (O- are started Specificpolysaccharide) or oligosaccharide (Oligosaccharide), to be covalently bonded on protein carrier The combined vaccine being made.Success based on this concept, medical biotechnology research circle opens by using same chemical synthesis process It has issued the combined vaccine of different bacterium；The combined vaccine of same bacterium is equally also developed with different synthetic technologys.

Capsular polysaccharide can shield bacterial cell surface functional component, make it from being recognized by host immune system, prevent Complement system is swallowed by the protein activation of bacterium surface and immunocyte.If bacterium is swallowed by immunocyte, capsular polysaccharide Bacterium can be avoided to be killed.Capsular polysaccharide is one of major antigen composition of meninx Neisseria, as vaccine to larger Children have certain protection.However, crowd of the capsular polysaccharide to 2 years old Infants Below, the elderly and B cell immune deficiency Immune effect is poor, and Inoculant can not reach antibody level of protection, and antibody disappears quickly.The polysaccharide vaccine and other polysaccharide epidemic diseases Seedling is the same, belongs to T cell independent antigen, with the immunogenicity that the age is related, and do not induce the reinforcement of T cell dependence should Answer.By the way that by polysaccharide and certain protein covalent bond, polysaccharide conversion can be made into T cell dependence antigen, so as to stimulate baby children The synthesis of the T cell dependence antibody of youngster, and booster response can be produced, while immunoglobulin (IgG) antibody can also be improved Ratio.This polysaccharide conjugate vaccine can not only protect infant (less than 2 years old children), additionally it is possible to resistance be significantly enhanced poor Patient to the resistance of bacterium infection, therefore with very wide application prospect., JohnRobbins brominations in 1980 Cyanogen activates Hib capsular polysaccharides at random, then, regard adipyl dihydrazide (Adipic Dihydrazide, ADH) as attachment (linker) it is added on the polysaccharide of activation, the polysaccharide covalent key after derivation is finally connected to EDC methods by the broken wound of carrier protein On wind toxoid, popular influenzae type polysaccharide-tetanus toxoid conjugate (PRP-TT) is synthesized.Due to Have on each polysaccharide chain it is same on multiple activation points, protein carrier have multiple tie points, the conjugate of formation is a kind of polysaccharide With the macromolecular of albumen interconnection, mean molecule quantity is about 5 × 106Da.1980, Harold Jennings were special in the U.S. In profit 4356170, set forth with bovine serum albumin (Bovine serum albumin, BSA) is carrier, will be meningococcal A, C groups of polysaccharide have synthesized epidemic meningitis polysaccharide-BAS and have combined epidemic disease by being connected on BSA with reducing amine method covalent bond Seedling.1987 and nineteen ninety, Porter Anderson in United States Patent (USP) 4673574 and 4902506, are described with change respectively Different avirulent strain diphtheria toxin 197 (Cross reaction material sub197, CRM197) is as carrier protein, to reduce Amine method has synthesized popular influenzae type oligosaccharide-variation avirulent strain diphtheria toxin 197 (HbOC-CRM197) combined vaccine. Specific method is to use sodium periodate oxidation Hib capsular polysaccharides, produces the oligosaccharide for aldehyde radical in two ends, passes through reducing agent cyanogen Base sodium borohydride (sodiumcyanoborohydride), oligosaccharide is covalently bonded on protein carrier.Form molecular weight About 90kDa lipopolysaccharides conjugate, has been made containing the combination with 6 glycan molecules on 30% polysaccharide and each albumen Vaccine.Then, Merck (Merck, Sharpe and Dohme) is scorching with the neisseria meningitis of purifying with mercapto chemistry B crowds of bacterial strain (Neisseria meningitidis groups B) bacterial surface protein compound (outer membrane Protein complex, OMP) as protein carrier, synthesize popular influenzae type polysaccharide-bacterial surface protein and be combined Thing (PRP-OMP) combined vaccine.With these synthetic technologys, three kinds of streams for being widely used in clinical inoculation are successively have developed Row influenzae type combined vaccine, i.e. PRP-TT, PRP-HbOC and PRP-OMP.The success of Hib combined vaccines is to develop it Its bacterium combined vaccine provides theory and technology basis, and subsequent exploitation enters the increasingly complex multivalence combined vaccine of technology Stage, its reason is some infectious diseases, meningitis as caused by pneumonia, epidemic meningitis coccus caused by pneumococcus etc., Can be caused by a variety of different serotypes or strain infection, and due to the chemistry of bacterial surface polysaccharides between each serotype or bacterial strain The difference of structure, its antibody does not have cross-immune reaction, therefore, the single serotype of inoculation or bacterial strain combined vaccine, it is impossible to protect Shield is vaccinated infection of the human body from other serotypes or bacterial strain.For this reason, multivalence combined vaccine is synthesized and prepares to come Expanding the protection coverage rate of vaccine turns into the main target of exploitation.

By effort for many years, the wide multivalence combined vaccine of a variety of coverage rates is have developed with combination technology.Bacterial capsule Polysaccharide-protein combined vaccine is appeared in earliest in the 1930s, 3 type pneumococal polysaccharides are connected to by Goebel and Avery On horse serum globulin, the conjugate of generation can produce the single-minded antibody of polysaccharide in animals, while providing corresponding immune Protection., first GL-PP combined vaccine in the world, Type B haemophilus influenzae (HiB) polysaccharide-tetanus poison in 1987 Plain (TT) combined vaccine, which is approved by the FDA in the United States, enters market.Merck ＆ Co., Inc., Pfizer and Novartis Co., Ltd develop HiB in succession Polysaccharide-tetanus toxoid conjugate and epidemic meningitis polysaccharide-tetanus toxoid conjugate, and successfully list. 2000, U.S. Hui Shi (Wyeth) company successfully developed and has listed 7 valency pneumococal polysaccharide-CRM197 combined vaccines, be by 7 different Pneumococcus serotypes polysaccharide are covalently bonded on CRM197 protein carriers respectively, and the one of mixed preparing Polyvaccine is planted, to prevent infantile pneumonia, it is popular that 7 Pneumococcal serotypes cover North America and Europe more than 90% Pneumococcus different serotypes bacterial strain.2006, Sanofi Pasteur have developed 4 valency meningococcal polysacharides-broken wound Wind toxoid combined vaccine, for preventing 4 kinds of epidemic meningitis coccus groups, i.e. A, C, Y, W135, caused meningitis.2009 Year, GlaxoSmithKline (GSK) have also been developed a kind of 10 valency pneumococcal polysaccharide-protein combined vaccine, to prevent Pneumonia caused by 10 kinds of Pneumococcus serotypes.The vaccine has used three kinds of protein as protein carrier, wherein most main The carrier wanted is albumen-D (Protein D, PD), and it is as carrier with this albumen to have 8 serotype polysaccharide.Albumen-D is to use The gene recombination method of the popular haemophilus of non-separable express without esterification surface albumen, body can be stimulated to produce Raw protection antibody, there is the otitis media acuta caused by the popular hemophilus infection for potentially preventing non-separable, and other are also There are tetanus toxoid and diphtheria endotoxin as carrier, be used separately as the egg of serotype 18C and 19F combined vaccine Bai Zaiti.From the design above in association with vaccine product, it can be seen that the exploitation of combined vaccine is transitioned into technically from univalent vaccine Increasingly complex polyvaccine, improves the coverage rate of bacterial vaccine.

GL-PP conjugate vaccines (combined vaccine) are current state-of-the-art vaccine technologies, and albumen is added on specific antigen Matter carrier, can increase its immunogenicity.Protein carrier has T cell dependency characteristic, and GL-PP conjugate vaccines can be thin by non-T The polysaccharide antigen of born of the same parents' pauper character is changed into the antigen of T cell pauper character, and the t helper cell generation of excitating organism is a series of Immune-enhancing effect.Capsular polysaccharide conjugate vaccine, protein carrier is added on polysaccharide, T cell is changed into from T-independent antigen Dependence antigen, increases its immunogenicity.The antibody produced after combined vaccine inoculation is generation vaccine in quality and quantity 400-1000 times, generation immunoprotection is wider stronger, and guard time is more permanent, reaches efficient protection.2000, Pfizer was public First 7 valent pneumococcal conjugate vaccines listing of department；Pfizer Inc. exploitation infant more targeted 7 (4,6B, 9V, 14th, 18C, 19F and 23F) valency pneumoprotein vaccine, it is also effective to less than 5 years old children.Capsular polysaccharide protein conjugate vaccines, Add protein carrier on polysaccharide, T cell dependence antigen is changed into from T-independent antigen, its immunogenicity can be increased, can For children more than 6 week old.Current Pfizer 13 valency combined vaccines of China's registration, add 6 serotypes (1,3, 5,7F, 6A, 19A).But the data of China shows that pneumococcal infection bacterial strain serotype is followed successively by：5、6、1、19、23、14、2、 4, the valent pneumococcal conjugate vaccine of Pfizer 7 only has 50% or so to China's common causative bacterial type coverage rate, and 13 valency combined vaccines Coverage rate also only have 70%.The valent pneumococcal conjugate vaccines of Hui Shi 7 need to be inoculated with 4 injections, expensive per 860 yuan of pin, no Beneficial to popularization.13 valency vaccines estimate that its price will be more expensive.Therefore the 7 and 13 valency combined vaccines of Pfizer Inc. are less fitted Close the large-scale children Streptococcus prevention of China.

