CN107412765A

CN107412765A - People's Rotavirus Vaccine and preparation method thereof

Info

Publication number: CN107412765A
Application number: CN201710278659.7A
Authority: CN
Inventors: 李东; 鲍路伟; 史晋; 吴克
Original assignee: BRAVOVAX Co Ltd
Current assignee: Bravovax Co ltd; SHANGHAI BOWO BIOTECHNOLOGY CO Ltd
Priority date: 2014-07-25
Filing date: 2014-09-23
Publication date: 2017-12-01
Anticipated expiration: 2034-09-23
Also published as: CN104258387A; CN104258387B; CN107412765B

Abstract

The invention discloses people's Rotavirus Vaccine, and it is the conjugate vaccines that multivalent pneumococcal polysaccharide forms with two or more carrier protein through connector, wherein, connector is that one of magnetic Nano microsphere, carrier protein is rotavirus protein.The preparation method of people's Rotavirus Vaccine forms multivalent pneumococcal polysaccharide with two kinds or more of carrier protein (one is rotavirus carrier protein) with magnetic Nano microsphere through coupling respectively.People provided by the invention is simple with Rotavirus Vaccine preparation technology, nanoparticle is used to strengthen mouse Th1 type immune responses for people's Rotavirus Vaccine of attachment, and immune lasting effect, specificity and the compatibility of polysaccharide specificity antibody, it additionally can induce mouse and produce rotavirus antibody；Possesses the preventive effect of two kinds of vaccines；Therefore there is very wide application prospect.

Description

People's Rotavirus Vaccine and preparation method thereof

Technical field

The present invention relates to a kind of vaccine, specifically, it is to be related to people with Rotavirus Vaccine vaccine and preparation method thereof, belongs to Biological technical field.

Background technology

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

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

And vaccine is divided into therapeutic and preventative two kinds, disease is treated by therapeutic vaccine, and passes through preventative epidemic disease Seedling protects human body not encroached on by invasive organism.Come prevention disease it is the mankind in generation more than one by inoculating against property vaccine In the clinical practice of discipline, it was demonstrated that be effective means.By effort for many years, medical field has been developed a variety of Vaccine is to prevent, bacterium, virus and fungi etc., various diseases caused by infection, drastically increases the health of the mankind It is horizontal.The continuous development of biotechnology, promote the variation of vaccine kind.Today, to infectious disease caused by pre- anti-virus There are the vaccine that inactivation of viruses technological development comes out, such as Vaccinum Encephalitidis Epidemicae, polio vaccine, influenza vaccines；Use attenuated virus Technological development come out attenuated live vaccine, as Rotavirus Vaccine, oral polio virus vaccine, measles virus vaccines, Mumps virus vaccine, rubella virus vaccine and chicken pox vaccine etc..It is raw to prevent useful proteins and polysaccharide of bacterial infectious disease etc. The technological development of thing macromolecule purifying come out bacterium class vaccine, as tetanus toxoid, diphtheria toxoid, DT-Pa and Its subcellular components, epidemic meningitis Streptococcus polysaccharides and 23 valency pneumococal polysaccharides etc..With gene recombinant protein technological development Vaccine out, such as hepatitis B surface antigen (prevention hepatitis B), human nipple shape viruslike particle virus (prevention cervix Cancer) etc..The prevention meningitis and the bacterial vaccine of pneumonia developed with half chemical combination technology, such as popular haemophilus b Type polysaccharide-protein combined vaccine, 7 valencys or 10 valency pneumococcal polysaccharide-protein combined vaccines and 4 valency meningococcal polysacharides- Protein conjugate vaccines.As can be seen here, the development of medical biotechnology, it is the motive power that vaccine product continues to develop, by life Thing technology is updated, and can develop more new generation vaccine products and human health is chosen to deal with different infectious diseases War.

2nd, combined vaccine is summarized

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

3rd, the harm and its epidemiological study of pneumococcus

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

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

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

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

23 valency Pnu-Imune 23s of Chengdu Inst. of Biological Products of Chinese biological technology group production are to have chosen 23 kinds Most common pathogenic bacteria (1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F), distinguish the various polysaccharide on fermented and cultured and separating-purifying pneumococcal capsule, be mixed by equal proportion Vaccine.The polysaccharide of bacterium is a kind of thymus independent antigen, and the main distinction of this antigen and thymus dependent antigen is The former does not need the auxiliary of T lymphocytes to produce antibody.Clinical practice proves that the vaccine that capsular polysaccharide makes is clearly effective, And widely used in multiple countries.But there is problems with this kind of polysaccharide vaccine：(1) in brood or infants only Faint immune response can be produced, does not produce immune response even, immune response strengthens with the growth at age；(2) produce low The antibody of affinity；(3) of short duration immune response is only produced, does not possess immunological memory and Immune-enhancing effect effect during inoculation repeatedly Should；(4) immune tolerance is easily produced；(5) common adjuvant is not easy to play a part of Immune-enhancing effect, 23 valency polysaccharide to this antigen The protective rate that vaccine infects for intrusion type lung chain is 50-70%, 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 immunogene of the class of T cell self 2, without T The participation of cell, they be can not inducing immunological memory effect, stimulate body caused by 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 1980s The technology of bacterial vaccine.John Robbins are by by popular influenzae type (Haemophilusinfluenzaetype B, Hib) capsular polysaccharide (Polyribosylribitolphosphate, PRP), it is connected to protein carrier with the form of covalent bond (tetanus toxoid) has synthesized popular influenzae type polysaccharide PRP- tetanus toxoid conjugates (PRP-TT), After immune animal, the protection antibody of sterilization can be produced, so as to start bacterial vaccine development technique of new generation.Especially have Meaning, for 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 stimulate body to be produced adult and long-acting be directed to the polysaccharide The bacterium specificity protection IgG antibody in source.Therefore, polysaccharide is a kind of haptens for the infant less than 2 years old, it is impossible to as Vaccine is inoculated with the children less than 2 years old.When bacterial polysaccharides is connected into protein carrier in the form of covalent bond, such as tetanus poison Plain (TetanusToxoid, TT), because protein is a kind of T cell dependence antigen, the polysaccharide of covalent key connection can be turned Become T cell dependence antigen, the specific IgG antibodies of the polysaccharide are directed to so as to stimulate body to produce, protect body not Infected by bacterium.The success of popular influenzae type polysaccharide-tetanus toxoid (PRP-TT) combined vaccine exploitation, is opened The technology platform of an exploitation bacterial vaccine is created, i.e., by bacterial polysaccharides, such as capsular polysaccharide, O- specific polysaccharides (O- Specificpolysaccharide) or oligosaccharide (Oligosaccharide), to be covalently bonded on protein carrier Manufactured combined vaccine.Success based on this concept, medical biotechnology research circle open by using same chemical synthesis process It has issued the combined vaccine of different bacterium；The combined vaccine of same bacterium is equally also developed with different synthetic technologys.

