CN106668853A

CN106668853A - Pneumococcus conjugate vaccine and preparation method thereof

Info

Publication number: CN106668853A
Application number: CN201710024067.2A
Authority: CN
Inventors: 吴克; 刘昊智; 程超; 王文灏
Original assignee: BRAVOVAX Co Ltd
Current assignee: BRAVOVAX Co Ltd
Priority date: 2014-07-25
Filing date: 2014-09-23
Publication date: 2017-05-17
Also published as: CN104189901B; CN104189901A; CN107412764A; CN107412764B

Abstract

The invention discloses a pneumococcus conjugated vaccine. The conjugate vaccine is a conjugate vaccine prepared by connecting a multivalent pneumococcus polysaccharide with two or more carrier proteins through a linker, wherein the linker is magnetic nanoparticles. The preparation method of the pneumococcus conjugated vaccine comprises the following step: coupling the multivalent pneumococcus polysaccharide and two or more carrier proteins with the nanoparticles respectively. The pneumococcus conjugated vaccine provided by the invention is simple in preparation process; by adopting the pneumococcus conjugated vaccine taking the nanoparticles as the linker, the Th1 immune response of a mouse and the immunity persistence, specificity and affinity of a polysaccharide specific antibody can be enhanced, and the mouse can be induced to produce a rotavirus antibody; the pneumococcus conjugated vaccine has the prevention effects of two kinds of vaccines, so that the pneumococcus conjugated vaccine has a very wide application prospect.

Description

A kind of streptococcus pneumoniae conjugate vaccines and preparation method thereof

Technical field

The present invention relates to a kind of vaccine, is to be related to a kind of streptococcus pneumoniae conjugate vaccines vaccine and preparation method thereof specifically, 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, their simple structures, mostly It is unicellular, also some even cellularity do not have yet, and we do not have all the time at one's side microorganism, it will usually by Microscope or ultramicroscope can just see their form and structure clearly；Wherein, pathogenic microorganism 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 The ability bred in vivo and resist host resistance and do not eliminated by it.Microorganism is pathogenic generic character, and pathogenecity is strong Weak degree is referred to as virulence.The establishment of infectious disease not by the virulence unilateral decision of microorganism, will also regard the strong of host Health situation and immune functional state.In general, the strong microorganism infection of the virulence body that immunity is not crossed, 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 Main resistance then can be susceptible and pathogenic to these microorganisms when reducing.The virulence of pathogen and host resistance between the two compared with Amount, draws the generation of infectious disease, 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 mortality 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 by 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.Through effort for many years, medical circle has been developed over a variety of To prevent, antibacterial, virus and funguses etc. infect the various diseases for causing to vaccine, drastically increase the health of the mankind Level.The continuous development of biotechnology, promotes the variation of vaccine kind.Today, to infectious disease caused by pre- anti-virus There are inactivation of viruses technological development vaccine out, such as Vaccinum Encephalitidis Epidemicae, poliomyelitis vaccine, influenza vaccines；Use attenuated virus Technological development attenuated live vaccine out, such as Rotavirus Vaccine, oral polio virus vaccine, measles virus vaccines, Mumps viruss vaccine, rubella virus vaccine and chickenpox vaccine etc..The useful proteins and polysaccharide of prevention bacterial infectious disease etc. are raw Thing macromolecule purifying technological development antibacterial class vaccine out, such as tetanus toxoid, diphtheria toxoid, DT-Pa and Its subcellular components, epidemic cerebrospinal 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 uteri Cancer) etc..The prevention meningitiss developed with half chemical combination technology and the bacterial vaccine of pneumonia, such as popular haemophiluss 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 of the continuous development of vaccine product, by opposite Thing technology is updated, and can be developed more new generation vaccine products and human health be chosen dealing with different infectious disease War.

2nd, combined vaccine general introduction

Polysaccharide is a kind of important immune effective 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 immunogen.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 Meningitiss, E.Coli O 157 that at present the serious disease such as hemophilus influenza (Hib) of many harm causes:It is little that H7 causes 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, and 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 (with albumen as carrier Bacterial polysaccharideses class) polysaccharide covalent combined and is prepared into polysaccharide-protein on protein carrier and combines epidemic disease by vaccine using chemical method Seedling, for improving the immunogenicity of bacterial vaccine polysaccharide antigen, such as Haemophilus Influenzae Type b Conjugate Vaccine, meningococcuss knot Vaccine and pneumococcal conjugated vaccine etc. are closed, short decades, its development was quite rapid, achieved with notable achievement.

3rd, pneumococcal harm and its epidemiological study

Infection by caused by streptococcus pneumoniae (lung chain) be whole world M ＆ M act primarily as because.Pneumonia, send out Hot bacteremia and meningitiss are the most common forms of expression of invasive pneumococcal disease, and the antibacterial 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 Jing is often in influenza infection Occur afterwards, the probability that pneumococcal disease shows effect during influenza further increases.

The disease caused by streptococcus pneumoniae has become a global important public health problem.Streptococcus pneumoniae becomes The number one killer of global child.The case fatality rate of China's pneumonia is 16.4%, wherein more than 50 years old middle-aged and elderly people and less than 1 years old baby children Youngster is up to respectively 28.6% and 22.0%.Carrying rate of China streptococcus pneumoniae in healthy children is higher, and statistics show, 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 child Dead major reason.Main reason is that the development of infant immunisation system is not perfect, immunity is weaker.And the age gets over Little baby, immunity is weaker.About 90 kinds serotypes (bacterial strain) of streptococcus pneumoniae, the statistics of China show streptococcus pneumoniae Several serotypes are followed successively by before infection strain：5th, 6,19,23,14,2,4 type.860 plants of streptococcus pneumoniae separation strains are have collected according to one Research show have 109 plants (12.7%) to show as sero-group 6, reach 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 lipopolysaccharide (Lipopolysaccharide, LPS) shell, or both have concurrently, its function is side Help pathogenic infection host.Capsular polysaccharide can shield bacterial cell surface functional component and avoid being recognized by host immune system, Prevent complement system by bacterial surface protein activation and immunocyte phagocytosis, if antibacterial is swallowed, capsular polysaccharide is prevented from Antibacterial is killed.In most of pathogen, different bacterial strains expresses the capsular polysaccharide and lipopolysaccharide of different structure, and generation is various not Same serological type strain.Pneumonia and meningitiss caused by streptococcus pneumoniae is by a big chunk bacterium in known 90 kinds of serotype Caused by strain infection institute.