Although 13 valent pneumococcal conjugate vaccines have been listed, wherein there is the immune effect of several serotypes with respect to it His serotype is relatively low, such as 3 types.And with the increase and the reuse of carrier protein of the same race of different serotypes so that carrier egg White consumption increases, and its immunogenicity is reduced on the contrary.GlaxoSmithKline is developing 10 valency pneumococcal Polysaccharide Conjugate Vaccines In design, albumen-D is have selected for carrier, its reason is the consideration based on two aspects.First, being in order to avoid reuse Carrier is used as the tetanus toxoid and diphtheria toxoid of DPT vaccine component.Clinical test shows, works as multivalence Pneumonia combined vaccine contains univalent vaccine or multiple vaccines with protein carrier same composition while when being inoculated with other, such as Hib-TT, whooping cough-Hib polysaccharide conjugate vaccines-IPV (inactivated polio vaccine)-hepatitis B vaccine (referred to as 6 vaccines), The immunogenicity of most of serotype polysaccharide in multivalent pneumococcal combined vaccine can be suppressed, particularly to lockjaw Toxoid is especially apparent as the serotype polysaccharide immunogenic influence of carrier.Reason is work in multivalent pneumococcal combined vaccine Tetanus toxoid and diphtheria toxoid total concentration for carrier is too high, and the combined vaccine containing whooping cough component is inoculated with the same time When, such as 6 vaccines can cause so-called vector receptors Reverse transcriptase effect, reduce the immunogene of combined vaccine saccharide portion Property.SanofiPasteur 11 valency pneumococal polysaccharide-TT and DT mixed carrier protein conjugate vaccines and Merck 7 valency pneumonia Streptococcus polysaccharides-OMP combined vaccines (PCV-OMP), are all that clinical test results are not good and cause the example of product development failure, former Because just with this.Second, being to confer to protein carrier with real protective immunity originality function.Clinical test proves albumen-D energy Enough stimulate body to produce protection antibody, there is the acute middle ear for potentially preventing the popular hemophilus infection of non-separable to cause It is scorching.GlaxoSmithKline 10 valency pneumococcal Polysaccharide Conjugate Vaccines selection albumen-D is used as carrier so that protein carrier is produced Raw antibody has the protective effect of clinical meaning, is the much progress on combined vaccine technology development process.

But, limit of the popular haemophilus actual clinical meaning of non-separable by the bacterial infection rate lowly System, the otitis media acuta incidence of disease is relatively low caused by it infects.It is exactly that albumen is carried but these combined vaccines have a common ground shortcoming Body does not assign the defencive function of immunogenicity, that is to say, that although combined vaccine carrier can stimulate body to produce antibody, It is that the protectiveness that the designer of vaccine does not have using its antibody keeps off infection, meanwhile, do not determine that its is produced yet Antibody whether reach protectiveness titre levels.Tetanus toxoid, diphtheria toxoid and the nontoxic variant toxin of diphtheria etc. are passed System albumen is selected as the main cause of carrier, be not because the antibody of their generation has a protectiveness, but from its Security and the immunogenicity of polysaccharide in conjugate can be strengthened to consider.It is clear that tetanus toxoid and diphtheria class poison Element has been two components of PertussisDiphtheriaTetanus triple vaccine, by traditional vaccination；So, tetanus toxoid in combined vaccine and white Whether larynx toxoid carrier, which can stimulate body to produce, reaches that protection antibody titre is unimportant, opposite, in combined vaccine Saccharide portion antibody titer be only vaccine design person need pay close attention to subject matter.In addition, some just knots under development Close the carrier used in vaccine product, the recombinant Pseudomonas aeruginosa exotoxin of the deletion mutant detoxification as expressed by E.coli A (rEPA), the recombinant cholera toxin of the deletion mutant detoxification expressed by E.coli etc. is also all based on identical and considered.

The species and type of new polysaccharide conjugate vaccine are increasing year by year, and alternative carrier protein species is less, no It is more that carrier protein is reused with vaccine.The different combined vaccines of inoculation same vehicle albumen may produce immunosupress effect Should, cause influencing each other for immune effect between different vaccines.Moreover, main carriers albumen such as TT of polysaccharide conjugate etc., its Itself is as vaccine ring vaccination infant, and original high titre specific antibody for carrier protein can in crowd's body It can suppress specific immune response of the body to polysaccharide in combined vaccine.

Chinese patent (ZL02159032.X) discloses a kind of preparation method of polysaccharide-protein combined vaccine, is also current Prepare one of most-often used technology of polysaccharide conjugate vaccine.In the technology, bridging agent is used as using adipic dihydrazide (ADH) With reference to polysaccharide and albumen.This combination through cyanogen bromide-activated, i.e., uses cyanogen bromide in the basic conditions firstly the need of by polysaccharide The hydroxyl acted on polysaccharide molecule, forms cyanate, is then reacted with ADH；A C―O bond cleavage in cyanate, with Addition reaction occurs for the amino of ADH one end, so that ester hydrazides (AH) group is imported into polysaccharide molecule, forms polysaccharide-AH derivatives； Polysaccharide-AH derivatives form stable conjugate under carbodiimide (EDAC) mediation with carrier protein.Such combination side Formula can reduce the steric hindrance that polysaccharide is combined with carrier protein, remain the epitope of polysaccharide, while avoiding polysaccharide in itself Dissolubility, reduce the side effect that polysaccharide and antiserum react.

However, above-mentioned traditional polysaccharide-protein combination technology has following weak point：(1) polysaccharide-AH derivatives meeting Continue to react with the polysaccharide of cyanogen bromide-activated, form the self-polymerization thing of polysaccharide, reduce the joint efficiency of polysaccharide-protein；(2) EDAC easily causes the self-crosslinking of polysaccharide and carrier protein while mediating ADH derivation polysaccharide to be combined with carrier protein, from And reduce the joint efficiency of polysaccharide-protein；(3) polysaccharide and carrier protein are macro-organism molecule, and centre is by only 6 carbon originals The ADH of sub- length is connected, and the structure of polysaccharide and protein will certainly influence each other so that the important epitope of some of polysaccharide is easy Shielded by protein, and then reduce the immunogenicity of polysaccharide.Therefore, the immunogenicity and antibody of polysaccharide-protein combined vaccine Lasting effect still needs further raising, and such as polysaccharide conjugate vaccine needs that immune effect could be produced immune three times.These deficiencies Place limits the further development of polysaccharide conjugate vaccine.

4th, application and prospect of the nanoparticle in biotechnology

Nano material refers to that crystallite dimension is less than 100 nanometers of monocrystal or polycrystal, unique small-size effect and table The effect such as face or interface, makes it possess many excellent or brand-new performance, and it is just being increasingly subject to the attention of people.For example receive Continuous infiltration and influence of the rice material on drug research field, the revolution for having triggered the field depth of drug field one remote.Medicine is The mankind be used to resisting with prophylactic important substance, for a long time, the research and development of medicine provide many to be clinical Treatment means, be that patient brings many benefits.But, existing medicine can not followed there are still many problems, such as medicine Be detained in loop system and reach valid density, specific therapeutic purpose can not be reached, can not by blood-brain barrier, can not be at some It is partially formed higher concentration and while not producing (application of Wu Xin honor drug-carried nanometers and the progress such as toxic side effect [J] Chinese Hospitals materia medica magazines, 2001,21 (3):171-173.).Magnetic Nano microsphere pharmaceutical carrier be nanometer technology with The product that modern medicine and pharmacology is combined, has small-size effect due to it, good targeting, biocompatibility, a biological degradability And the advantages of functional group, therefore it is expected to these defects for overcoming conventional medicament to be brought.Magnetic nanometer particles can also be used for egg Purifying, recovery and the enzyme immobilizatio of white matter and enzyme, it is simple to operate, and improve the stability of enzyme.Utilize magnetic nanometer particles Carry out immunoassay, with specificity is good, separation fast, favorable reproducibility the characteristics of.PCI is carried out using Magnetic Microspheres-Carrier, In the intravascular carry out embolism of magnetic control, then there is magnetic control guiding, target position embolism.