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

By effort for many years, the wide multivalence combined vaccine of a variety of coverage rates is have developed with combination technology.Bacterial capsule Polysaccharide-protein combined vaccine appears in earliest in the 1930s, 3 type pneumococal polysaccharides are connected to by Goebel and Avery On horse serum globulin, caused conjugate can produce the single-minded antibody of polysaccharide in animals, while provide corresponding immune Protection.1987, first GL-PP combined vaccine, Type B haemophilus influenzae (HiB) polysaccharide-tetanus were malicious in the world Plain (TT) combined vaccine is approved by the FDA in the United States into market.Merck ＆ Co., Inc., Pfizer and Novartis Co., Ltd develop HiB in succession Polysaccharide-tetanus toxoid conjugate and epidemic meningitis polysaccharide-tetanus toxoid conjugate, and successfully list. 2000, U.S. Hui Shi (Wyeth) company successfully developed and has listed 7 valency pneumococal polysaccharide-CRM197 combined vaccines, be by 7 different Pneumococcus serotypes polysaccharide are covalently bonded on CRM197 protein carriers respectively, mixed preparing form one Kind polyvaccine, to prevent infantile pneumonia, it is popular that 7 Pneumococcal serotypes cover North America and Europe more than 90% Pneumococcus different serotypes bacterial strain.2006, Sanofi Pasteur have developed 4 valency meningococcal polysacharides-broken wound Wind toxoid combined vaccine, for preventing 4 kinds of epidemic meningitis coccus groups, i.e. A, C, Y, W135, caused meningitis.2009 Year, GlaxoSmithKline (GSK) have also been developed a kind of 10 valency pneumococcal polysaccharide-protein combined vaccines, to prevent Pneumonia caused by 10 kinds of Pneumococcus serotypes.The vaccine has used three kinds of protein as protein carrier, wherein most main The carrier wanted is albumen-D (Protein D, PD), and it is to be used as carrier by the use of this albumen to have 8 serotype polysaccharide.Albumen-D is to use The gene recombination method of the popular haemophilus of non-separable express without esterification surface albumen, body can be stimulated to produce Raw protection antibody, there is the otitis media acuta caused by the popular hemophilus infection for potentially preventing non-separable, and other are also There are tetanus toxoid and diphtheria endotoxin to be used as carrier, be used separately as the egg of serotype 18C and 19F combined vaccine Bai Zaiti.From the design above in association with vaccine product, it can be seen that the exploitation of combined vaccine is transitioned into technically from univalent vaccine Increasingly complex polyvaccine, improve 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, its immunogenicity can be increased.Protein carrier has T cell dependency characteristic, and GL-PP conjugate vaccines can be thin by non-T The polysaccharide antigen of born of the same parents' pauper character is changed into the antigen of T cell pauper character, and the t helper cell generation of excitating organism is a series of Immune-enhancing effect.Capsular polysaccharide conjugate vaccine, protein carrier is added on polysaccharide, T cell is changed into from T-independent antigen Dependence antigen, increase its immunogenicity.Caused antibody is generation vaccine in quality and quantity after combined vaccine inoculation 400-1000 times, generation immunoprotection is wider stronger, and guard time is more permanent, reaches efficient protection.2000, Pfizer was public First 7 valent pneumococcal conjugate vaccines listing of department；Pfizer Inc. exploitation infant more targeted 7 (4,6B, 9V, 14th, 18C, 19F and 23F) valency pneumoprotein vaccine, it is also effective to less than 5 years old children.Capsular polysaccharide protein conjugate vaccines, Add protein carrier on polysaccharide, T cell dependence antigen is changed into from T-independent antigen, its immunogenicity can be increased, can For children more than 6 week old.At present Pfizer China registration 13 valency combined vaccines, add 6 serotypes (1,3, 5,7F, 6A, 19A).But the data in China shows that pneumococcal infection bacterial strain serotype is followed successively by：5、6、1、19、23、14、2、 4, the valent pneumococcal conjugate vaccine of Pfizer 7 only has 50% or so to China's common causative bacterial type coverage rate, and 13 valency combined vaccines Coverage rate also only have 70%.The valent pneumococcal conjugate vaccines of Hui Shi 7 need to be inoculated with 4 injections, expensive per 860 yuan of pin, no Beneficial to popularization.13 valency vaccines estimate that its price will be more expensive.Therefore the 7 and 13 valency combined vaccines of Pfizer Inc. are less suitable Close the large-scale children Streptococcus prevention in China.

Although 13 valent pneumococcal conjugate vaccines have 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 of different serotypes and the reuse of carrier protein of the same race so that carrier egg White dosage increases, and its immunogenicity reduces 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, it is in order to avoid reusing The tetanus toxoid and diphtheria toxoid as DPT vaccine component are as carrier.Clinical test shows, works as multivalence Pneumonia combined vaccine contains with the univalent vaccine of protein carrier same composition or multiple vaccines while when being inoculated with other, such as Hib-TT, whooping cough-Hib polysaccharide conjugate vaccines-IPV (inactivated polio vaccine)-hepatitis B vaccine (referred to as 6 vaccines), The immunogenicity of most of serotype polysaccharide in multivalent pneumococcal combined vaccine can be suppressed, particularly to lockjaw Toxoid influences 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, is inoculated with the combined vaccine containing whooping cough component at the same time When, such as 6 vaccines, so-called vector receptors Reverse transcriptase effect can be caused, reduces the immunogene of combined vaccine saccharide portion Property.SanofiPasteur 11 valency pneumococal polysaccharide-TT and DT mixed carrier protein conjugate vaccines and Merck 7 valency pneumonia Streptococcus polysaccharides-OMP combined vaccines (PCV-OMP), all it is that clinical test results are bad and cause the example of product development failure, it is former Because just with this.Second, protein carrier is to confer to real protective immunity originality function.Clinical test proves albumen-D energy Enough stimulate body to produce protection antibody, there is acute middle ear caused by potentially preventing non-separable popularity hemophilus infection It is scorching.GlaxoSmithKline 10 valency pneumococcal Polysaccharide Conjugate Vaccines selection albumen-D is used as carrier so that protein carrier produces Raw antibody has the protective effect of clinical meaning, is the much progress on combined vaccine technology development process.

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

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

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

However, there is following weak point for above-mentioned traditional polysaccharide-protein combination technology：(1) polysaccharide-AH derivatives meeting Continue to react with the polysaccharide of cyanogen bromide-activated, form the self-polymerization thing of polysaccharide, reduce the joint efficiency of polysaccharide-protein；(2) EDAC easily causes the self-crosslinking of polysaccharide and carrier protein while mediating ADH derivation polysaccharide to be combined with carrier protein, from And reduce the joint efficiency of polysaccharide-protein；(3) polysaccharide and carrier protein are macro-organism molecule, and 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 to be improved, and immune effect could be produced three times as polysaccharide conjugate vaccine needs to be immunized.These deficiencies Place limits the further development of polysaccharide conjugate vaccine.