As can be seen here, most of bacterial polysaccharideses class vaccines must be pathogenic to improve containing various different types of bacterial polysaccharideses Bacterial strain coverage rate, optimization and selection are an extremely complex epidemic diseases in vaccine comprising which kind of antibacterial or serotype polysaccharide Knowledge is inscribed.Once specifying which kind of polysaccharide antibody has protective effect, then vaccine can be produced as immunogen 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 Modal pathogenic bacterium (1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F), the various polysaccharide in difference fermentation culture and separating-purifying pneumococcal capsule is mixed by equal proportion Vaccine.The polysaccharide of antibacterial is a kind of thymus independent antigen, and this antigen is differred primarily in that with thymus dependent antigen The former does not need the auxiliary of T lymphocytes producing antibody.Clinical practice proves that the vaccine that capsular polysaccharide makes is clearly effective, And widely use in multiple countries.But there is problems with this kind of polysaccharide vaccine：(1) in brood or infants only Faint immunoreation can be produced, immunoreation is not even produced, immunoreation strengthens with the growth at age；(2) produce low The antibody of affinity；(3) of short duration immunoreation is only produced, the immunological memory and immunostimulant when not possessing inoculation repeatedly is imitated Should；(4) immunologic tolerance is easily produced；(5) common adjuvant is difficult to play a part of immunostimulant, 23 valency polysaccharide to this antigen Vaccine is 50-70% for the protective rate that intrusion type lung chain infects, and is only used for crowd's inoculation of more than 2 years old, and pneumonia Peak age of onset be the 6-12 monthly ages；(6) polysaccharide with repetitive structure is the immunogen of the class of T cell self reliance type 2, without T The participation of cell, they be cannot 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 the eighties in 20th century 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 infant of the age less than 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 enough stimulate body to produce as being grown up and long-acting be directed to the polysaccharide The antibacterial specificity protection IgG antibody in source.Therefore, polysaccharide is for being a kind of hapten less than the infant of 2 years old, it is impossible to enough conducts Vaccine is being inoculated with the child less than 2 years old.When bacterial polysaccharideses to be connected in the form of covalent bond protein carrier, such as tetanus poison Plain (Tetanus Toxoid, TT), because protein is a kind of T cell dependence antigen, can be by covalently bonded polysaccharide 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 antibacterial.The success of popular influenzae type polysaccharide-tetanus toxoid (PRP-TT) combined vaccine exploitation, The technology platform of an exploitation bacterial vaccine has been started, will bacterial polysaccharideses, such as capsular polysaccharide, O- specific polysaccharide (O- Specificpolysaccharide) or oligosaccharide (Oligosaccharide), to be covalently bonded on protein carrier Made by combined vaccine.Based on the success of this concept, medical biotechnology research circle using same chemical synthesis process by being opened Have issued the combined vaccine of different bacterium；Equally also with different synthetic technologys develop the combined vaccine of same antibacterial.

Capsular polysaccharide can shield bacterial cell surface functional component so as to avoid being recognized by host immune system, prevent Complement system is swallowed by the protein activation of bacterium surface and immunocyte.If antibacterial is swallowed by immunocyte, capsular polysaccharide Antibacterial can be avoided to be killed.Capsular polysaccharide is one of major antigen composition of meninges Neisseria, as vaccine to larger Child has certain protection.However, crowd of the capsular polysaccharide to 2 years old Infants Below, old people and B cell immunodeficiency 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 dependent reinforcement of T cell should Answer.By the way that polysaccharide conversion by polysaccharide and certain protein covalent bond, can be made into T cell dependence antigen, so as to stimulate baby children The synthesis of the T cell dependency antibody of youngster, and booster response can be produced, while the antibody of immunoglobulin (IgG) can also be improved Ratio.This polysaccharide conjugate vaccine can not only protect infant (less than 2 years old child), additionally it is possible to resistance be significantly enhanced poor Resistance of the patient to bacterium infection, therefore with very wide application prospect.1980, JohnRobbins brominations Cyanogen activating Hib capsular polysaccharides at random, then, by adipyl dihydrazide (Adipic Dihydrazide, ADH) as junctional complex (linker) it is added on the polysaccharide of activation, finally the polysaccharide covalent key after derivation is connected to EDC methods for carrier protein broken wound On wind toxoid, popular influenzae type polysaccharide-tetanus toxoid conjugate (PRP-TT) is synthesized.Due to There are multiple activation points on each polysaccharide chain, same on protein carrier there are multiple junction points, the conjugate of formation is a kind of polysaccharide With the cross-coupled macromole of albumen, 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 group polysaccharide is connected on BSA by reduction amine method covalent bond, has been synthesized epidemic cerebrospinal meningitis polysaccharide-BAS and has been combined epidemic disease 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, diphtherotoxin 197 (Cross reaction material sub197, CRM197) as carrier protein, to reduce Amine method has synthesized popular influenzae type oligosaccharide-variation avirulent strain diphtheria toxin, diphtherotoxin 197 (HbOC-CRM197) combined vaccine. Specific method is, with sodium periodate oxidation Hib capsular polysaccharides, the oligosaccharide for aldehyde radical in two ends to be produced, by reducing agent cyanogen Base sodium borohydride (sodiumcyanoborohydride), oligosaccharide is covalently bonded on protein carrier.Form molecular weight The combined with lipopolysaccharide thing of about 90kDa, has made containing the combination that 6 glycan molecules are carried on 30% polysaccharide and each albumen Vaccine.Subsequently, Merck (Merck, Sharpe and Dohme) is with mercapto chemistry, and the neisseria meningitis with purification are scorching B group's bacterial strain (Neisseria meningitidis groups B) bacterial surface protein complex (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 antibacterial 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 disease, the meningitiss such as caused by streptococcus pneumoniae caused by pneumonia, epidemic cerebrospinal meningitis coccus, Can be caused by various 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, it is inoculated with single serotype or bacterial strain combined vaccine, it is impossible to protect Shield is vaccinated the infection that human body avoids other serotypes or bacterial strain.For this reason, synthesize and prepare multivalence combined vaccine to come Expanding the protection coverage rate of vaccine becomes the main target of exploitation.