Application of the magnetic Nano microsphere in biomedicine specifically has with the deeply increasingly extensive of research：

1st, immobilised enzymes

Boiomacromolecule all has many functional groups such as enzyme molecule, can pass through physical absorption, crosslinking, covalent coupling Etc. the surface that they are fixed on magnetic particle by mode.Advantage with magnetic Nano microsphere immobilised enzymes is：It is easy to enzyme and bottom Thing and product separation；Improve the biocompatibility and immunocompetence of enzyme；The stability of enzyme is improved, and simple to operate is reduced into This.Bendkiene etc. is prepared for chitosan magnetic microsphere, as fixation support., can after enzyme is fixed on this carrier Easily to be separated and recovered with magnetic devices from the mixed liquor of reaction.Domestic researcher also explores to this respect, Ding little Bin etc. uses dispersion copolymerization method, synthesizes Fe3O4/ (St-MPEO) (St- styrene, MPEO- PEOs polymeric monomer) Microballoon, the microballoon has amphiphilic structure, all has good swelling behavior in most of polarity, apolar medium so that The immobilized compound of microballoon all has higher activity (Ding XB, Wei L, Zhao HZ.Synthesis in medium and characterization of aliphatic polycarbonatediols[J].Applied Polymer Science, 2001,79 (3):1847-1851).The magnetic Nano microsphere carrier is expected purifying for protein and enzyme, returned The field such as receipts and enzyme immobilizatio, cell separation.

2nd, targeted drug

Medicament carrier microspheres with targeting refer to that drug bearing microsphere can highly selectively be distributed in effective object, so as to strengthen Curative effect, reduction side effect.Initial target medicine carrier microballoon be according to clinical needs, by from it is various to body tissue or The different carrier of diseased region affinity makes drug bearing microsphere, or monoclonal antibody is combined with carrier, defeated to allow medicament to It is sent to the privileged site that treatment is expected to reach.Requirement more and more higher with people to treatment, targeting positioning is also because by matrix Limit and be not entirely satisfactory, therefore occurred as soon as magnetic Nano microsphere drug-loading system.This system is in externally-applied magnetic field Under effect, people is noted to pathological tissues by dynamic (quiet) arteries and veins, carrier is directed to diseased region (target position), contained drug is determined Position release, focuses on diseased region and has an effect (A Paul, Alivisatos.Ultrasensitive magnetic Biosensor for homogeneous immunoassay [J] .Science, 2001,12 (5):53-60).Germany Lubbe etc. completes the clinical trial of first case applied magnetic drug targeting treatment in the world.Suffer to 14 advanced solid tumors In the magnetic target therapy of person, it is found that patient is fine to the tolerance of magnetic targeted drug.Lexion etc. also make by applied magnetic microballoon Squamous carcinoma (Lexion C, Amold W, Klein RJ, the et al.Locotegional cancer of rabbit are treated for pharmaceutical carrier Treatment with magnetic drug targeting [J] .Cancer Res, 2000,60 (23):6641-6648), Domestic Tao Kaixiong etc. treats mouse implanted gastric tumor with adriamycin magnetic albumin microspheres, and (Tao Kaixiong, Sun Hongwu, Chen Daoda wait Targeting Treatment with Adriamycin Magnetic Albumin Microspheres in Human mouse implanted gastric tumor [J] China experimental surgery magazine, 2000,17 (1):63- 64)), the bleomycin A5 magnetic microsphere treatment oral cavity top collar portion cvernous hemangioma 25 such as Guo Jun.In addition, high-intensity magnetic field also has Cancer suppressing action (Guo Jun, Li Cheng, Wu Hanjiang bleomycin A5 magnetic microsphere targeted therapy oromaxillo-facial regions cvernous hemangioma 25 Clinical report [J] Nanjing Railway College of Medicine journal, 2000,19 (2):112-114)).The glucose magnetic microsphere such as Xu Huixian Immobilization L- asparagus ferns phthalein amine enzyme treats acute lymphatic leukaemia, also achieves good therapeutic effect.These results all show, Increase with the magnetic and medicated aggregation in tumor locus of raising of magnetic field intensity, acted on using the magnetic steering of externally-applied magnetic field, make medicine Pinpoint in target site, play concentration, efficient antitumor action.Targeting and surface just because of magnetic Nano microsphere are combined Idiosyncratic carrier, make people be expected to be utilized to follow the trail of and eliminate the cancer cell that is shifting, so that as being eliminated in human body " biological missile " of cancer cell.But, being located at body surface the tumour of current applied magnetic drug therapy more or in vitro table is nearer, therefore The intensity of externally-applied magnetic field can be weaker, and easily controllable, if the tumour for the treatment of deep organ or tissue, is needed to be optimized Magnetic field intensity, positioning and Pharmaceutical carrier particles size etc..And the intensity of magnetization that improve magnetic microsphere then has certain difficulty, because Its magnetic property can be substantially reduced for the clad on magnetic particle surface, in addition, the controllable of granular size is also to have to be solved one Individual problem.

3rd, cell separation and immunoassay

If magnetic particle surface, which is drawn, connects the specific antibodies with bioactivity, in the presence of externally-applied magnetic field, utilize The specific binding of antibody and cell, it is possible to obtain immune magnetic microsphere (Immunomagnetic microspheres, IMMS) or immune magnetic pearl (Immunomagnetic beads, IMBS), using they can fast and effeciently by cell separation or Carry out immunoassay.When being separated in particular by IMBS to antigenic substance specific to cell, organelle surface, with letter Just quick, separation purity is high, retain the features such as target substance is active.Mccole etc. separates the adult infected by liver fluke with IMBS T lymphocytes in ox peripheral blood, the cell separated is pure, is worked well for liver fluke infection mechanism.John is with being connected with list Anti- IMMS detections salmonella, whole detection process only needs 2-3h, and sensitivity is 103-104 thalline/ml, a certain amount of blood and The presence of excrement is noiseless to analyzing, compared with agglutination and immunofluorescence technique, and sensitivity improves 103 times.Glenn is with IMBS points Inclusion virus is closed from breathing, with reference to ELISA, the diffusion reduced in conventional tube and micropore analysis is inhaled with non-specific Attached, the formation of compound only needs 7min, and conventional microporous rule needs 120min.The isolation technics of cell can be additionally used in controlling for cancer Treat.Kang Ji is superfine with the covalently bound method of physical absorption combination chemical bond, anti-human wing moon bright carcinoma monoclonal antibody is connected to pre- The surface of the Magnetic Polystyrene Microsphere carrier first prepared, constructing can specifically be combined with target cell and assign it with magnetic response The immune magnetic microsphere of property.As a result show, constructed IMMS can be combined effectively with target cell, with IMMS from marrow The preliminary experiment of separation cancer cell shows that IMM can effectively remove cancer cell, and bone marrow cell only has minimal amount of loss.It is immune Analysis in modern biotechnology analytical technology is a kind of important method, its quantitative analysis to protein, antigen, antibody and cell Play huge effect.Immunoassay is carried out using the carrier-bound antigen of magnetic nanometer particles or antibody, with specificity The features such as height, fast separation, favorable reproducibility.Jing Xiaoyan etc. uses agalactosis polymerization, in the aqueous systems of alcohol one, using potassium persulfate as Initiator, in Fe3O4 magnetic fluids particle surface formation initiation point, with acrylic acid (AA) for stabilizer, passes through styrene (ST) With propylene phthalein amine copolymer, prepare monodispersed amido magnetic microsphere, the microballoon can directly, quickly with antibody (antigen) albumen Matter is crosslinked, it is to avoid need to use protein during other functional group microballoon combination immunoreagents for shortcoming (Jing Xiaoyan, king of crosslinking agent Monarch, Li Rumin waits preparation research [J] applicating technologies of magnetic function polymer microspheres, 2000,27 (1):16-17)).