4th, the harm and its epidemiological study of human rotavirus

Worldwide, rotavirus (Rotavirus) is the main pathogens for causing children's seriousness to be suffered from diarrhoea, and is being sent out Up to country, in the children being in hospital by acute diarrhea, the recall rate of rotavirus accounts for 35~52%.In the U.S., estimation has 300 every year Ten thousand children suffer from rotavirus diarrhea, cause 82000 people's hospitalizations, 150 people death.In developing country, rotavirus is also Cause 2 years old most common pathogen of Infants Below severe gastro-enteritis, the annual children for having more than less than 1.25 hundred million 5 years old of estimation suffer from Diarrhea Caused by Porcine rotavirus, wherein 1,008,000,000 children suffer from mild diarrhea, 870,000 is dead.In China, Child birth rat about 1,000 7000000, estimate that annual about 350,000 children cause death due to suffering from rotavirus diarrhea, arrange second place of the world.Due to described The increase of the incidence of disease and fatal rate that the infection of virus was suffered from diarrhoea to 2 years old Infants Below has remarkable effect, exploitation prevention colyliform disease Poison infection, effective and safe vaccine is the task of top priority.

Rotavirus Reoviridae (Reoviridae genus), is the disease for causing the mankind and numerous animal diarrheas Substance.Totivirus diameter 70nm, there are three special shell structures, the core shell structure of innermost layer is nucleocapsid protein, is enclosed with virus Gene.The gene of virus is made up of 11 segmented fragments of AMPLIGEN, is that 6 structural proteins and 5 non-structural proteins are compiled Code.Nucleocapsid protein is made up of tri- virus proteins of VP1, VP2 and VP3；Middle glutelin is VP6, coat protein be by VP4 and VP7 is formed.Because the gene of rotavirus is bifilar RNA, multi-disc segment structure, the gene of this structure can be carried out between gene Reconfiguring to a certain degree, i.e. rotavirus gene matches somebody with somebody (reassortment) again.When two or multiple viruses are same When infect a host cell after, in the packing stage of reovirion, each viral genetic fragment will in the cell again Combination, causes to match somebody with somebody gene again.This heavy ability with gene of rotavirus result in human body to the various of its immune response Property, also increase the difficulty for making specific Rotavirus Vaccine.

Rotavirus is divided into different types, hypotype and serotype according to the difference of virus antigenicity.7 are had now found that Serotype (A types~G types), most of human pathogens belong to A, B and c-type.Epidemiology survey finds to cause human and animal It is most common with A types in ill rotavirus, it is the main target of vaccine development.A types rotavirus is according to its VP6 antigenic characteristic Difference, be categorized further, as hypotype, one kind that most Strain belong in hypotype I or II.Different rotavirus tables Face-piece albumen VP7 and VP4 neutralization epitope cluster are different, can independently induce respective neutrality antibody, viral blood Clear type can be determined by the specificity of VP4 and VP7 antigens.A types rotavirus can be further categorized into according to VP7 and VP4 difference G serotypes and P serotypes.Because rotavirus gene is made up of 11 fragments, VP7 and VP4 encoding gene can be independent Separation combination, generate the gene resortment that a kind of binary mode is carried out.VP7 is a kind of glycoprotein (glycoprotein), because Its antigenic characteristic is divided into 15 kinds of different G serotypes, relative with its specific nucleotide sequence, i.e. genotype (genotype) Should；That is, every G serotypes have its special genotype, therefore, usual G serotypes and G genotype are generally applicable.Entirely In human disease's rotavirus strain that ball identifies, what it is more than 90% is G1, G2, G3, G4, and G9 strain, shares 10 serotypes Separated from human body and (be shown in Table 1).VP4 is a kind of two different virus proteins, i.e. VP8 can be cut into by trypsase It is P serotypes by its antigenic serotypes that are different and determining, some P serotypes can further divide with VP5 virus proteins For 2 sub- serotypes.The serum or monoclonal antibody of different VP4 Serotypes are distinguished due to lacking, hinders VP4 serum Type parting.RT-PCR application just makes it possible VP4 genotyping, and applies to the epidemiology survey of sample. Therefore, VP4 is substantially classified according to gene order, finds 14 P serotypes and at least 26 P genotype altogether at present (being marked in bracket).P serotypes and P genotype may not be corresponded to, it is necessary to mark, for example, Rotavirus Wa strain strain is marked simultaneously It is shown as P1A [8] G1 viruses.In the strain of human disease, G1, G3, G4 and G9 P and G serotypes combination are typically P1A [8], and G2 is typically P1B [4].As can be seen here, worldwide the rotavirus of prevalence enjoys the epitope cluster of identical cross-neutralization (epitopes) P1 serotypes, at least 7 VP4 serotypes are found in human rotavirus.On epidemiology intentionally The G serotypes 1 of mankind's strain of justice, 3, and 4 be the 1A Asias serotype for belonging to P serotypes, and G serotypes 2 are the 1B of P serotypes Sub- serotype.

Because natural rotavirus infection can induce immunoprotection, at least infection to severe rotavirus well, Therefore, the effort of most of vaccine developments is placed on attenuated live vaccine.Initial research, which concentrates on, uses animal rotavirus strain, Carried out by being referred to as Jennerian methods, reason is that the animal strain of nature attenuation in human body is safe, and is mainly produced Raw is mixed type immunoprotection.