By effort for many years, with combination technology the wide multivalence combined vaccine of various coverage rates is have developed.Bacterial capsule Polysaccharide-protein combined vaccine occurs in earliest the thirties in 20th century, and Goebel and Avery is connected to 3 type pneumococal polysaccharides On horse serum globulin, the conjugate of generation can in animals produce the single-minded antibody of polysaccharide, while providing corresponding immunity Protection.1987, first GL-PP combined vaccine in the world, Type B hemophilus influenza (HiB) polysaccharide-tetanus poison Plain (TT) combined vaccine is approved by the FDA in the United States and enters market.Merck ＆ Co., Inc., Pfizer and Novartis Co., Ltd develop HiB in succession Polysaccharide-tetanus toxoid conjugate and epidemic cerebrospinal 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 to respectively on CRM197 protein carriers, and the one of mixed preparing Polyvalent vaccine is planted, to prevent infantile pneumonia, it is popular that 7 Pneumococcal serotypes cover North America and Europe more than 90% Streptococcus pneumoniae 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 cerebrospinal meningitis coccus groups, i.e. A, C, Y, W135, caused meningitiss.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 institutes.The vaccine has used three kinds of protein as protein carrier, wherein most main The carrier wanted is albumen-D (Protein D, PD), has 8 serotype polysaccharide to be as carrier with this albumen.Albumen-D is to use The gene recombination method of the popular haemophiluss of non-separable express without esterification surface albumen, body can be stimulated to produce Raw protection antibody, there is the acute otitis media 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 the combined vaccine of serotype 18C and 19F 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 polyvalent vaccine, 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, adds protein carrier on polysaccharide, and from T-independent antigen T cell is changed into 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 higher, and guard time is more permanent, reaches efficiently protection.2000, Pfizer was public First 7 valent pneumococcal conjugate vaccines listing of department；Pfizer Inc.'s exploitation infant it is more targeted 7 (4,6B, 9V, 14th, 18C, 19F and 23F) valency pneumoprotein vaccine, it is also effective to less than 5 years old child.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.13 valency combined vaccines of at present Pfizer's China's registration, increased 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 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, have selected albumen-D is carrier, and its reason is the consideration based on two aspects.First, being in order to avoid reusing As DPT vaccine component tetanus toxoid and diphtheria toxoid as carrier.Clinical trial 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 vaccine-IPV (PKV)-Hepatitis B virus vaccine (referred to as 6 vaccines), The immunogenicity of the most of serotype polysaccharide in multivalent pneumococcal combined vaccine can be suppressed, particularly to tetanus Toxoid affects to be especially apparent as the serotype polysaccharide immunogenic of carrier.Reason is made 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 competitive inhibition effect, reduce the immunogen of combined vaccine saccharide portion Property.The 11 valency pneumococal polysaccharide-TT and DT mixed carrier protein conjugate vaccines and the 7 valency pneumonia of Merck of SanofiPasteur Streptococcus polysaccharides-OMP combined vaccines (PCV-OMP), are all that clinical test results are good and example that cause product development failure, former Because just with this.Second, being to confer to protein carrier with real protective immunity originality function.Clinical trial proves albumen-D energy Enough stimulate body to produce protection antibody, there is the acute middle ear for potentially preventing non-separable popularity hemophilus infection to cause It is scorching.The 10 valency pneumococcal Polysaccharide Conjugate Vaccines of GlaxoSmithKline select albumen-D 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, the popular haemophiluss actual clinical meaning of non-separable is subject to that the bacterial infection rate is low limits System, acute otitis media sickness rate is relatively low caused by its infection.It is exactly that albumen is carried but these combined vaccines have a common ground shortcoming Body does not give immunogenic defencive function, that is to say, that although combined vaccine carrier can stimulate body to produce antibody, Be, the protectiveness that the designer of vaccine does not have using its antibody keeping off infection, meanwhile, do not determine its generation yet Antibody whether reach protectiveness titre levels.Nontoxic variant toxin of tetanus toxoid, diphtheria toxoid and diphtheria etc. is passed System albumen is selected as the main cause of carrier, is not because the antibody of their generation has protectiveness, but from it Safety considers with the immunogenicity that can strengthen polysaccharide in conjugate.It is clear that tetanus toxoid and diphtheria class poison Element has been two components of PertussisDiphtheriaTetanus triple vaccine, by traditional vaccination；So, the tetanus toxoid in combined vaccine and white Larynx toxoid carrier body whether can be stimulated to produce to reach protection antibody titre unimportant, it is contrary, in combined vaccine Saccharide portion antibody titer be only vaccine design person need concern subject matter.In addition, some knots just under development Close the carrier used by vaccine product, the recombinant Pseudomonas aeruginosa extracellular toxin of the deletion mutant detoxification as expressed by E.coli A (rEPA), recombinant cholera toxin of the deletion mutant detoxification expressed by E.coli etc. are also all based on identical and consider.

The species and type of new polysaccharide conjugate vaccine is increasing year by year, and alternative carrier protein species is less, no It is more carrier protein to be reused with vaccine.The different combined vaccines of inoculation same vehicle albumen may produce immunosuppressant effect Should, cause influencing each other for immune effect between different vaccines.And, main carriers albumen such as TT of polysaccharide conjugate etc., its , used as vaccine ring vaccination infant, original high titre specific antibody for carrier protein can in crowd's body for itself 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, using adipic dihydrazide (ADH) as bridging agent With reference to polysaccharide and albumen.This combination uses in the basic conditions Bromine cyanide. firstly the need of by polysaccharide Jing cyanogen bromide-activateds The hydroxyl on polysaccharide molecule is acted on, cyanate is formed, is then reacted with ADH；A C―O bond cleavage in cyanate, with There is additive reaction in the amino of ADH one end, so as to ester hydrazides (AH) group is imported into polysaccharide molecule, form polysaccharide-AH derivatives； Polysaccharide-AH derivatives form stable conjugate under the mediation of carbodiimide (EDAC) with carrier protein.Such combination side It is sterically hindered that formula can reduce that polysaccharide combined with carrier protein, the epitope of polysaccharide is remained, while avoiding polysaccharide itself Dissolubility, reduce the side effect of polysaccharide and antiserum reaction.

However, above-mentioned traditional polysaccharide-protein combination technology has following weak point：(1) polysaccharide-AH derivatives meeting Continue to be reacted 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 only 6 carbon originals are leaned in centre 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 immunity to produce immune effect three times.These deficiencies Place limits the further development of polysaccharide conjugate vaccine.

4th, application and prospect of the Nano microsphere in biotechnology

Nano material refers to monocrystal of the crystallite dimension less than 100 nanometers or polycrystal, the small-size effect and table of uniqueness The effect such as face or interface so as to possess many excellent or brand-new performance, it is just being increasingly subject to the attention of people.For example receive Continuous infiltration and impact of the rice material on drug research field, the revolution for having caused the field depth of drug world one remote.Medicine is The mankind are used to resist and prophylactic important substance, and for a long time, the research and development of medicine provide many for clinic Treatment meanss, be that patient brings many benefits.But, existing medicine there are still many problems, and such as medicine cannot followed Be detained in loop systems and reach valid density, cannot reach specific therapeutic goal, cannot by blood brain barrier, cannot be at certain Be partially formed higher concentration and while do not produce toxic and side effects etc. (Wu Xinrong. the application of drug-carried nanometer and progress [J]. Chinese Hospitals materia medica magazine, 2001,21 (3):171-173.).Magnetic Nano microsphere pharmaceutical carrier be nanotechnology with The product that modern medicine and pharmacology is combined, because it has small-size effect, good targeting, biocompatibility, 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 The purification of white matter and enzyme, reclaim and enzyme immobilizatio, it is simple to operate, and improve the stability of enzyme.Using magnetic nanometer particles Carry out immunoassay, with specificity it is good, separate fast, favorable reproducibility the characteristics of.Interventional therapy is carried out using Magnetic Microspheres-Carrier, Thromboembolism is carried out in magnetic control Ink vessel transfusing, then there is magnetic control guiding, target position thromboembolism.