4th, magnetic control inspection plug

During common people Jie treats, it may occur that the phenomenon such as dystopy embolism and infarct, and cause serious complication, This is the thorny problem for being clinically badly in need of solving, and uses people Jie of Magnetic Microspheres-Carrier to treat, in the intravascular carry out bolt of magnetic control Plug then has the advantages that magnetic control guiding, target position embolism, and approach is provided to solve above problem.Scholars focus on magnetic spherolite Footpath, the magnetic control time, magnetic field intensity, magnetic microsphere lapping (it is coarse, carry positive charge, with hydrophobic property) in terms of do Careful research.The therapy that Minalnimura etc. is combined with thermotherapy and arterial embolism is used for the research of mouse liver cancer model.He Have developed DM-MS arterial ducts locally administration, additional 500kHz magnetic field.Treat after 3d, tumour growth rate (embolism-thermotherapy Group, simple embolization group and control group) be respectively 28%, 124% and 385% (Minalnimura T, Sato H, Kasaoka S, et al.Tumor regression by inductive hyperthermia combined with hepatic embolization using dextran magnetite incorporated microspheres in rats[J].Int J Oncol, 2000,16 (6):1153-1160).It is a kind of antitumor that this research, which shows that DM-MS thermotherapies and embolism are combined therapy, Feasible sex therapy, with wide research and application prospect.Goodwin etc. is to adriamycin magnetic microsphere hepatic artery embolism and medicine Target and (Goodwin SC, Bittner CA, Peterson CL, et are studied to the toxicity of antitumor therapy al.Single-dose toxicity study of hepatic intra-arterial infusion of doxorubicin coupled to a novel magnetically targeted drug carrier[J].Toxical, 2001,60(1):117-183).The pork liver cancer model result that they set up shows the nontoxic secondary work of adriamycin magnetic microsphere low dosage With.Only when the content of magnetic microsphere>Just there is a preferable effect=75mg (with or without adriamycin) target area, liver cancer cells it is bad Dead degree is directly proportional to embolism degree, and adriamycin can not freely circulate in whole body and successfully be controlled in target area.Hui Xuhui etc. Endovascular Embolization is inquired into homemade poly-methyl methacrylate vinegar magnetic microsphere, experiment shows, 30-50um PMMA Magnetic microsphere has that magnetic response ability is strong, magnetic control embolization effect is good, remain to realize that target position embolism etc. is excellent in the case of high Hemodynamic environment Point, is that (polymethyl methacrylate magnetic is micro- before Hui Xuhui, Gao Lida, what energy for a kind of preferable magnetic control Endovascular Embolization material Ball Endovascular Embolization experimental study [J] Sichuan medical science, 2001,22 (10):928-929)).In magnetic control embolism, magnetic microsphere The size of carrier is the influence most important factor of Target localization.If particle diameter is smaller, magnetic responsiveness is weak, and magnetic control degree is poor, no High Hemodynamic environment can be used for or compared with the endovascular magnetic control embolism of Large Diameter Pipeline.Therefore, with magnetic microsphere application in other respects Difference, in magnetic control embolism people Jie treats, the general magnetic microsphere larger using particle diameter.

Also there is report to point out, nanoparticle can as DNA vaccination carrier protein and adjuvant, but applied to preventative polysaccharide And/or protide vaccine has no report.

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 a kind of stronger pneumonia of immunogenicity is many Valency combined vaccine.

For achieving the above object, the technical solution adopted by the present invention is as follows：

A kind of pneumonia multivalence combined vaccine, it is that multivalent pneumococcal polysaccharide is passed through with two or more carrier protein The pneumonia multivalence combined vaccine of connector, wherein, connector is magnetic Nano microsphere.

As a kind of preferred scheme, the magnetic particle of the magnetic Nano microsphere is located at the inside of magnetic Nano microsphere for core, High polymer material is wrapped in the outside of magnetic particle.

As further preferred scheme, magnetic particle is Fe₃O₄。

As further preferred scheme, magnetic Nano microsphere particle diameter is 0.1-10 μm, preferably 0.1-5 μm.

As another preferred scheme, high polymer material is bioabsorbable polymer material, selected from chitosan, polyethylene glycol, is gathered One or more of mixtures in poly lactic coglycolic acid (PLGA), PLA-PEG copolymer (PELA)； Most preferably PLGA or PELA.

As another preferred scheme, multivalent pneumococcal polysaccharide is a variety of pneumococcal capsular polysaccharides, is preferably separated Purify the capsular polysaccharide on Pneumococcal serotype pod membrane, the serotype of the Pneumococcal serotype includes 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F and/or 33F.

As further preferred scheme, the mass ratio of multivalent pneumococcal polysaccharide and two kinds of carrier proteins is (0.5~2)： 1, be preferably (0.5~1)：1, wherein the mass ratio between each carrier protein is preferably 1：1.

As a kind of preferred preferred scheme, the carrier protein is selected from recombined human rotavirus protein, diphtheria toxoid, broken wound Wind toxoid, carrier protein CRM197, bloodthirsty Bacillus influenzae surface protein HiD, pertussis Prn surface proteins, pertussis Fha resist Former and/or Pneumococal surface protein A (rPspA).

As further preferred scheme, it is bloodthirsty Bacillus influenzae surface protein HiD or pneumonia to have in the carrier protein a kind of Coccus surface protein A (rPspA).

As further preferred scheme, recombined human rotavirus protein is the part amino of P genotype rotavirus proteins One kind in acid sequence or complete sequence, preferably P genotype rotavirus strain P [8], P [4], P [6] or P [11]；Wherein, P Genotype rotavirus strain is selected from one in P [8] G1, P [4] G2, P [8] G3, P [8] G4, P [8] G9, P [8] G5 or P [6] G8 Kind.

As still more preferably scheme, P genotype P [8] rotavirus strain be selected from Wa, Ku, P, YO, MO, VA70, D, AU32, CH-32, CH-55, CHW2, CH927A, W161, F45, Ai-75, Hochi, Hosokawa, BR1054, WT78 or One kind in WI79 strains.

As still more preferably scheme, P genotype P [4] rotavirus strain be selected from DS-1, RV-5, S2, L26, One kind in KUN, E210, CHW17, AU64,107E18, MW333 or TB-Chen strain.

As still more preferably scheme, P genotype P [6] rotavirus strain be selected from M37,1076, RV-3, ST3, One kind in SC2, BrB, McN13, US1205, MW023, US585 or AU19 strain.

As another further preferred scheme, recombined human rotavirus protein be selected from VP8, VP4, VP8 polypeptide chain fragment, One kind in core VP8, VP4 polypeptide chain fragment, VP8 specific antigen cluster peptide chains or VP4 specific antigen cluster peptide chains.

As another further preferred scheme, recombinant rotavirus albumen is the VP7 albumen of G serotype rotavirus Partial amino-acid series or complete sequence.

As still more preferably scheme, G serotypes rotavirus strain is selected from G1, G2, G3, G4, G9, G5, G8, G10 Or one kind in G11 serotypes.

As still more preferably scheme, the G serotypes rotavirus strain be selected from P [8] G1, P [4] G2, P [8] G3, One kind in P [8] G4, P [8] G9, P [8] G5 or P [6] G8 serotypes.

It is specifically by multivalence pneumonia ball it is a further object of the present invention to provide the preparation method of the pneumonia multivalence combined vaccine Granulose is formed with magnetic Nano microsphere coupling respectively with two kinds or more of carrier protein.

As a kind of preferred scheme, the preparation method of the pneumonia multivalence combined vaccine specifically includes following steps：

A) capsular polysaccharide respectively on separating-purifying various serotype pneumococcal capsule；

B) carrier protein simultaneously selected by separating-purifying is prepared respectively；

C) various capsular polysaccharides and magnetic Nano microsphere are coupled into polysaccharide-magnetic Nano microsphere couplet respectively；

D) polysaccharide-magnetic Nano microsphere couplet is combined with variety carrier albumen coupling respectively again；

E) couplet obtained by purification procedures d) is into the pneumonia multivalence combined vaccinogen liquid.