After finding that rotavirus is the pathogen 10 years for causing children's seriousness to be suffered from diarrhoea, in nineteen eighty-three, start to use pig Rotavirus strain RIT4237 (G6P [1] type), manufactured first rotavirus attenuated live vaccine has carried out clinical test, in sweet smell The result of orchid experiment shows that the vaccine is safely effectively, to pass through mixed type human rotavirus The effect of (heterotypichuman rotaviruses), the protective rate of prevention severe rotavirus diarrhoea is up to 80%.But Disappointing in the clinical trial result that subsequent other countries are carried out, display is low or without protective effect, the experiment with End up in failure.In 1987, monkey rotavirus RRV strains were used to develop attenuated live vaccine, were the rotavirus of first exploitation Another in vaccine.Clinical test shows, although the vaccine can induce body to produce protection antibody, result is unstable It is fixed, trace it to its cause and be, the G serotypes of RRV strains are G3P [3], when the rotavirus of human infection is the G serotypes of homotype When, i.e. G3, the significant effect of vaccine；If during the G serotypes infection of different shaped, then it is ineffective.Then, RRV strains pass through VP7 genes in mankind's strain are incorporated into the strain by the method for gene resortment so that common in human rotavirus Other three kinds of G serotypes G1, G2 and G4 can also be expressed on RRV recombined strains, and which results in 4 valency Rotashield vaccines Succeed in developing.The vaccine obtained FDA approval list marketings in 1998, but due to being found that minority after large-scale inoculation, But the generation of the significantly raised entembole side effect case of quantity, in 1999 by production company under city.In 1988, open Begin use another rotavirus strain, i.e. WC3 pigs strain (G6P [5] type), carry out clinical test when, start result prove effectively, But significant protectiveness is displayed without in a subsequent experiment, the vaccine, which also stopped, to be continued to test.To nineteen ninety, in order to So that the antigenic structure of WC3 strains passes through gene resortment (gene reassortment) side closer to human rotavirus Method, it will be incorporated into for the gene of VP4 and VP7 encoding histones from human rotavirus on WC3 recombined strains, this method is referred to as For improved Jennerian methods.The strain and method are exactly developing RotaTeq 5 valencys by Merck (pentavalent) development approach of vaccine.2006, the WC3- gene resortment vaccines RotaTeq of 5 valencys is produced by Merck Go through to list, the vaccine contains the two human rotavirus's albumen substituents of VP7 and VP4 because of i.e. G1, G2, in G3, G4 Corresponding VP7 GFPs, and P [8] corresponding VP4.Clinical test shows that the vaccine does not have entembole side effect, It is 74% to the protective rate of the gastroenteritis caused by G1-G4 rotavirus, to the protective rate of severe gastro-enteritis up to 98%, to living The protective rate that institute and acute disease access is 94.5%.The same year, human rotavirus's attenuated live vaccine Rotarix of GSK productions are also obtained Approval must be listed, this vaccine is the mankind strain 89-12 based on attenuation, and serotype is G1P [8] strain, be in world wide most Common serotype.The virus is separated from the patient clinical samples for suffering from rotavirus gastroenteritis, and is being organized Cell obtains after repeatedly passing on variation attenuation.Clinical test shows, after two dosage are injected, to all rotavirus infections Protective rate to the protective rate of serious gastroenteritis up to 96%, is reached to the protective rate for needing gastroenteritis case in hospital up to more than 87% 100%.Further experiment shows, the scope protection of the vaccine not only includes the gastroenteritis as caused by G1P [8] strain, and Include the G3P relevant with VP4 [8], gastroenteritis caused by G4 [8] and G9P [8] strain.To G3, G4 and G9 rotavirus infections are protected The validity of shield is identical with G1's, has exceeded 95%, and the validity to G2 strains is 75%, and gastroenteritis is caused to all rotavirus Protective rate in hospital is 75%.

Statistics shows that G1 strains are worldwide most commonly detected strains, in Asia, North America and Europe, G1- G4 strains account for the 97.5% of total infection rotavirus.In South America, Africa, Australia account for 83.5%-90.4%, meanwhile, at this A little regional G5, G8 and G9 strains start to become important.From the point of view of P serotypes, P1A [8] strain is most common, ensuing to be P1B [4] strain.This result is predictable, is most commonly detected VP7 serum because VP7 serotypes G1 is P1A [8] Type；The important VP7 serotypes of other two epidemiology, G3 and G4, it may have identical VP4 serotypes.Another main blood Clear type G2 VP4 has the feature of P1B [4].Although G and P serotypes or genotype can have a variety of different combinations, 4 kinds of P- G combination, i.e. P [8] G1, P [4] G2, P [8] G3, and P [8] G4 constitute 88.5% common pathogenic strain.

As can be seen here, if configuring vaccine, it is necessary to comprising a variety of different G serotypes poison with the VP7 albumen of G serotypes Strain improves the coverage rate of vaccine, i.e. G1, G2, G3, G4, G9, G8 and G5, and the rotavirus attenuated live vaccine of 7 valencys is also current Develop the striving direction of vaccine of new generation.If developing Rotavirus Vaccine from another strategy, albumen is determined with P serotypes VP4 difference can then greatly reduce the strain kind included in vaccine and be covered to reach identical hair to prepare the vaccine of a new generation Rate.Analysis more than, if preparing vaccine with P [8] and P [4] two P serotypes strains, immune effect will be with 7 The G serotypes of valency are suitable.From the point of view of Rotarix the and Rotateq effects clinically used, almost it is not different, moreover, existing Statistics on see, Rotarix seems more more effective than Rotateq.From analyzing the strain serotype that is included of the two vaccines From the point of view of the composition for determining albumen, Rotarix is only containing a kind of this VP7 virus protein of G1；Although Rotateq contain G1, G2, G3, With G4 four kinds of VP7 virus proteins, immune effect does not strengthen；And the P serotypes contained from both determine albumen VP4 number From the point of view of amount, Rotarix contains P [8] one kind, and Rotateq contains P [8] (one of strain) and P [5] (the original strains of WC-3 For G6P [5]) two kinds；But the content of the important antigenic component P [8] in Rotarix will be above the content in Rotateq.Thus It can be seen that being assessed from the angle of P serotypes, the VP4 strain Rotavirus Vaccines containing P [8] have great meaning.If new one For P [8], the P [4] and [6] three kinds of VP4 antigenic components of P for including P serotypes in vaccine, you can cover global most area Popular strain.

5th, application and prospect of the nanoparticle in biotechnology

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

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

1st, immobilised enzymes

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

2nd, targeted drug

Medicament carrier microspheres with targeting refer to that drug bearing microsphere can highly selectively be distributed in effective object, so as to strengthen Curative effect, reduce side effect.Initial target medicine carrier microballoon be according to clinical needs, by from it is various to body tissue or The different carrier of diseased region affinity makes drug bearing microsphere, or monoclonal antibody is combined with carrier, defeated to allow medicament to It is sent to the privileged site that treatment it is expected to reach.With requirement more and more higher of the people to treatment, targeting positioning is also because by matrix Limit and be not entirely satisfactory, therefore there have been 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), is 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 trial of first case applied magnetic drug targeting treatment in the world.Suffer to 14 advanced solid tumors In the magnetic target therapy of person, it is found that patient is fine to the tolerance of magnetic targeted drug.Lexion etc. also make by applied magnetic microballoon Squamous carcinoma (Lexion C, Amold W, Klein RJ, the et al.Locotegional cancer of rabbit are treated for pharmaceutical carrier Treatment with magnetic drugtargeting [J] .CancerRes, 2000,60 (23)：6641-6648), state Interior Tao Kaixiong etc. with adriamycin magnetic albumin microspheres treat mouse implanted gastric tumor (Tao Kaixiong, Sun Hongwu, Chen Daoda, wait Ah Mycin Magnetic albumin microsphcres targeted therapy mouse implanted gastric tumor [J] China experimental surgery magazine, 2000,17 (1)：63- 64)), the bleomycin A5 magnetic microsphere treatment oral cavity top collar portion cvernous hemangioma 25 such as Guo Jun.In addition, high-intensity magnetic field also has Cancer suppressing action (Guo Jun, Li Cheng, Wu Hanjiang bleomycin A5 magnetic microsphere targeted therapy oromaxillo-facial regions cvernous hemangioma 25 Clinical report [J] Nanjing Railway College of Medicine journal, 2000,19 (2)：112-114)).The glucose magnetic microsphere such as Xu Huixian Immobilization L- asparagus ferns phthalein amine enzyme treats acute lymphatic leukaemia, also achieves good therapeutic effect.These results all show, Increase with the magnetic and medicated aggregation in tumor locus of raising of magnetic field intensity, acted on using the magnetic steering of externally-applied magnetic field, make medicine Pinpoint in target site, play concentration, efficient antitumor action.Targeting and surface just because of magnetic Nano microsphere combine Idiosyncratic carrier, make people be expected to be utilized to follow the trail of and eliminate the cancer cell that is shifting, so as to be eliminated as in human body " biological missile " of cancer cell.But the tumour of applied magnetic drug therapy is much nearer positioned at body surface or in vitro table at present, therefore The intensity of externally-applied magnetic field can be weaker, and easily controllable, if the tumour for the treatment of deep organ or tissue, 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 Its magnetic property can be substantially reduced for the clad on magnetic particle surface, in addition, the controllable of granular size is also to have to be solved one Individual problem.