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

1st, immobilized enzyme

Biopolymer such as enzyme molecule etc. all has many functional groups, can pass through physical absorption, crosslinking, covalent coupling They are fixed on into the surface of magnetic particle etc. mode.Advantage with magnetic Nano microsphere immobilized enzyme is：It is easy to enzyme and bottom Thing and product are separated；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.After enzyme is fixed on this carrier, can Easily to use magnetic devices to separate and recover from the mixed liquor of reaction.Domestic researcher has also been made to explore to this respect, Ding little Bin etc. adopts dispersion copolymerization method, synthesizes Fe3O4/ (St-MPEO) (St- styrene, MPEO- poly(ethylene oxide) polymeric monomers) Microsphere, the microsphere has amphiphilic structure, all has good swelling behavior in most of polarity, apolar medium so that The immobilized compound of microsphere 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 the purification for protein and enzyme, returns The field such as receipts and enzyme immobilizatio, cell separation.

2nd, targeted drug

Medicament carrier microspheres with targeting are distributed in effective object with referring to medicine carrying microballoonss energy high selection, so as to strengthen Curative effect, reduction side effect.Initial target medicine carrier microsphere be according to clinical needs, by from it is various to body tissue or The different carrier of diseased region affinity makes medicine carrying microballoonss, or monoclonal antibody is combined with carrier, defeated to allow medicament to It is sent to the specific part that treatment is expected to reach.With requirement more and more higher of the people to treatment, targeting positioning is also because by substrate 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), determined contained drug 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 experiment of first case applied magnetic drug targeting treatment in the world.14 advanced solid tumor are being suffered from In the magnetic target therapy of person, it is found that patient is fine to the toleration of magnetic targeted drug.Lexion etc. also make by applied magnetic microsphere For scale cancer (Lexion C, Amold W, Klein RJ, the et al.Locotegional cancer that pharmaceutical carrier treats rabbit Treatment with magnetic drug targeting [J] .Cancer Res, 2000,60 (23):6641-6648), Domestic Tao Kaixiong etc. with adriamycin magnetic albumin microspheres treatment Mus implanted gastric tumor (Tao Kaixiong, Sun Hongwu, Chen Daoda, etc. Targeting Treatment with Adriamycin Magnetic Albumin Microspheres in Human Mus implanted gastric tumor [J]. Chinese experimental surgery magazine, 2000,17 (1):63- 64)), Guo Jun etc. treats oral cavity top collar portion cavernous hemangioma 25 with Bleomycin A5 magnetic microsphere.Additionally, high-intensity magnetic field also has Cancer suppressing action (Guo Jun, Li Cheng, Wu Hanjiang. Bleomycin A5 magnetic microsphere targeted therapy oromaxillo-facial region cavernous hemangioma 25 Clinical report [J]. Nanjing Railway College of Medicine's journal, 2000,19 (2):112-114)).The glucose magnetic microsphere such as Xu Huixian Immobilization L- Radix Asparagi phthalein amine enzyme also achieves good therapeutic effect treating acute lymphatic leukaemia.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 is combined Idiosyncratic carrier, make people be expected to be utilized to follow the trail of and eliminate the cancerous cell for shifting, so as to become in human body eliminate " biological missile " of cancerous cell.But, being located at body surface more the tumor of current applied magnetic Drug therapy or in vitro table is nearer, therefore The intensity of externally-applied magnetic field can be weaker, and easily controllable, if the tumor for the treatment of deep organ or tissue, needs 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 Clad for magnetic particle surface can substantially reduce its magnetic property, in addition, the controllable of granular size is also have to be solved one Individual problem.

3rd, cell separation and immunoassay

If magnetic particle surface is drawn connects the specific antibodies with biological activity, 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 separating to cell, antigenic substance specific to organelle surface in particular by IMBS, with letter Just the features such as quick, separation purity is high, reservation target substance is active.The IMBS such as Mccole is separated by growing up that Liver fluke infected T lymphocytes in cattle peripheral blood, the cell for separating is pure, works 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 feces is noiseless to analyzing, and compares with agglutination with immunofluorescence, and sensitivity improves 103 times.Glenn is with IMBS point Inclusion virus is closed from breathing, with reference to enzyme immunoassay, the diffusion and non-specific suction in conventional tube and micropore analysis is reduced Attached, the formation of complex only needs 7min, and conventional microporous rule needs 120min.The isolation technics of cell can be additionally used in controlling for cancer Treat.The superfine physical absorptions of Kang Ji combine the covalently bound method of chemical bond, anti-human wing moon bright carcinoma monoclonal antibody are connected to pre- The surface of the Magnetic Polystyrene Microsphere carrier for first preparing, constructing specifically can be combined with target cell and be given 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 animal bone marrow The preliminary experiment for separating cancerous cell shows that IMM can effectively remove cancerous cell, and medullary cell only has minimal amount of loss.Immunity It is a kind of important method to analyze in modern biotechnology analytical technology, 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. adopts agalactosis polymerization, in the aqueous systems of alcohol one, is with potassium persulfate Initiator, forms initiation point, with acrylic acid (AA) as stabilizer, by styrene (ST) in Fe3O4 magnetic fluids particle surface With propylene phthalein amine copolymer, prepare monodispersed amido magnetic microsphere, the microsphere can directly, quickly with antibody (antigen) albumen Matter is crosslinked, it is to avoid other functional group microspheres need to adopt protein for shortcoming (Jing Xiaoyan, the king of cross-linking agent when combining immunoreagent Monarch, Li Rumin, etc. the preparation research [J] of magnetic function polymer microsphere. applicating technology, 2000,27 (1):16-17)).