As further preferred scheme, the preparation method of the pneumonia multivalence combined vaccine specifically includes following steps：

B) variety carrier albumen respectively selected by separating-purifying；

C) various capsular polysaccharides and magnetic Nano microsphere are coupled into polysaccharide-magnetic Nano microsphere couplet respectively, then passed through Chemical modification the, so that-OH that the polysaccharide-magnetic Nano microsphere couplet surface is not reacted with polysaccharide is modified as into-CHO；

As further preferred scheme, the preparation method also includes preparing magnetic Nano microsphere, including first prepares nano magnetic Property particle prepares the process of magnetic Nano microsphere again；Wherein magnetic nanoparticle can pass through chemical coprecipitation, hydro-thermal method or molten Prepared by glue method pyrolysismethod, magnetic Nano microsphere can be prepared by investment, monomer polymerization method or in-situ method, preferably using chemistry Coprecipitation prepares magnetic nanoparticle and magnetic nanoparticle and high polymer material is combined into solvent extraction through fast film emulsification again Method and/or investment or monomer polymerization method prepare magnetic Nano microsphere；Nano magnetic is most preferably prepared using chemical coprecipitation Property particle magnetic nanoparticle and high polymer material are combined into solvent extraction through fast film emulsification again and/or investment prepares grain The homogeneous magnetic Nano microsphere in footpath；Wherein magnetic particle is preferably Fe₃O₄。

As still more preferably scheme, the preparation method of above-mentioned nano-magnetic ion can refer to Fe in the prior art₃O₄ Prepared by the method for magnetic Nano microsphere, for details, reference can be made to following embodiments, whether achievable is not intended as the present invention Key factor.

As still more preferably scheme, the separation purifying technique of polysaccharide can be according to required polysaccharide and albumen in step a Species is operated according to prior art.

As still more preferably scheme, the preparation of the albumen in step b and separation purifying technique can be according to required more Sugar and protein classes are operated according to prior art.

As still more preferably scheme, step c concrete operations are：In coupling medium reaction buffer, at room temperature, Under pH4.0-9.0, polysaccharide obtained by step a and magnetic Nano microsphere coreaction are obtained into polysaccharide-magnetic Nano microsphere in 6-24 hours even It is conjuncted, then it is further modified by 25% glutaraldehyde so that polysaccharide-magnetic Nano microsphere couplet surface not with it is many - the OH of sugar reaction is modified as-CHO；Wherein coupling medium is preferably PB, PBS or TBS, most preferably 0.1M TBS solution；Reaction is slow The Optimal pH of fliud flushing is 6；Optimum reacting time is 12-16 hours.

As still more preferably scheme, step d concrete operations are：In coupling medium reaction buffer, 4 DEG C, Under pH4.0-9.0, by polysaccharide-magnetic Nano microsphere couplet chemically modified obtained by step c and carrier protein coreaction 12- 24 hours；Wherein coupling medium is preferably PB, PBS or TBS, most preferably 0.1M TBS solution；The Optimal pH of reaction buffer is 6；Optimum reacting time is 24 hours.

As still more preferably scheme, isolating and purifying for step e is by coupling conjugate and unreacted polysaccharide and load Body protein is isolated and purified, and purification process can use chromatography or ultrafiltration；Can be according to the molecule of the pneumonia multivalence combined vaccine of acquisition Size is isolated and purified, chromatography be preferred to use Superdex200 solvent resistant columns, Sepharose CL-4B or Sepharose CL-6B are carried out；And ultrafiltration is to separate conjugate and unreacted reactant using different retention molecular weight film.

The pneumonia multivalence combined vaccine preparation can use aqua or freeze-dried.In order to strengthen its immunogenicity, assistant can be added Agent, conventional adjuvant has aluminium adjuvant, such as aluminium hydroxide, aluminum phosphate, prioritizing selection aluminum phosphate of the present invention.The solvent of conjugate can be 0.2 sodium chloride solution, 1 × PBS other can stablize the buffer solution of polysaccharide or conjugate.The pneumonia of the present invention is more The preparation method of each preparation of valency combined vaccine is prepared using the conventional meanses of the art.Wherein, preferably in sugared (such as sucrose Or lactose) in the presence of freezed.

The pneumonia multivalence combined vaccine that the present invention is provided can be immunized with any existing approach, including skin corium or The forms such as percutaneous drug delivery, intramuscular delivery.Wherein, the amount given is that those skilled in the art are confirmable according to general knowledge.

Term is defined

Core VP8：It is that one section in rotavirus protein VP8 has and cell surface contains sialic acid (sialic acid) The polypeptide chain of adhesive function, usually contains 160 amino acid residues.

VP8 polypeptide chain fragments：It is less than total length VP8 polypeptide chain for any molecular weight.

VP4 polypeptide chain fragments：It is less than total length VP4 polypeptide chain for any molecular weight.

VP8 specific antigen cluster chains：Full VP8 polypeptide chains contain multiple antigenic determinants, are cut by the method for genetic recombination Cut without important amino acid, and retain the polypeptide chain containing specific antigen cluster, molecular weight is typically smaller than total length VP8 polypeptides Chain.

VP4 specific antigen cluster chains：Full VP4 polypeptide chains contain multiple antigenic determinants, are cut by the method for genetic recombination Cut without important amino acid, and retain the polypeptide chain containing specific antigen cluster, molecular weight is typically smaller than total length VP4 polypeptides Chain.

VP8 fusion proteins：With gene recombination method, by VP8 albumen and other soluble protein polypeptide chain amalgamation and expressions, with Improve the solubilities of VP8 in aqueous；Or enhancing VP8 immunogenicity.

NSP4 albumen：It is non-structural protein, with enterotoxin characteristic, molecular weight is 28kDa, contains 175 amino acid.

VP8-NSP4 fusion proteins：With the method for genetic recombination, by VP8 and NSP4 polypeptide chain amalgamation and expressions, to improve VP8 Solubility in aqueous, while so that fusion protein has the protection antibody for stimulating body to produce anti-NSP4.

Capsular polysaccharide fragment：Be tod by physics (such as ultrasonic wave, particle spray), chemistry (such as acid, alkali, enzymic digestion) method It is many that polysaccharide (the claiming full polysaccharide or original polysaccharide) degraded (depolymerization) purified in inoculum is obtained Bglii fragment, molecular weight is usually less than original polysaccharide.Pod membrane oligosaccharide：By physics (such as ultrasonic wave, particle spray), chemistry (such as Acid, alkali, enzymic digestion) etc. method by the polysaccharide purified in inoculum (claim full polysaccharide or original polysaccharide) degraded (depolymerization) monosaccharide residue in the polysaccharide fragment obtained, molecular structure is usually less than 10.But monose is residual The definition of radix amount is variant, and the polysaccharide chain more than 10 less than 20 monosaccharide residues is also turned into oligosaccharide by some documents.

The present invention has the advantage that compared with prior art：

It is and existing 1. the pneumonia multivalence combined vaccine is the immunoconjugates containing two or more different carriers albumen Pneumococcal conjugated vaccine compare, its immunogenicity is stronger, and the polysaccharide antibody level of induction is higher than single carrier conjugates, can be with Cause immune response, especially infant in broader crowd；Carrier epitope can be avoided by reducing each carrier dosage Overload；T-helper cell activity can be strengthened by two kinds of carriers；

2. because the Protein Epitopes with protectiveness also can two kinds of albumen mixing notes of induction ratio in two kinds of carrier proteins Higher immune response when penetrating, mutually synergy, further enhance the immunogenicity of carrier protein, add body to many The immune response of sugar；Another is adjuvant effect carrier protein, immunogenicity can be increased further, and be immunized with certain Memory；

3. the pioneering use nanoparticle of the present invention is effectively prevented from system as polysaccharide and the connector of overloading body protein The autoimmunity syndrome of capsular polysaccharide and protein during standby, it is possible to increase with reference to the yield of product, and beneficial to the quality of product Control；The space length that can effectively extend between capsular polysaccharide and carrier protein, reduces carrier protein to capsular polysaccharide antigen The spatial masking effect of epitope, is conducive to improving the immunogenicity of capsular polysaccharide；

4.-the OH by nanoparticle surface initiated in the preparation process of the pneumonia multivalence combined vaccine is first carried out with polysaccharide Coupling reaction, then by couplet it is chemically modified by unreacted-OH be modified as-CHO with-the NH in carrier protein₂Carry out even It is coupled and closes, associated methods more of the prior art is more stable；

5. its preparation method is simple, the need for being adapted to scale industrial production, capsular polysaccharide and carrier egg are not significantly changed White architectural feature.

To sum up state, the pneumonia multivalence combined vaccine preparation technology that the present invention is provided is simple, uses nanoparticle for attachment Pneumonia multivalence combined vaccine can strengthen mouse Th1 type immune responses, and polysaccharide specificity antibody immune lasting effect, spy The opposite sex and compatibility, additionally can induce mouse and produce rotavirus antibody；Possesses the preventive effect of two kinds of vaccines；Therefore have Very wide application prospect.