3rd, cell separation and immunoassay

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

4th, magnetic control inspection plug

During common people Jie treats, it may occur that phenomena such as dystopy embolism and infarct, and cause serious complication, This is the thorny problem for being clinically badly in need of solving, and uses people Jie of Magnetic Microspheres-Carrier to treat, in the intravascular carry out bolt of magnetic control Plug then has the advantages that magnetic control guiding, target position embolism, and approach is provided to solve above problem.Scholars focus on magnetic spherolite Footpath, magnetic control time, magnetic field intensity, magnetic microsphere lapping (coarse, carrying positive charge, having hydrophobic property) etc. are done Careful research.The therapy that Minalnimura etc. is combined with thermotherapy and arterial embolism is used for the research of mouse liver cancer model.He Have developed DM-MS arterial ducts locally be administered, additional 500kHz magnetic field.After treating 3d, tumour growth rate (embolism-thermotherapy Group, simple embolization group and control group) be respectively 28%, 124% and 385% (Minalnimura T, Sato H, Kasaoka S, et a1.Tumor regression by inductive hyperthermia combined with hepatic embolization using dextran magnetite incorporated microspheres in rats[J].Int J Oncol, 2000,16 (6)：1153-1160).It is a kind of antitumor that this research display DM-MS thermotherapy and embolism, which are combined therapy, Feasible sex therapy, there is wide research and application prospect.Goodwin etc. is to adriamycin magnetic microsphere hepatic artery embolism and medicine Target and (Goodwin SC, Bittner CA, Peterson CL, et are studied to the toxicity of antitumor therapy al.Single-dose toxicity study ofhepatic intra-arterial infusion ofdoxorubicin Coupled to a novel magnetically targeted drug carrier [J] .Toxical, 2001,60 (1)： 117-183).The pork liver cancer model result that they establish shows that adriamycin magnetic microsphere low dosage has no toxic side effect.Only work as magnetic Just there are preferable effect, the degree of necrosis and bolt of liver cancer cells in content ＞=75mg (with or without adriamycin) target area of property microballoon Plug degree is directly proportional, and adriamycin can not freely circulate in whole body and successfully be controlled in target area.Hui Xuhui etc. is gathered with homemade Methyl methacrylate vinegar magnetic microsphere is inquired into Endovascular Embolization, and experiment shows, 30-50um PMMA magnetic microspheres tool Have the advantages that magnetic response ability is strong, magnetic control embolization effect is good, remain to realize target position embolism in the case of high Hemodynamic environment, be it is a kind of compared with Good magnetic control Endovascular Embolization material (Hui Xuhui, Gao Lida, polymethyl methacrylate magnetic microsphere vascular peg stays before what energy Fill in experimental study [J] Sichuan medical science, 2001,22 (10)：928-929)).In magnetic control embolism, the size of Magnetic Microspheres-Carrier It is to influence the most important factor of Target localization.If particle diameter is smaller, magnetic responsiveness is weak, and magnetic control degree is poor, it is impossible to be used in high blood Flow velocity or compared with the endovascular magnetic control embolism of Large Diameter Pipeline.Therefore, the application with magnetic microsphere in other respects is different, in magnetic control In embolism people Jie treatment, the general magnetic microsphere larger using particle diameter.

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

The content of the invention

In view of the above-mentioned problems existing in the prior art, it is an object of the invention to provide a kind of stronger people's wheel of immunogenicity Shape viral vaccine.

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

People's Rotavirus Vaccine, it is for multivalent pneumococcal polysaccharide with two or more carrier protein through being connected The conjugate vaccines that body forms, wherein, connector is that one of magnetic Nano microsphere, carrier protein is rotavirus protein.

As a kind of preferred scheme, the inside that the magnetic particle of the magnetic Nano microsphere is located at magnetic Nano microsphere is Core, high polymer material are wrapped in the outside of magnetic particle.

As further preferred scheme, magnetic particle Fe₃O₄。

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

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

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

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

As further preferred scheme, the rotavirus protein is restructuring human rotavirus albumen.

As a kind of preferred preferred scheme, the carrier protein is selected from diphtheria toxoid, tetanus toxoid, carrier protein CRM197, bloodthirsty Bacillus influenzae surface protein HiD, pertussis Pm surface proteins, pertussis Fha antigens and/or pneumococcus table Face albumin A (rPspA).

As further preferred scheme, it is bloodthirsty Bacillus influenzae surface protein HiD or lung to have one kind in the carrier protein Scorching coccus surface protein A (rPspA).

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

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

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

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

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

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

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

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

It is a further object of the present invention to provide the preparation method of people's Rotavirus Vaccine, specifically by multivalence pneumonia ball Granulose forms with two kinds or more of carrier protein through coupling.

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

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

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

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

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

E) couplet obtained by purification procedures d) is into the conjugate vaccines stoste.

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

B) the variety carrier albumen selected by difference separating-purifying；

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

As further preferred scheme, the preparation method also includes preparing magnetic Nano microsphere, including first prepares nanometer Magnetic particle prepares the process of magnetic Nano microsphere again；Wherein magnetic nanoparticle can by chemical coprecipitation, hydro-thermal method or Prepared by sol method pyrolysismethod, magnetic Nano microsphere can be prepared by investment, monomer polymerization method or in-situ method, preferably useization Learn coprecipitation and prepare magnetic nanoparticle and again emulsify magnetic nanoparticle and high polymer material through fast film and extract with reference to solvent Follow the example of and/or investment or monomer polymerization method prepare magnetic Nano microsphere；Nanometer is most preferably prepared using chemical coprecipitation Magnetic particle is again prepared magnetic nanoparticle and high polymer material through fast film emulsification with reference to solvent extraction and/or investment The magnetic Nano microsphere of uniform particle diameter；Wherein magnetic particle is preferably Fe₃O₄。

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

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

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

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

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

As still more preferably scheme, isolating and purifying for step e is by coupling conjugate and unreacted polysaccharide and load Body protein isolates and purifies, and purification process can use chromatography or ultrafiltration；It can be carried out according to the molecular size of the conjugate vaccines of acquisition Isolate and purify, chromatography is preferably using Superdex200 solvent resistant columns, Sepharose CL-4B or Sepharose CL-6B Carry out；And ultrafiltration is to separate conjugate and unreacted reactant using different retention molecular weight film.