4th, magnetic control inspection plug

During common people Jie treatment, it may occur that the phenomenon such as dystopy thromboembolism and infarction, 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, and in magnetic control Ink vessel transfusing bolt is carried out Plug then has the advantages that magnetic control guiding, target position thromboembolism, and to solve the above difficult problem approach is provided.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) etc. aspect do Careful research.Minalnimura etc. is used for the research of Mus liver cancer model with the therapy that thermotherapy and arterial thrombosiss combine.He Have developed DM-MS ductus arteriosuss local be administered, the magnetic field of additional 500kHz.After treatment 3d, tumor rate of increase (thromboembolism-thermotherapy Group, simple embolization group and matched 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).This research shows that the DM-MS thermotherapies and thromboembolism therapy that combines is a kind of antitumor Feasible Sex therapy, with wide research and application prospect.Goodwin etc. is to amycin magnetic microsphere hepatic artery embolism and medicine Targeting is studied (Goodwin SC, Bittner CA, Peterson CL, et 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 Hepar Sus domestica cancer model result that they set up shows the nontoxic secondary work of amycin magnetic microsphere low dosage With.Only when the content of magnetic microsphere>Just there is a preferable effect=75mg (with or without amycin) target area, hepatoma carcinoma cell it is bad Dead degree is directly proportional to thromboembolism degree, and amycin can not freely be circulated 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, the PMMA of 30-50um Magnetic microsphere has that magnetic response ability is strong, magnetic control effect of embolization is good, remains to realize that target position thromboembolism etc. is excellent in the case of high Hemodynamic environment Point, be a kind of preferable magnetic control Endovascular Embolization material (Hui Xuhui, Gao Lida, He Nengqian. polymethyl methacrylate magnetic is micro- Ball Endovascular Embolization experimentation [J]. Sichuan medical science, 2001,22 (10):928-929)).In magnetic control thromboembolism, magnetic microsphere The size of carrier is to affect the most important factor of Target localization.If particle diameter is less, 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 thromboembolism of Large Diameter Pipeline.Therefore, with magnetic microsphere application in other respects Difference, in magnetic control thromboembolism people Jie treatment, the general magnetic microsphere larger using particle diameter.

Also report points out, Nano microsphere as the carrier protein of DNA vaccination and adjuvant, but can be applied to preventative polysaccharide And/or protide vaccine has no report.

The content of the invention

For the problems referred to above that prior art is present, it is an object of the invention to provide a kind of higher pneumonia ball of immunogenicity Bacterium conjugate vaccines.

For achieving the above object, the technical solution used in the present invention is as follows：

A kind of streptococcus pneumoniae conjugate vaccines, it is multivalent pneumococcal polysaccharide and two or more carrier protein Jing The streptococcus pneumoniae conjugate vaccines of connector, wherein, connector is magnetic Nano microsphere.

Used as a kind of preferred version, the magnetic particle of the magnetic Nano microsphere is core positioned at the inside of magnetic Nano microsphere, Macromolecular material is wrapped in the outside of magnetic particle.

Used as further preferred scheme, magnetic particle is Fe₃O₄。

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

Used as another kind of preferred version, macromolecular material is bioabsorbable polymer material, selected from shitosan, Polyethylene Glycol, is gathered One or more mixture in poly lactic coglycolic acid (PLGA), PLA-PEG copolymer (PELA)； Most preferably PLGA or PELA.

Used as another kind of preferred version, multivalent pneumococcal polysaccharide is various pneumococcal capsular polysaccharides, is preferably separated Capsular polysaccharide on purification 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.

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

Used as a kind of preferred preferred version, the carrier protein is selected from recombined human rotavirus protein, diphtheria toxoid, broken wound Wind toxoid, carrier protein CRM197, bloodthirsty hemophilus influenza surface protein HiD, pertussis Prn surface protein, pertussis Fha resist Former and/or Pneumococal surface protein A (rPspA).

Used as further preferred scheme, it is bloodthirsty hemophilus influenza surface protein HiD or pneumonia to have in the carrier protein a kind of Coccus surface protein A (rPspA).

Used 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 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, the rotavirus strain of P genotype P [8] 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, the rotavirus strain of P genotype P [4] 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, the rotavirus strain of P genotype P [6] selected from M37,1076, RV-3, ST3, One kind in SC2, BrB, McN13, US1205, MW023, US585 or AU19 strain.

As another kind of further preferably scheme, recombined human rotavirus protein 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.

Used as another kind of further preferably scheme, recombinant rotavirus albumen is the VP7 albumen of G serotype rotavirus Partial amino-acid series or complete sequence.

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

As still more preferably scheme, the G serotypes rotavirus strain 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 a further object of the present invention to provide the preparation method of the streptococcus pneumoniae conjugate vaccines, specifically by multivalence pneumonia ball Carrier protein of the granulose with two kinds or more is coupled with magnetic Nano microsphere forms respectively.

As a kind of preferred version, the preparation method of the streptococcus pneumoniae conjugate vaccines, following steps are specifically included：

A) capsular polysaccharide being individually separated on purification various serotype pneumococcal capsule；

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

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

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

E) purification procedures d) gained couplet is into the streptococcus pneumoniae conjugate vaccines stock solution.

As further preferred scheme, the preparation method of the streptococcus pneumoniae conjugate vaccines, following steps are specifically included：

B) it is individually separated the selected variety carrier albumen of purification；

C) respectively various capsular polysaccharides and magnetic Nano microsphere be coupled into into polysaccharide-magnetic Nano microsphere couplet, then Jing Chemical modification, so as to the polysaccharide-magnetic Nano microsphere couplet surface is not modified as into-CHO with the-OH of polysaccharide reaction；

Used as further preferred scheme, the preparation method also includes preparing magnetic Nano microsphere, including first preparing nano magnetic Property particle prepares again the process of magnetic Nano microsphere；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 again magnetic nanoparticle is combined into solvent extraction with macromolecular material Jing fast film emulsifyings Method and/or investment or monomer polymerization method prepare magnetic Nano microsphere；Most preferably nano magnetic is prepared using chemical coprecipitation Property particle again with macromolecular material Jing fast film emulsifyings magnetic nanoparticle is combined into solvent extraction and/or investment prepares grain The homogeneous magnetic Nano microsphere in footpath；Wherein magnetic particle is preferably Fe₃O₄。

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

Used 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.

Used 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.

Used as still more preferably scheme, the concrete operations of step c are：In coupling medium reaction buffer, under room temperature, Under pH4.0-9.0, step a gained polysaccharide is obtained into polysaccharide-magnetic Nano microsphere idol with magnetic Nano microsphere coreaction 6-24 hours 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 for rushing liquid is 6；Optimum reacting time is 12-16 hours.

Used as still more preferably scheme, the concrete operations of step d are：In coupling medium reaction buffer, 4 DEG C, Under pH4.0-9.0, by the chemically modified polysaccharide-magnetic Nano microsphere couplet of step c gained 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.

Used as still more preferably scheme, isolating and purifying for step e is will to be coupled conjugate and unreacted polysaccharide and load Body protein is isolated and purified, and purification process can be with chromatography or ultrafiltration；Can be according to the molecule of the streptococcus pneumoniae conjugate vaccines for obtaining Size is isolated and purified, chromatography preferably using Superdex200 solvent resistant columns, Sepharose CL-4B or Sepharose CL-6B are carried out；And ultrafiltration is come separating and combining thing and unreacted reactant using different retention molecular weight film.