Brief description of the drawings

Fig. 1 is the pneumonia multivalence combined vaccine obtained by the embodiment of the present invention 1¹H-NMR spectrum；

The immune response experimental result of the polysaccharide specificity antibody for the pneumonia multivalence combined vaccine that Fig. 2 provides for the present invention is shown It is intended to；

The immune lasting effect experimental result of the polysaccharide specificity antibody for the pneumonia multivalence combined vaccine that Fig. 3 provides for the present invention Schematic diagram.

Embodiment

The present invention is made with reference to embodiment further in detail, intactly to illustrate.Reagent used below or equipment are Commercially available kind, unless otherwise specified, operates, will not be described here to specifications.

Below for the present invention is further illustrated in conjunction with specific embodiments, but it is not construed as limitation of the invention.

Embodiment 1

First, magnetic Nano microsphere is prepared

1. take 2.24gFeSO₄-7H₂O and 3.24gFeCl₃-6H₂O is dissolved in the dddH that 10mL and 15mL filters deoxygenation respectively₂O In, mix and hook after dissolving, add the dddH that 100mL filters deoxygenation₂O；

2. in N₂Protection under stir 5min, it is disposable to add 50mL1mol/LNaOH solution, then adjust pH value of solution to 9- 10, accelerate mixing speed to 200-250r/min, continuously stir 30min；

3. reaction vessel is transferred in 65-70 DEG C of water-bath, continue in N₂Protection under stirring ageing 30min；

4. reaction, which is finished, is settled to 100mL, micro- Microscopic observation magnetic particle synthesizes situation；

5. 400mgPLGA is dissolved in 10mLEA solvents as oil phase (O), the above-mentioned magnetic particle solution conducts of 3mL are added Interior aqueous phase (W₁), just emulsificationization is carried out in ice-water bath using ultrasonic cell disintegration instrument (120W, 60s) and prepares colostrum, then will just Breast pours into a certain amount of aqueous solution (outer aqueous phase, W containing 15g/LPVA and 0.9% (ω) NaCl₂), magnetic agitation (300r/min, 2min) prepare pre- double emulsion (W₁/0/W₂), then pre- emulsion is poured into the storage tank of fast film emulsification, with certain N₂Pressure by its SPG films are pressed through repeatedly, obtain the nanoparticle emulsion drop of uniform particle diameter.In addition, unspent nanoparticle can be made into it is freeze-dried Continue to employ.

Or by taking Fe₃O₄Magnetic particle is added with 50mL with absolute ethyl alcohol as isometric, after ultrasonic activation 30min, puts 60 In DEG C water-bath, the slow 10mLPELA that is added dropwise is to magnetic Nano microsphere progress-NH₂It is terminal-modified and PELA is wrapped in Fe₃O₄Magnetic Outside property particle, stirring reaction l0h, is made magnetic Nano microsphere under nitrogen protection；After completion of the reaction, washed with 50mL absolute ethyl alcohols Paint 3 times, then washed with 0.01MPBS after paint three times, it is settled to 50mL, micro- Microscopic observation magnetic bead is modified situation, micro- to magnetic Nano Ball surface-NH₂End is changed to-OH ends.

2nd, the preparation of pneumococal polysaccharide

1. choose 24 kinds of serotypes (1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F) pneumococcus culture；

2. the strong capsular polysaccharide of antigenicity in any of the above Pneumococcal serotype is purified respectively：Pneumococcus, after inactivation Supernatant is collected by centrifugation, through being concentrated by ultrafiltration, is separately added into right amount that (volume fraction is 70% according to each Pneumococcus serotypes characteristic ) pre-cooled ethanol, it is collected by centrifugation, obtains rough polysaccharide；Rough polysaccharide is dissolved in sodium acetate solution, then by 1：2 ratios are with cold Phenol is mixed, and removing protein is removed in centrifugation, and phenol is carried 5-6 times repeatedly, is collected supernatant, is dialysed with distilled water, liquid adds 2mol/L chlorine after dialysis Change calcium solution, add ethanol stirring, centrifugation removes nucleic acid, collects supernatant, adds ethanol (final concentration 80% is stirred), be collected by centrifugation Precipitation, precipitation is washed with ethanol, acetone, is that multivalence refines capsular polysaccharide after dehydrating, is put -20 DEG C and save backup.

3rd, the preparation of rotavirus carrier protein

The preparation of rotavirus carrier protein can be found in the preparation method of a variety of recombinant proteins of the prior art, this implementation Example is prepared using the method referred in CN 101972475, can be reduced to following steps, design parameter is not repeated：

1st, the cDNA storehouses of rotavirus are set up

The VP4 genes for Wa strain VP8 albumen selected, amplimer design is as follows：Sense primer HWaVP4 ρ ET28：

5 '-TTACATATGGCTTCGCTCATTTATAG-3 ', anti-sense primer AHWaVP4 ρ ET28：

5’-CCGGATCCCTAGTCTTCATTAACTTGTGCT-3’。

2nd, pET28aWaVP8 expression total length VP8 plasmids are built

3rd, expression restructuring VP8 albumen

1) obtained pET28aWaVP8 plasmids are transformed into BL21 (DE3) competent cells, inoculating cell to 50 μ G/mL kanamycins LB culture dishes, in CO at 37 DEG C₂In incubator overnight.

2) bacterium colony is picked out, 10 milliliters of kanamycins LB nutrient solution (1% tryptoses containing 50 μ g/mL are inoculated into Peptone, 0.5% yeast extract, 1%NaCl, pH7.5) middle amplification, in 37 DEG C of overnight incubations.

3) nutrient solution is turned to be inoculated into 100 milliliters of 50 μ g/mL kanamycins LB nutrient solutions, continues to cultivate.Treat absorbance When 600nm OD reaches 1.0, nutrient solution turn is inoculated into 6 and is raised in 50 μ g/mL kanamycins LB nutrient solutions, is continued at 37 DEG C, Shake in fast 200rpm shaking table and cultivate, when absorbance 600nm OD reaches 0.6~0.8, add 0.3mM IPTG (isopropyls Base-β-D- thiogalactosides) induction VP8 expression.

4) under identical condition of culture, after inducing 4 hours, with 4000g, after 10 DEG C centrifuge 20 minutes, bacterium is collected Body.

5) bacterial suspension is crushed after bacterium in 20mL 1 × PBS solution with French press filtration kettle (Frenchpress), Under 10000g, centrifuged 30 minutes at 10 DEG C, abandon supernatant, collect the inclusion body of precipitation.Before being further purified, storage is forgiven Body is in -40 DEG C.

3rd, VP8 albumen is purified from inclusion body

1) 0.5 gram of the VP8 inclusion bodys (weight in wet base) of preparation are weighed, (10mMTris, 100mM phosphate delay with cleaning buffer solution Fliud flushing, 2Murea, pH8.0) suspension inclusion body, it is incubated 30 minutes, is centrifuged 10 minutes with 10,000g, collection is forgiven at room temperature Body.Above step is repeated three times except the foreign protein depolluted.

2) by inclusion body precipitation be dissolved in dissolving buffer solution (10mMTris-HCl, 100mM phosphate buffer, 8M urea, PH8.0 in), it is incubated 1 hour in stirring on ice.Centrifuged 30 minutes with 16,000g, collect supernatant, abandon insoluble sediment.

3) with fixing metal ions affinity chromatography chromatography (IMAC) purifying His-tagged restructuring VP8 albumen.

4) eluent containing VP8 is collected, is transferred in bag filter in the TBS containing 20mM beta -mercaptoethanols 1 and 8M urea (pH4.0) dialysed in, and gradually reduce the concentration (i.e. 8,6,4,2, and 1M) of urea, the dialysed overnight in 4 DEG C.Then containing Dialyse twice, finally dialysed in TBS solution in the TBSpH5.5 solution of 2mM beta -mercaptoethanols.According to restructuring VP8 albumen sources Strain it is different, finally to be dialysed.

4th, the preparation of Pneumococal surface protein A (rPspA)

PspA albumen is cloned into Escherichia coli to be expressed and isolated and purified, specifically included：

1. the optimization of target gene and the structure of recombinant expression plasmid

PspA gene orders (GI is obtained in GenBank：193804931) and optimize, add and carried out after His labels Full genome is synthesized, by the sequence of synthesis after Sac I and the double digestions of Nde I, the expression vector of directed cloning to same double digestion In pET-30a (+), transformed competence colibacillus e. coli bl21 Star (DE3), 37 DEG C of incubated overnights, picking positive monoclonal bacterium colony, Plasmid is extracted after amplification cultivation, is identified with Nde I and the double digestions of Sac I, and send sequencing, correct recombinant expression plasmid will be sequenced and orders Entitled pET-30a-rPspA.