The conjugate vaccines preparation can use aqua or freeze-dried.In order to strengthen its immunogenicity, adjuvant can be added, is commonly used Adjuvant have aluminium adjuvant, such as aluminium hydroxide, aluminum phosphate, prioritizing selection aluminum phosphate of the present invention.The solvent of conjugate can be 0.2 chlorination Sodium solution, 1 × PBS or other buffer solutions that can stablize polysaccharide or conjugate.The conjugate vaccines of the present invention are respectively made The preparation method of agent is prepared using the conventional meanses of the art.Wherein, preferably in the presence of sugared (such as sucrose or lactose) Freezed.

Conjugate vaccines provided by the invention can be immunized with any existing approach, including skin corium or skin are given The forms such as medicine, intramuscular delivery.Wherein, the amount given is that those skilled in the art are confirmable according to general knowledge.

Term defines

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

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

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

VP8 specific antigen cluster chains：Full VP8 polypeptide chains contain multiple antigenic determinants, are cut by the method for genetic recombination Cut and be free of important amino acid, and retain the polypeptide chain containing specific antigen cluster, molecular weight is typically smaller than total length VP8 polypeptides Chain.

VP4 specific antigen cluster chains：Full VP4 polypeptide chains contain multiple antigenic determinants, are cut by the method for genetic recombination Cut and be free of important amino acid, and retain the polypeptide chain containing specific antigen cluster, molecular weight is typically smaller than total length VP4 polypeptides Chain.

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

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

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

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

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

1. the conjugate vaccines are the immunoconjugates containing two or more different carriers albumen, with existing pneumonia Coccus combined vaccine is compared, and its immunogenicity is stronger, and the polysaccharide antibody of induction is horizontal to be higher than single carrier conjugates, can be wider Cause immune response, especially infant in wealthy crowd；By reducing each carrier dosage carrier epitope can be avoided to overload；Can Strengthen T-helper cell activity by two kinds of carriers；

2. due to having the Protein Epitopes of protectiveness also can two kinds of albumen mixing notes of induction ratio in two kinds of carrier proteins Higher immune response when penetrating, mutually synergy, further enhance the immunogenicity of carrier protein, add body to more The immune response of sugar；

3. the present invention it is pioneering using magnetic Nano microsphere as polysaccharide and the connector of overloading body protein, be effectively prevented from Autoimmunity syndrome in preparation process between capsular polysaccharide and protein, it is possible to increase with reference to the yield of product, and be beneficial to product Quality control；The space length that can effectively extend between capsular polysaccharide and carrier protein, it is more to pod membrane to reduce carrier protein The spatial masking effect of sugar antigens epitope, be advantageous to improve the immunogenicity of capsular polysaccharide；

4.-the OH by magnetic Nano microsphere surface initiated in the preparation process of the conjugate vaccines first carries out even with polysaccharide Connection reaction, then by couplet it is chemically modified by unreacted-OH be modified as-CHO with-the NH in carrier protein₂It is coupled With reference to associated methods more of the prior art are more stable；

5. its preparation method is simple, it 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, conjugate vaccines preparation technology provided by the invention is simple, uses magnetic Nano microsphere sewing for attachment Mouse Th1 type immune responses can be strengthened by closing vaccine, and the immune lasting effect of polysaccharide specificity antibody, specificity and affine Property, it additionally can induce mouse and produce rotavirus antibody；Possesses the preventive effect of two kinds of vaccines；Therefore with very wide Application prospect.

Brief description of the drawings

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

Fig. 2 is the immune response experimental result schematic diagram of the polysaccharide specificity antibody of conjugate vaccines provided by the invention；

Fig. 3 is the immune lasting effect experimental result schematic diagram of the polysaccharide specificity antibody of conjugate vaccines provided by the invention；

Fig. 4 is the rotavirus neutralization test result schematic diagram of conjugate vaccines provided by the invention.

Embodiment

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

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

Embodiment 1

First, magnetic Nano microsphere is prepared

1. take 2.24gFeSO₄-7H₂O and 3.24gFeCl₃-6H₂O is dissolved in the dddH of 10mL and 15mL filtering deoxygenations respectively₂O In, mix after dissolving and hook, add the dddH of 100mL filtering deoxygenations₂O；

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

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

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

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

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

2nd, the preparation of pneumococal polysaccharide

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

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

3rd, the preparation of rotavirus carrier protein

The preparation of rotavirus carrier protein can be found in the preparation method of a variety of recombinant proteins of the prior art, this implementation Example uses the method referred in CN 101972475 to prepare, and can be reduced to following steps, design parameter does not repeat：

1st, the cDNA storehouses of rotavirus are established

Human rotavirus Wa strains (2018-VR, ATCC) are in MA104 cells (the monkey embryo added with 1 μ g/mL trypsase Foetus renal cells) in, 36 DEG C of temperature expands 1 to 2 day.Low-speed centrifugal removes cell fragment, adds hydroxyapatite (hydroxyapatite, HA) purified virus RNA into supernatant.Concretely comprise the following steps：600 μ l 6M guanidinium isothiocyanates (GITC) Solution is added in 400 μ l culture supernatant, is mixed.Then 50 μ l (two drops) HA suspension is added, is turned upside down at room temperature 20 minutes are placed so as to by nucleic acid absorption to HA on mixing test tube or earthquake device.Centrifuged 1 minute with 13,000g, in discarding Clear liquid.RNA-HA is washed with 1mL 10mM potassium phosphates pH6.8 twice.The 200mM potassium phosphate pH6.8 solution for adding 40 μ l is tied from HA Eluted rna on crystalline substance, 3 minutes RNA for collecting purifying are centrifuged with 13000g.Reverse transcription-polymerase chain reaction is carried out immediately, by sample Product solution is stored in -20 DEG C.

In order to which amplification coding has a VP4 genes of Wa strain VP8 albumen, design of primers is as follows：Sense primers HWaVP4pET28：5 '-TTACATATGGCTTCGCTCATTTATAG-3 ', anti-sense primer AHWaVP4 ρ ET28：5’- CCGGATCCCTAGTCTTCATTAACTTGTGCT-3’。

With SuperScriptOne-StepRT-PCR systems, RT- is carried out containing PlatinumTaqDNA polymerase kits PCR is expanded.

Comprise the following steps that：The RNA of 8 μ l purifying is added to DMSO added with 4 μ l, 2 μ l 20mMHWaVP4 ρ ET28 and The 0.2-mL test tubes of AHWaVP4pET28 primers, heat 2 minutes at 94 DEG C, annealed on ice bath.Then containing for 86 μ l is added 0.2mM various dNTP, 1.5mM MgCl₂With 2 μ l reverse transcriptase and Taq enzyme reaction mixture.Carried out such as in PCR instrument The reaction of lower condition：42 DEG C hatch 60 minutes, are reacted subsequently into the PCR in 35 cycles, 94 DEG C 30 seconds, 42 DEG C 50 seconds, 72 DEG C 70 seconds, last 72 DEG C hatched 7 minutes.RT-PCR amplified productions are purified on GFX posts.