The streptococcus pneumoniae conjugate vaccines preparation can be using water preparation or lyophilized preparation.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 or other can stablize the buffer of polysaccharide or conjugate.The pneumonia ball of the present invention The preparation method of each preparation of bacterium conjugate vaccines is prepared using the conventional meanses of the art.Wherein, preferably in sugared (such as sucrose Or Lactose) in the presence of carry out lyophilizing.

The streptococcus pneumoniae conjugate vaccines that the present invention is provided can carry out immunity with any existing approach, including skin corium or The forms such as percutaneous drug delivery, intramuscular delivery.Wherein, the amount for being given is that those skilled in the art are confirmable according to general knowledge.

Term is defined

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

VP8 polypeptide chain fragments：For the polypeptide chain that any molecular weight is less than total length VP8.

VP4 polypeptide chain fragments：For the polypeptide chain that any molecular weight is less than total length VP4.

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

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

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

NSP4 albumen：It is non-structural protein, with enterotoxin characteristic, molecular weight is 28kDa, containing 175 aminoacid.

VP8-NSP4 fusion protein：With the method for gene recombinaton, 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：By physics (such as ultrasound wave, particle spray), chemistry (such as acid, alkali, enzymic digestion) method will By the polysaccharide (claim full polysaccharide or original polysaccharide) degraded (depolymerization) of purification in inoculum obtained it is many Bglii fragment, molecular weight is usually less than original polysaccharide.Pod membrane oligosaccharide：By physics (such as ultrasound wave, particle spray), chemistry (such as Acid, alkali, enzymic digestion) etc. method will be by the polysaccharide of purification in inoculum (claim full polysaccharide or original polysaccharide) degraded (depolymerization) polysaccharide fragment for being obtained, the monosaccharide residue in molecular structure is usually less than 10.But monosaccharide is residual The definition of radix amount is variant, and the polysaccharide chain more than 10 less than 20 monosaccharide residues is also become oligosaccharide by some documents.

Compared with prior art the present invention has the advantage that：

1. the streptococcus pneumoniae conjugate vaccines are the immunoconjugates containing two or more different carriers albumen, and existing Pneumococcal conjugated vaccine compare, its immunogenicity is higher, and the polysaccharide antibody level of induction, can be with higher than single carrier conjugates Cause immunne response in broader crowd, especially infant；Carrier epi-position 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 in two kinds of carrier proteins also can two kinds of albumen mixing notes of induction ratio Higher immunoreation when penetrating, mutual synergism further enhances the immunogenicity of carrier protein, increased body to many The immunoreation of sugar；Another kind is adjuvant effect carrier protein, and immunogenicity can be increased further, and with certain immunity Memory；

3. the pioneering employing Nano microsphere 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 epi-position, is conducive to improving the immunogenicity of capsular polysaccharide；

4.-the OH by Nano microsphere surface for initiating in the preparation process of the streptococcus pneumoniae conjugate vaccines is first carried out with polysaccharide Coupling reaction, then unreacted-OH is modified as into-NHs of-the CHO in carrier protein by couplet is chemically modified₂Carry out idol It is coupled and closes, associated methods more of the prior art is more stable；

5. its preparation method is simple, is adapted to the needs of scale industrial production, does not significantly change capsular polysaccharide and carrier egg White architectural feature.

To sum up state, the streptococcus pneumoniae conjugate vaccines preparation process is simple that the present invention is provided adopts Nano microsphere for junctional complex Streptococcus pneumoniae conjugate vaccines can strengthen mice Th1 type immunne response, and immune lasting effect, the spy of polysaccharide specificity antibody The opposite sex and affinity, additionally can induce mice and produce rotavirus antibody；Possesses the preventive effect of two kinds of vaccines；Therefore have Very wide application prospect.

Description of the drawings

Fig. 1 is the streptococcus pneumoniae conjugate vaccines obtained by the embodiment of the present invention 1¹H-NMR spectrum；

The immunne response experimental result of the polysaccharide specificity antibody of the streptococcus pneumoniae conjugate vaccines that Fig. 2 is provided for the present invention is shown It is intended to；

The immune lasting effect experimental result of the polysaccharide specificity antibody of the streptococcus pneumoniae conjugate vaccines that Fig. 3 is provided for the present invention Schematic diagram.

Specific embodiment

The present invention is made with reference to embodiment further illustrate in detail, intactly.Reagent used below or equipment are Commercially available kind, if no special instructions, operates to specifications, will not be described here.

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

Embodiment 1

First, magnetic Nano microsphere is prepared

1. 2.24gFeSO is taken₄-7H₂O and 3.24gFeCl₃-6H₂O is dissolved in respectively the dddH that 10mL and 15mL filters deoxygenation₂O In, mix after dissolving and hook, add 100mL to filter the dddH of 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, continuous stirring 30min；

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

4. reaction is finished and is settled to 100mL, basis of microscopic observation magnetic particle synthesis situation；

5. 400mgPLGA is dissolved in 10mLEA solvents as oil phase (O), adds the above-mentioned magnetic particle solution conducts of 3mL Interior water phase (W₁), just emulsifying is carried out in ice-water bath using ultrasonic cell disintegration instrument (120W, 60s) and prepares colostrum, then will just Breast pours a certain amount of aqueous solution (outer water phase, W containing 15g/LPVA and 0.9% (ω) NaCl into₂), magnetic agitation (300r/min, 2min) prepare pre- double emulsion (W₁/0/W₂), then pre- emulsion is poured in the storage tank of fast film emulsifying, with certain N₂Pressure by its SPG films are pressed through repeatedly, obtain the Nano microsphere emulsion drop of uniform particle diameter.Additionally, unspent Nano microsphere can be made into lyophilized preparation Continue to employ.

Or by taking Fe₃O₄Magnetic particle is added with dehydrated alcohol with 50mL by equal-volume, after ultrasonic activation 30min, puts 60 In DEG C water-bath, slow Deca 10mLPELA carries out-NH to magnetic Nano microsphere₂It is terminal-modified and PELA is wrapped in into Fe₃O₄Magnetic Outside property particle, under nitrogen protection stirring reaction l0h, makes magnetic Nano microsphere；After completion of the reaction, washed with 50mL dehydrated alcohol Paint 3 times, then washed after paint three times with 0.01MPBS, is settled to 50mL, and basis of 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) streptococcus pneumoniae culture；

2. the strong capsular polysaccharide of antigenicity in any of the above Pneumococcal serotype is purified respectively：Streptococcus pneumoniae, after inactivation It is collected by centrifugation supernatant, Jing is concentrated by ultrafiltration, is separately added in right amount that (volume fraction is 70% according to each Pneumococcus serotypes characteristic ) pre-cooled ethanol, it is collected by centrifugation, obtain 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 repeatedly phenol is carried 5-6 time, collects supernatant, is dialysed with distilled water, and liquid adds 2mol/L chlorine after dialysis Change calcium solution, add ethanol stirring, centrifugation to remove nucleic acid, collect supernatant, add ethanol (final concentration 80% is stirred), be collected by centrifugation Precipitation, is precipitated with ethanol, washing with acetone, and the refined capsular polysaccharide of multivalence is after dehydrate, is put -20 DEG C and is saved backup.