2. induced expression and the purifying of recombinant protein

Recovery engineering bacteria, with 1：100 ratio inoculation 2 × LB culture mediums, at 37 DEG C, expand culture under 237r/min, when When thalline value is about 12, IPTG to final concentration of 1mmol/L is added, 37 DEG C of induction 4h, sampling carries out SDS-PAGE analysis centrifugals Induction thalline is collected, physiological saline is added and washing 2 times is resuspended, with 1:10 (g/mL) ratio adds 05mol/LNaCl5mmol/L Imidazoles 20mmol/LPB (pH7.4) buffer solution is resuspended in thalline, ultrasonic disruption thalline, 8000 × g centrifugation 40min, collection Clearly, purified in nickel ion chromatographic column, by specification operation purified product and analyze by carrier pET-30a (+) conversion E. coli bl21 Star (DE3) whole cell (control) and after upper step purification of samples is separated through SDS-PAGE electrotransfer to nitre On acid cellulose film, 2h is closed with 5% skimmed milk power shaking table slight oscillatory；Add His mouse source monoclonal antibody (1: 800 dilution), 4 DEG C of mistakes Night；TBST is cleaned 3 times, is added the sheep anti-mouse igg (1: 2000 dilution) of HRP marks, is incubated at room temperature 1h；Washing 3 times, DAB colour developings.

5th, the preparation of pneumonia multivalence combined vaccine

1. polysaccharide-magnetic Nano microsphere coupling

Under 0.1MTBS solution buffer solutions, pH6.0 adds obtained one or more capsular polysaccharides of above-mentioned steps and magnetic is received In meter Wei Qiu, the present embodiment using 13 valency capsular polysaccharides (1,3,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F), pod membrane The mass ratio of polysaccharide and magnetic Nano microsphere is 1:(0.5-1), reacts 6-24 hours at room temperature；The mass ratio wherein optimized is 1:1, aldolisation 16 hours at room temperature.After reaction terminates, fully dialysed with bag filter, remove unreacted magnetic Nano micro- Ball.

2. coupling is modifies

Take and 2mL25% glutaric acids be slowly added dropwise under step 1 polysaccharide-magnetic Nano microsphere couplet 30mL, stirring condition, 200r/min stirring reactions 6 hours；After completion of the reaction, washed with 0.01MPBS after three times, be settled to 30mL, micro- Microscopic observation Magnetic Nano microsphere be modified situation so that polysaccharide-magnetic Nano microsphere couplet surface with polysaccharide react-OH be not modified as- CHO；N₂, 4 DEG C save backup.

3. it is coupled altogether with PspA carrier proteins and rotavirus protein

Under 0.1MTBS solution buffer solutions, 4 DEG C, under pH4.0-9.0, by modified polysaccharide-magnetic Nano microsphere respectively with Rotavirus protein and PspA albumen coreaction (to ensure enough carrier proteins and magnetic Nano microsphere, were used for 24 hours Nano-carrier albumen addition is measured, such as mass ratio uses polysaccharide：Magnetic Nano microsphere：PspA：Rotavirus protein=1：1：2： 2)。

4. pneumonia multivalence combined vaccine is isolated and purified

Isolated and purified with Superdex200 solvent resistant columns (2.6cm × 60cm), eluent delays for 20mM phosphoric acid Fliud flushing (pH7.4), flow velocity is 3mL/min.Collect the eluting peak corresponding to polysaccharide-magnetic Nano microsphere-complex carries albumen.

The composition analysis of obtained pneumonia multivalence combined vaccine

With¹H-NMR carries out the detection pneumonia multivalence combined vaccine, and testing result is shown in Fig. 1.As shown in figure 1, many with pod membrane Glycan molecule is compared, and conjugate occurs in that characteristic peak at 0.4-1.4ppm, corresponding to the aliphatic chain amino acid residue of carrier protein. The characteristic peak of the aromatic amino acid residue corresponding to carrier protein is occurred in that at 7.2ppm.This show capsular polysaccharide molecule into It has been coupled two kinds of carrier proteins of rotavirus protein and PspA carrier proteins work(.Occurred in that at 6.2ppm corresponding to succinyl The characteristic peak of imines, this shows to contain succinimide in the connecting bridge of polysaccharide conjugate vaccine.In addition, 5.2,1.6,3.6 outlets PELA characteristic peak proves that magnetic Nano microsphere PELA is used as attachment.Therefore, capsular polysaccharide is combining two kinds of carrier proteins Front and rear, obvious change does not occur for its structure.

It is the molecular weight distribution situation that SEC-MALLS methods detect GL-PP conjugate by CL-4B；By immune double The method of expansion determines albumen and many sugar types in GL-PP conjugate using different antibody serums；Detected by anthrone method The polyoses content of GL-PP conjugate；Lowry methods protein content detects the total protein content of GL-PP conjugate, then passes through The GL-PP for obtaining conjugate is calculated with reference to than (Ratio)；Rotavirus protein, PspA protein concentrations pass through ELISA Detection.As a result show：In the pneumonia multivalence combined vaccinogen liquid, the concentration ratio of each material is about：Polysaccharide：Magnetic Nano is micro- Ball：PspA：Rotavirus protein=1：1：1：1).

Comparative example 1

This comparative example is differed only in embodiment 1：Nonmagnetic nanoparticle is as connector in obtained vaccine, and And complex carries albumen and polysaccharide are conjugated by the method referred in the prior art and obtained.

Comparative example 2

This comparative example is differed only in embodiment 1：Without PspA carrier proteins in obtained vaccine, only with polysaccharide- Magnetic nanometer particles-rotavirus carrier protein is pneumonia multivalence combined vaccine.

Embodiment 2

The present embodiment and the carrier protein for differing only in the pneumonia multivalence combined vaccine of embodiment 1 are tetanus Toxin vector albumen and PspA.

There is theoretical error scope with the acquired results of embodiment 1 in the composition analysis result of obtained pneumonia multivalence combined vaccine Interior uniformity.

Embodiment 3

The present embodiment is differed only in embodiment 1：The carrier protein of the pneumonia multivalence combined vaccine is colyliform disease Poisonous carrier albumen and tetanus toxoid carrier albumen.

Embodiment 4

The present embodiment is differed only in embodiment 1：The carrier protein of the pneumonia multivalence combined vaccine is colyliform disease Poisonous carrier albumen and bloodthirsty Bacillus influenzae surface protein HiD.

Embodiment 5

The present embodiment is differed only in embodiment 1：The carrier protein of the pneumonia multivalence combined vaccine is carrier egg White CRM197 and PspA.

Embodiment 6

The present embodiment is differed only in embodiment 1：Connector is PLGA magnetic nanoparticles.

Embodiment 7

The present embodiment is differed only in embodiment 1：Connector is PEG magnetic nanoparticles.

Vaccine evaluation

Assessment experimental evaluation embodiment 1~7 and the institute of comparative example 1~2 of the conventional potency of the vaccines such as immunogenicity are used below Obtain the immune effect of vaccine：

A. pneumonia multivalence combined vaccine immunogenicity experiments

The female Blab/C mouse of 100 5 week old are chosen, body weight is 15-22 grams.Be randomly divided into 10 groups, i.e. embodiment 1~ 7th, comparative example 1~2 and positive controls (Prevnar 13), every group of 10 mouse.Intraperitoneal injection, every per injection is 5 micro- Gram, inject 1 time weekly, co-injection 3 times.Posterior orbit takes blood within 21 days.With ELISA method detection mice plasma moderate resistance capsular polysaccharide IgG, IgG1 and IgG2a.Experimental result is shown in Table 1

Table 1

As shown in table 1, the pneumonia multivalence combined vaccine of gained can be obviously improved antibody titer (p in embodiment 1~7< 0.0001**), as shown in Fig. 2 and being substantially better than comparative example 1 and comparative example 2 and positive controls；As can be seen here, using double Carrier protein and can significantly increase that Th1 types are immune with magnetic Nano microsphere and pneumonia multivalence combined vaccine obtained by pneumonia polysaccharide should Answer, and immune effect is substantially better than obtained by single carrier protein without nano-magnetic microsphere connector and comparative example 2 of comparative example 1 Pneumonia multivalence combined vaccine；But it will also realize that according to the immune response result of the gained of embodiment 1~7, be complex carries by PspA or HiD The immune response effect of one of albumen is preferable, and PELA and PLGA is good compared with PEG immune responses effect in magnetic nano-carrier.