PCR primer is incorporated into pGEM-TEasy carrier with the method that is directly connected to.The amplification plasmid of structure is transformed into To in Competent cells E.coliTop10.With WizardSVMiniprepDNA, the conversion bacterium colony of the positive is selected, is trained in LB Support in base and expand bacterium, collecting bacterial body, purification system recombinant dna plasmid.The plasmid for containing VP8 protein expression sequences is referred to as PGEM-TWaVP8, plasmid is stored in -20 DEG C.

2nd, pET28aWaVP8 expression total length VP8 plasmids are built

With nucleic acid restriction endonuclease NdeI and BamHI come pGEM-TWaVP8 made from digestion step 1, to obtain VP8 bases Because of insetion sequence.Equally with nucleic acid restriction endonuclease NdeI and BamHI come digested vector pET28a (+).Add 10 μ g VP8 Gene inserts, T4DNA ligases and the pET28a (+) that cuts through are in test tube, in 16 DEG C of reactions overnight.What the structure was completed Plasmid is referred to as pET28aWaVP8.The plasmid expression come out albumen contain 6- histidine marks (hexa-histidinetag, 6 × His-Tag), and have thrombin recognition site (thrombinrecognitionsite) in 5 ' ends.

PET28aWaVP8 plasmids are transformed into competent cells E.coliTOP10, this is cloned in 50 μ g/mL's The LB culture mediums ware screening pET28aWaVP8 positive bacterium colonies of kanamycins (Kanamycin).It is inoculated in LB nutrient solutions and carries out Amplification, preserve below -20 DEG C of strain of expression, while ρ ET28aWaVP8 plasmids are extracted from bacterium, it was demonstrated that VP8 nucleotide sequence (SEQIDNO：1).

3rd, expression restructuring VP8 albumen

1) pET28aWaVP8 plasmids made from step 2 are transformed into BL21 (DE3) competent cells by, and inoculation is thin Born of the same parents are to 50 μ g/mL kanamycins LB culture dishes, in CO at 37 DEG C₂In incubator overnight.

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

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

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

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

4th, VP8 albumen is purified from inclusion body

1) weighs the VP8 inclusion bodys 0.5 gram (weight in wet base) of step 3 preparation, with cleaning buffer solution (10mMTris, 100mM phosphorus Phthalate buffer, 2Murea, pH8.0) suspension inclusion body, it is incubated 30 minutes, is centrifuged 10 minutes with 10,000g at room temperature, is received Collect inclusion body.Above step is repeated three times except the foreign protein to depollute.

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

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

Specific method is：SepharoseFF glue will be chelated in advance in 0.1M NiSO4 solution, mixing is flat at room temperature Weighing apparatus, shake for several times so that Ni²⁺After being sequestered on glue, Ni-Sepharose is cleaned twice with deionized water, removes what is do not chelated Ni²⁺Ion.By colloidal suspension in buffer B, IMAC posts are made in dress post., then will be dissolved with VP8 with buffer B pre-balance post The buffer B upper prop of albumen, at room temperature, treat that solution balances the His-tag and Ni caused for 30 minutes in restructuring VP8 albumen on post Ionic adsorption.Post is washed with buffer B, post is washed with the imidazole buffer Bs containing 10mM.Further use buffer solution C (10mMTris-HCl, 100mM phosphate buffer, 8M urea, pH6.3) washes post.Then (contained with elution buffer 100mMEDTA, 2mM beta -mercaptoethanol and buffer B) elution VP8 albumen.

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

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

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

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

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

2. induced expression and the purifying of recombinant protein

Recovery engineering bacteria, 2 × LB culture mediums are inoculated with 1: 100 ratio, expands culture under 37 DEG C of 237r/min, works as bacterium When body value is about 12, IPTG to final concentration of 1mmol/L, 37 DEG C of induction 4h are added, sampling carries out SDS-PAGE analysis centrifugal receipts Collection induction thalline, adds physiological saline and washing 2 times is resuspended, and 05mol/LNaCl5mmol/L miaows are added with 1: 10 (g/mL) ratio Thalline is resuspended in azoles 20mmol/LPB (pH7.4) buffer solution, ultrasonic disruption thalline, 8000 × g centrifugation 40min, collects supernatant, Purified in nickel ion chromatographic column, by specification operation purified product and analysis by carrier pET-30a (+) conversion it is big Enterobacteria BL21Star (DE3) whole cell (control) and electrotransfer is fine to nitric acid after SDS-PAGE is separated by upper step purification of samples Tie up on plain film, 2h is closed with 5% skimmed milk power shaking table slight oscillatory；His mouse source monoclonal antibody (1: 800 dilution) is added, 4 DEG C overnight； TBST is cleaned 3 times, is added the sheep anti-mouse igg (1: 2000 dilution) of HRP marks, is incubated at room temperature 1h；Washing 3 times, DAB colour developings.

5th, the preparation of people's Rotavirus Vaccine

1. polysaccharide-magnetic Nano microsphere coupling

Under 0.1MTBS solution buffer solutions, pH6.0 adds obtained one or more capsular polysaccharides of above-mentioned steps and magnetic is received 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 The mass ratio of polysaccharide and magnetic Nano microsphere is 1: (0.5-1), reacts 6-24 hours at room temperature；The mass ratio wherein optimized is 1: 1, aldolisation 16 hours at room temperature.After reaction terminates, fully dialysed with bag filter, it is micro- to remove unreacted magnetic Nano Ball.

2. coupling is modifies

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

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

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

4. people's Rotavirus Vaccine isolates and purifies

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

The composition analysis of obtained people's Rotavirus Vaccine

With¹H-NMR carries out detecting people's Rotavirus Vaccine, and testing result is shown in Fig. 1.It is as shown in figure 1, more with pod membrane Glycan molecule is compared, and characteristic peak occurs at 0.4-1.4ppm in conjugate, corresponding to the aliphatic chain amino acid residue of carrier protein. Occurs the characteristic peak of the aromatic amino acid residue corresponding to carrier protein 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 corresponding to succinyl at 6.2ppm The characteristic peak of imines, this shows to contain succinimide in the connecting bridge of polysaccharide conjugate vaccine.In addition, 5.2,1.6,3.6 outlets PELA characteristic peak proves magnetic Nano microsphere PELA as attachment.Therefore, capsular polysaccharide is combining two kinds of carrier proteins Front and rear, its structure does not occur substantially to change.

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 the albumen in GL-PP conjugate and more sugar types 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 that conjugate is calculated is combined than (Ratio)；Rotavirus protein, PspA protein concentrations pass through ELISA Detection.As a result show：In the people with Rotavirus Vaccine stoste, 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 differs only in embodiment 1：Nonmagnetic nanoparticle is as connector in obtained vaccine, and And the method by referring in the prior art complex carries albumen and polysaccharide is conjugated to obtain.