3rd, the preparation of rotavirus carrier protein

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

1st, the cDNA storehouses of rotavirus are set up

From the VP4 genes for Wa strain VP8 albumen, amplimer design it 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 in BL21 (DE3) competent cells, inoculating cell to 50 μ G/mL kanamycin LB culture dishs, in CO at 37 DEG C₂In incubator overnight.

2) bacterium colony is picked out, 10 milliliters of kanamycin LB culture fluid (1% Trypsins 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) culture fluid is turned to be inoculated into 100 milliliters of 50 μ g/mL kanamycin LB culture fluid, continues to cultivate.Treat absorbance When the OD of 600nm reaches 1.0, culture fluid turn is inoculated into into 6 and is raised in 50 μ g/mL kanamycin LB culture fluid, continued at 37 DEG C, Culture in the shaking table of fast 200rpm is shaken, when the OD of absorbance 600nm reaches 0.6~0.8, the IPTG (isopropyls of 0.3mM is added Base-β-D- thiogalactosides) induction VP8 expression.

4) under identical condition of culture, induction 4 hours after, with 4000g under, 10 DEG C centrifugation 20 minutes after, collect bacterium Body.

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

3rd, the purification VP8 albumen from inclusion body

1) 0.5 gram of the VP8 inclusion bodys (weight in wet base) of preparation are weighed, (10mMTris, 100mM phosphate delays with cleaning buffer solution Rush liquid, 2Murea, pH8.0) suspension inclusion body, it is incubated 30 minutes at room temperature, it is centrifuged 10 minutes with 10,000g, collection is forgiven Body.Repeat above step and remove the foreign protein for depolluting three times.

2) by inclusion body precipitation be dissolved in dissolving buffer (10mMTris-HCl, 100mM phosphate buffer, 8M carbamide, PH8.0 in), in stirring incubation on ice 1 hour.It is centrifuged 30 minutes with 16,000g, collects supernatant, abandons insoluble precipitate.

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

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

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

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

1. the structure of the optimization of genes of interest and recombinant expression plasmid

PspA gene order (GI are obtained in GenBank：193804931) and it is optimized, adds and carried out after His labels Full genome synthesizes, by the sequence of synthesis Jing 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 overnight, 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 life will be sequenced Entitled pET-30a-rPspA.

2. the abduction delivering and purification of recombiant protein

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

5th, the preparation of streptococcus pneumoniae conjugate vaccines

1. polysaccharide-magnetic Nano microsphere is coupled

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

2. it is coupled modifies

Step 1 polysaccharide-magnetic Nano microsphere couplet 30mL is taken, 2mL25% 1,3-propanedicarboxylic acids is slowly added dropwise under stirring condition, 200r/min stirring reactions 6 hours；After completion of the reaction, after washing three times with 0.01MPBS, 30mL, basis of microscopic observation are settled to Magnetic Nano microsphere is modified situation so that and polysaccharide-magnetic Nano microsphere couplet surface is not modified as with-the OH of polysaccharide reaction- CHO；N₂, 4 DEG C save backup.

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

Under 0.1MTBS solution buffer, 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 adopted for 24 hours Amount nano-carrier albumen addition, such as mass ratio adopts polysaccharide：Magnetic Nano microsphere：PspA：Rotavirus protein=1：1：2： 2)。

4. streptococcus pneumoniae conjugate vaccines are isolated and purified

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

The composition analysis of obtained streptococcus pneumoniae conjugate vaccines

With¹H-NMR carries out detecting the streptococcus pneumoniae conjugate vaccines that 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(.Occur in that corresponding to succinyl at 6.2ppm The characteristic peak of imines, this shows to contain butanimide in the cross structure of polysaccharide conjugate vaccine.Additionally, 5.2,1.6,3.6 outlets The characteristic peak of PELA proves magnetic Nano microsphere PELA as junctional complex.Therefore, capsular polysaccharide is with reference to two kinds of carrier proteins In front and back, there is no substantially change in 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 being calculated conjugate is combined than (Ratio)；Rotavirus protein, PspA protein concentrations pass through euzymelinked immunosorbent assay (ELISA) Detection.As a result show：In the streptococcus pneumoniae conjugate vaccines stock solution, 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 used as connector in obtained vaccine, and And complex carries albumen and polysaccharide are conjugated by the method referred in prior art and are 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 streptococcus pneumoniae conjugate vaccines.

Embodiment 2

The present embodiment is tetanus with the carrier protein for differing only in the streptococcus pneumoniae conjugate vaccines of embodiment 1 Toxin vector albumen and PspA.

There is theoretical error scope with the acquired results of embodiment 1 in the composition analysis result of obtained streptococcus pneumoniae conjugate vaccines Interior concordance.

Embodiment 3

The present embodiment is differed only in embodiment 1：The carrier protein of the streptococcus pneumoniae conjugate vaccines 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 streptococcus pneumoniae conjugate vaccines is colyliform disease Poisonous carrier albumen and bloodthirsty hemophilus influenza surface protein HiD.

Embodiment 5

The present embodiment is differed only in embodiment 1：The carrier protein of the streptococcus pneumoniae conjugate vaccines 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

Hereinafter adopt assessment experimental evaluation embodiment 1~7 and the institute of comparative example 1~2 of the vaccines such as immunogenicity routine potency Obtain the immune effect of vaccine：

A. streptococcus pneumoniae conjugate vaccines immunogenicity experiments

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

Table 1

As shown in table 1, the streptococcus pneumoniae conjugate vaccines 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 the immunity of Th1 types with magnetic Nano microsphere and streptococcus pneumoniae conjugate vaccines obtained by pneumonia polysaccharide should Answer, and immune effect is substantially better than obtained by the single carrier protein without nano-magnetic microsphere connector and comparative example 2 of comparative example 1 Streptococcus pneumoniae conjugate vaccines；But will also realize that according to the immunne response result of the gained of embodiment 1~7, be complex carries by PspA or HiD The immunne response effect of one of albumen is preferable, and PELA and PLGA is good compared with PEG immunne response effect in magnetic nano-carrier.B. it is many The experiment of the immune lasting effect of sugar specificity antibody, specificity and affinity

Once tested with embodiment 1, comparative example 1 and 2 and positive control gained streptococcus pneumoniae conjugate vaccines respectively.