B. the immune lasting effect of polysaccharide specificity antibody, specificity and affinity experiment

Once tested with pneumonia multivalence combined vaccine obtained by embodiment 1, comparative example 1 and 2 and positive control respectively.

1st, lasting effect is immunized

By determining the titre of immune latter 20 weeks interior polysaccharide specificity antibody of three pins, to study exempting from for polysaccharide specificity antibody Epidemic disease lasting effect.Fig. 3 is the immune lasting effect schematic diagram, as shown in figure 3, its polysaccharide specificity IgG titres are relatively low, with injection Time increases and gradually reduced, and the 4th Zhou Houyi can not be detected.The polysaccharide specificity IgG titres that comparative example 1 and 2 is produced are low In 1 group of embodiment, and higher than positive controls.Polysaccharide specificity IgG titres in the 2nd Zhou Dafeng, in the 4-20 weeks gradually under Drop, the decrease speed of wherein positive controls is most fast.After 18th week, 1 group of embodiment, comparative example 1, comparative example 2 and positive control The polysaccharide specificity IgG titres of group are 20%, 15%, 12% and the 10% of its peak value respectively.Therefore, the pneumonia that the present invention is provided Multivalence combined vaccine can strengthen the immune lasting effect of polysaccharide specificity antibody.

2nd, specificity and compatibility

Added into 200 times of PS-TT groups, PS-PLGA-TT groups and PS-PELA-TT group mice plasmas diluted different amounts of Capsular polysaccharide, the antibody level of mice plasma moderate resistance capsular polysaccharide is detected with ELISA method, 2 are the results are shown in Table.

Table 2

As shown in table 2, with the increase of polysaccharide addition, in the orifice plate of polysaccharide specificity antibody binding 96 ability of polysaccharide by Gradually reduce.When the polysaccharide of addition reaches 20 μ g, the Disability of antibody binding polysaccharide.This shows pneumonia provided by the present invention The anti-capsular polysaccharide antibody that multivalence combined vaccine inducing mouse is produced can specifically combine capsular polysaccharide.

The Antibody Avidity of anti-capsular polysaccharide is determined with ammonium thiocyanate.The polysaccharide of capsular polysaccharide group (negative control) is special Property antibody index of affinity be 1.18mol/L, and positive controls, 1 group of comparative example, the antibody of 1 group of 2 groups of groups of comparative example and embodiment Index of affinity is respectively 2.65mol/L, 2.80mol/L, 3.02mol/L and 3.21mol/L.This shows the pneumonia that the present invention is provided Multivalence combined vaccine can significantly improve the affinity of polysaccharide specificity antibody.

The immunogenicity comparative experiments of monotype pneumococal polysaccharide in C, multivalence pneumonia combined vaccine

Using the method described in embodiment 1, each monotype pneumococcal Polysaccharide Conjugate Vaccine totally 13 class envelope antigen is prepared Type (is combined into conjugate vaccines) only with a kind of pneumococal polysaccharide through magnetic nanoparticle and two kinds of carrier proteins, by Connect ELISA method and detect antibody titer respectively, various pneumonia polysaccharide conjugate vaccine is dissolved in 0.05mol/L pH9.6 carbonate In cushioning liquid, with 20ug/ml concentration coated elisa plates, 2% BSA fluid-tights are closed.During experiment by test serum 100,200, 400th, 800,1600,3200,6400,12800,25600,51200,102400,204800, enzyme mark is added after 409600 times of dilutions Plate, puts the sheep anti mouse secondary antibody that horseradish peroxidase-labeled is added after 37 DEG C of reaction 40min, conventional wash.Add and be free of simultaneously The buffer solution of serum is used as negative control.Developed the color with tetra-amino-biphenyl amine, A values are read at ELIASA 450nm wavelength.Calculate per hole Middle A values are worth ratio with negative hole A, but the ratio extension rate maximum more than 2.1 epoch is the antibody titer in serum, to 10 The titre of mouse carries out geometric average, and is that bottom calculates logarithm value with 10, and concrete outcome is shown in Table 3.

Each monotype pneumococcal conjugated vaccine Evaluation of Immunogenicity of table 3

As shown in Table 3：It is many as the pneumonia obtained by the complex carries albumen of connector using the magnetic nanoparticle that the present invention is provided Sugared combined vaccine can significantly increase immune response of the mouse to lung chain polysaccharide, wherein 1,3,5 and 6A/B types it is especially notable, tool There is more preferably immune response effect；Such population of China that is more suitable for is used.

In D, rotavirus and experimental result

Using method difference obtained mice serum each group (2 groups of embodiment 1,1 group of comparative example and comparative example in embodiment 1 Inject a pin, two pins and three pins respectively) each mice serum respectively take 10 μ L mix, for neutralization test Sample serum.

The neutralization test for the anti-WaVP8 antibody that injection small white mouse produces uses BSC-1 cells on microplate (microplate) Carry out.After heat inactivated serum is serially diluted, after being mixed with 100TCID50 Wa rotavirus strains, in 4 DEG C of cultures 1 hour.Then BSC-1 cells are inoculated on microplate, hatched 1 hour.Plus DMEM (being free of serum) is added to each hole, 37 DEG C Hatching 1 hour.Finally, dilution antiserum can prevent the concentration that rotavirus cytopathic effect (CPE) occurs from being dripped to neutralize Degree.Polysaccharide control group serum is used as negative control.Experimental result is shown in Table 4.

Table 4：With Rotavirus Wa strain strain result of the test in pneumonia multivalence combined vaccine injection small white mouse antibody

As shown in table 3, comparative example 1 and comparative example 2 are substantially better than with rotavirus effect in pneumonia multivalence combined vaccine, can Significantly induction rotavirus antibody is produced.

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.

Claims

1. a kind of pneumonia multivalence combined vaccine, it is characterised in that：The pneumonia multivalence combined vaccine is multivalent pneumococcal polysaccharide With pneumonia multivalence combined vaccine of two or more the carrier protein through connector, wherein, connector is received for magnetic Meter Wei Qiu.

2. pneumonia multivalence combined vaccine according to claim 1, it is characterised in that：The magnetic of the magnetic Nano microsphere is micro- The inside that grain is located at magnetic Nano microsphere is core, and high polymer material is wrapped in the outside of magnetic particle.

3. pneumonia multivalence combined vaccine according to claim 2, it is characterised in that：High polymer material is boiomacromolecule material Material, selected from chitosan, polyethylene glycol, Poly(D,L-lactide-co-glycolide (PLGA), PLA-PEG copolymer (PELA) one or more of mixtures in.

4. pneumonia multivalence combined vaccine according to claim 1, it is characterised in that：Multivalent pneumococcal polysaccharide is a variety of lungs Scorching coccus capsular polysaccharide, preferably separates the capsular polysaccharide on purification Pneumococcal serotype pod membrane, the serotype pneumonia ball The serotype of bacterium include 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20th, 22F, 23F and/or 33F.

5. pneumonia multivalence combined vaccine according to claim 1, it is characterised in that：Multivalent pneumococcal polysaccharide and two kinds of loads The mass ratio of body protein is (0.5~2)：1.

6. pneumonia multivalence combined vaccine according to claim 1, it is characterised in that：The carrier protein is selected from recombined human wheel Shape virus protein, diphtheria toxoid, tetanus toxoid, carrier protein CRM197, bloodthirsty Bacillus influenzae surface protein HiD, hundred Day coughs Prn surface proteins, pertussis Fha antigens and/or Pneumococal surface protein A (rPspA).

7. pneumonia multivalence combined vaccine according to claim 1, it is characterised in that：Have a kind of to be thermophilic in the carrier protein Blood Bacillus influenzae surface protein HiD or Pneumococal surface protein A (rPspA).

8. the preparation method of any pneumonia multivalence combined vaccine of claim 1~7, it is characterised in that：By multivalence pneumonia ball Granulose is formed with magnetic Nano microsphere through coupling respectively with two kinds or more of carrier protein.

9. the preparation method of pneumonia multivalence combined vaccine according to claim 8, it is characterised in that specifically include following step Suddenly：

B) the variety carrier albumen simultaneously selected by separating-purifying is prepared respectively；

10. the preparation method of pneumonia multivalence combined vaccine according to claim 8, it is characterised in that specifically include following Step：

B) variety carrier albumen respectively selected by separating-purifying；

C) various capsular polysaccharides and magnetic Nano microsphere are coupled into polysaccharide-magnetic Nano microsphere couplet respectively, then through chemistry It is modified, so that-the OH that the polysaccharide-magnetic Nano microsphere couplet surface is not reacted with polysaccharide is modified as into-CHO；