Comparative example 2

This comparative example differs only in embodiment 1：Without PspA carrier proteins in obtained vaccine, only with polysaccharide- Magnetic nanometer particles-rotavirus carrier protein behaviour Rotavirus Vaccine.

Embodiment 2

The present embodiment differs only in embodiment 1：The people is colyliform disease with the carrier protein of Rotavirus Vaccine Poisonous carrier albumen and tetanus toxoid carrier albumen.

Theoretical error scope be present with the acquired results of embodiment 1 in the composition analysis result of obtained people's Rotavirus Vaccine Interior uniformity.

Embodiment 3

The present embodiment differs only in embodiment 1：The people is colyliform disease with the carrier protein of Rotavirus Vaccine Poisonous carrier albumen and bloodthirsty Bacillus influenzae surface protein HiD.

Embodiment 4

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

Embodiment 5

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

Vaccine evaluation

Assessment experimental evaluation embodiment 1~7 using vaccine routine potency such as immunogenicities and the institute of comparative example 1~2 below Obtain the immune effect of vaccine：

A. people is with Rotavirus Vaccine immunogenicity experiments

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

Table 1

As shown in table 1, the people of gained can be obviously improved antibody titer (p ＜ with Rotavirus Vaccine in embodiment 1~7 0.0001**), as shown in Fig. 2 and being substantially better than comparative example 1 and comparative example 2 and positive controls；As can be seen here, using double Carrier protein and can significantly increase that Th1 types are immune with Rotavirus Vaccine with people obtained by magnetic Nano microsphere and pneumonia polysaccharide should Answer, and immune effect is substantially better than obtained by single carrier protein without nano-magnetic microsphere connector and comparative example 2 of comparative example 1 People's Rotavirus Vaccine；But it will also realize that according to the immune response result of the gained of embodiment 1~5, be complex carries by PspA or HiD The immune response effect of one of albumen is preferable, and PELA and PLGA is good compared with PEG immune responses effect in magnetic nano-carrier.

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

Once tested with Rotavirus Vaccine with embodiment 1, comparative example 1 and 2 and positive control income earner respectively.

1st, lasting effect is immunized

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

2nd, specificity and compatibility

Added into 200 times of the PS-TT groups diluted, PS-PLGA-TT groups and PS-PELA-TT group mice plasmas different amounts of Capsular polysaccharide, the antibody level of mice plasma moderate resistance capsular polysaccharide is detected with ELISA method, 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 antibody binding 96 ability of polysaccharide by Gradually reduce.When the polysaccharide of addition reaches 20 μ g, the Disability of antibody binding polysaccharide.This shows people provided by the present invention Anti- capsular polysaccharide antibody can specifically combine capsular polysaccharide caused by Rotavirus Vaccine inducing mouse.

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 is 1.18mol/L, and positive controls, 1 group of comparative example, the antibody of 1 group of 2 groups of groups of comparative example and embodiment Index of affinity is respectively 2.65mol/L, 2.80mol/L, 3.02mol/L and 3.21mol/L.This shows that people provided by the invention uses Rotavirus Vaccine can significantly improve the affinity of polysaccharide specificity antibody.

C, rotavirus neutralization test result

Using mice serum each group (embodiment 1~5,1 group of comparative example and comparative example 2 made from method difference in embodiment 1 Group inject a pin, two pins and three pins respectively) each mice serum respectively take 10 μ L mix, for neutralization test Sample serum.

The neutralization test of anti-WaVP8 antibody uses BSC-1 cells on microplate (microplate) caused by injection small white mouse Carry out.After heat inactivated serum is serially diluted, after being mixed with 100TCID50 Wa rotavirus strains, in 4 DEG C of cultures 1 hour.Then BSC-1 cells are inoculated on microplate, hatched 1 hour.DMEM (being free of serum) is added to be added to each hole, 37 DEG C Hatching 1 hour.Finally, dilution antiserum can prevent the concentration that rotavirus cytopathic effect (CPE) occurs from being dripped to neutralize Degree.Polysaccharide control group serum is used as negative control.Experimental result is shown in Table 3 and Fig. 4.

Table 3：People is injected with Rotavirus Vaccine in small white mouse antibody and Rotavirus Wa strain strain result of the test

As shown in table 3, it is better than contrast with rotavirus positive effect in the income earner's Rotavirus Vaccine of embodiment 1~5 Example 1 and comparative example 2, significantly can induce rotavirus antibody to produce；In addition, in embodiment 1,3 and 5 and rotavirus effect will It is in other words, preferable for the neutralization virus effectiveness of one of complex carries albumen by PspA or HiD better than other embodiment, and magnetic is received PELA and PLGA is good with virus effectiveness compared with PEG in meter Zai Ti..

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

Claims

1. people's Rotavirus Vaccine, it is characterised in that：The people is multivalent pneumococcal polysaccharide and two with Rotavirus Vaccine People's Rotavirus Vaccine that kind or two or more carrier proteins form through connector, wherein, connector is that magnetic Nano is micro- Ball, the one in two or more carrier protein is rotavirus protein.

2. people's Rotavirus Vaccine according to claim 1, it is characterised in that：The micro- magnetic particle of the magnetic Nano It is core positioned at the inside of nanoparticle, high polymer material is wrapped in the outside of magnetic particle.

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

4. people's Rotavirus Vaccine according to claim 1, it is characterised in that：Multivalent pneumococcal polysaccharide and two kinds of loads The mass ratio of body protein is (0.5~2)：1.

5. people's Rotavirus Vaccine according to claim 1, it is characterised in that：In two or more carrier protein Another carrier protein be selected from diphtheria toxoid, tetanus toxoid, carrier protein CRM197, bloodthirsty Bacillus influenzae surface protein HiD, pertussis Prn surface proteins, pertussis Fha antigens and/or Pneumococal surface protein A (rPspA).

6. people's Rotavirus Vaccine according to claim 1, it is characterised in that：Rotavirus protein is recombined human colyliform Virus protein.

7. people's Rotavirus Vaccine according to claim 1, it is characterised in that：Recombined human rotavirus protein is P bases Because of the partial amino-acid series or complete sequence of type rotavirus protein.

8. people's Rotavirus Vaccine according to claim 1, it is characterised in that：The virus of recombined human rotavirus protein Strain is one kind in P genotype rotavirus strain P [8], P [4], P [6] or P [11].

9. the preparation method of any people's Rotavirus Vaccine of claim 1~8, it is characterised in that：By multivalence pneumonia ball Granulose forms with nanoparticle through coupling respectively with two kinds or more of carrier protein.

10. the preparation method of people's Rotavirus Vaccine according to claim 9, it is characterised in that specifically include following Step：

B) simultaneously the rotavirus carrier protein selected by separating-purifying and another carrier protein are prepared respectively；

C) various capsular polysaccharides and nanoparticle are coupled into polysaccharide-nanoparticle couplet respectively；

D) polysaccharide-nanoparticle couplet is combined with rotavirus carrier protein and another carrier protein couplet respectively again；

E) couplet obtained by purification procedures d) into the people with Rotavirus Vaccine stoste.