1st, immune lasting effect

The titre of polysaccharide specificity antibody in 20 weeks after by determining the immunity 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 is gradually lowered, and the 4th Zhou Houyi cannot be detected.The polysaccharide specificity IgG titres that comparative example 1 and 2 is produced are low In 1 group of embodiment, and it is higher than positive controls.Polysaccharide specificity IgG titres in the 2nd Zhou Dafeng, in 4-20 is all 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 respectively 20%, 15%, 12% and the 10% of its peak value.Therefore, the pneumonia that the present invention is provided Coccus conjugate vaccines can strengthen the immune lasting effect of polysaccharide specificity antibody.

2nd, specificity and affinity

Add in PS-TT groups, PS-PLGA-TT groups and PS-PELA-TT group mice plasma to 200 times of dilutions different amounts of Capsular polysaccharide, with ELISA method the antibody horizontal of anti-capsular polysaccharide in mice plasma is detected, the results are shown in Table 2.

Table 2

As shown in table 2, with the increase of polysaccharide addition, in the orifice plate of polysaccharide specificity antibodies 96 ability of polysaccharide by Gradually reduce.When the polysaccharide for adding reaches 20 μ g, the Disability of antibodies polysaccharide.This shows pneumonia provided by the present invention The anti-capsular polysaccharide antibody that coccus conjugate vaccines 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 the antibody of 1 group of positive controls, 1 group of comparative example, 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 of present invention offer Coccus conjugate vaccines can significantly improve the affinity of polysaccharide specificity antibody.

The immunogenicity comparative experimentss of monotype pneumococal polysaccharide in C, streptococcus pneumoniae conjugate vaccines

Totally 13 class envelope antigen types are (i.e. to prepare each monotype streptococcus pneumoniae conjugate vaccines using the method described in embodiment 1 Conjugate vaccines are combined into only with a kind of pneumococal polysaccharide Jing magnetic nanoparticle and two kinds of carrier proteins), by indirect ELISA method distinguishes detection antibody titre, and the carbonate that various pneumonia polysaccharide conjugate vaccine is dissolved in 0.05mol/L pH9.6 is delayed In rushing solution, with 20ug/ml concentration coated elisa plates, 2% BSA fluid-tights are closed.During experiment by test serum 100,200,400, 800th, ELISA Plate is added after 1600,3200,6400,12800,25600,51200,102400,204800,409600 times of dilutions, 37 DEG C of reaction 40min are put, the sheep anti mouse two that horseradish peroxidase-labeled is added after conventional wash resists.Add simultaneously and do not contain serum Buffer as negative control.Developed the color with tetra-amino-biphenyl amine, A values are read at microplate reader 450nm wavelength.A in calculating per hole Value is 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 mice carries out geometric average, and calculates logarithm value with 10 the bottom of as, and concrete outcome is shown in Table 3.

The each monotype streptococcus pneumoniae conjugate vaccines Evaluation of Immunogenicity of table 3

As shown in Table 3：The pneumonia obtained by complex carries albumen with the magnetic nanoparticle of present invention offer as connector is more Sugared combined vaccine significantly increases can immunoreation of the mice to lung chain polysaccharide, wherein 1,3,5 and 6A/B types it is especially notable, tool There is more preferably immunne 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 mixing, for neutralization test Sample serum.

The neutralization test of the anti-WaVP8 antibody that injection white mice produces uses BSC-1 cells on microplate (microplate) Carry out.After heat inactivated serum is serially diluted, after mixing with the Wa rotavirus strains of 100TCID50, in 4 DEG C of cultures 1 hour.Then BSC-1 cells are inoculated on microplate, are hatched 1 hour.Plus DMEM (do not contain serum) is added to each hole, 37 DEG C Hatching 1 hour.Finally, dilute antiserum and be prevented from the concentration of rotavirus cytopathic effect (CPE) generation to neutralize drop Degree.Polysaccharide matched 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 streptococcus pneumoniae conjugate vaccines injection white mice antibody

As shown in table 3, comparative example 1 and comparative example 2 are substantially better than with rotavirus effect in streptococcus pneumoniae conjugate vaccines, can Significantly induction rotavirus antibody is produced.

Finally be necessary described herein be：Above example is served only for making further in detail technical scheme Ground explanation, it is impossible to be interpreted as limiting the scope of the invention, those skilled in the art's 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 streptococcus pneumoniae conjugate vaccines, it is characterised in that：The streptococcus pneumoniae conjugate vaccines are multivalent pneumococcal polysaccharide With the streptococcus pneumoniae conjugate vaccines of two or more carrier protein Jing connectors, wherein, connector is received for magnetic Meter Wei Qiu.

2. streptococcus pneumoniae conjugate vaccines according to claim 1, it is characterised in that：The magnetic of the magnetic Nano microsphere is micro- It is core that grain is located at the inside of magnetic Nano microsphere, and macromolecular material is wrapped in the outside of magnetic particle.

3. streptococcus pneumoniae conjugate vaccines according to claim 2, it is characterised in that：Macromolecular material is biopolymer material Material, selected from shitosan, Polyethylene Glycol, Poly(D,L-lactide-co-glycolide (PLGA), PLA-PEG copolymer (PELA) one or more the mixture in.

4. streptococcus pneumoniae conjugate vaccines according to claim 1, it is characterised in that：Multivalent pneumococcal polysaccharide is various 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. streptococcus pneumoniae conjugate vaccines 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. streptococcus pneumoniae conjugate vaccines 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 hemophilus influenza surface protein HiD, hundred Day coughs Prn surface proteins, pertussis Fha antigen and/or Pneumococal surface protein A (rPspA).

7. streptococcus pneumoniae conjugate vaccines according to claim 1, it is characterised in that：It is thermophilic to have in the carrier protein a kind of Blood hemophilus influenza surface protein HiD or Pneumococal surface protein A (rPspA).

8. the preparation method of the arbitrary streptococcus pneumoniae conjugate vaccines of claim 1~7, it is characterised in that：By multivalence pneumonia ball Carrier protein of the granulose with two kinds or more is coupled with magnetic Nano microsphere Jing forms respectively.

9. the preparation method of streptococcus pneumoniae conjugate vaccines 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 streptococcus pneumoniae conjugate vaccines according to claim 8, it is characterised in that specifically include following Step：

C) respectively various capsular polysaccharides and magnetic Nano microsphere be coupled into into polysaccharide-magnetic Nano microsphere couplet, then Jing chemical It is modified, so as to the polysaccharide-magnetic Nano microsphere couplet surface is not modified as into-CHO with the-OH of polysaccharide reaction；