CN104606674A - Rotavirus polysaccharide-protein conjugate vaccine and preparation method thereof - Google Patents

Rotavirus polysaccharide-protein conjugate vaccine and preparation method thereof Download PDF

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CN104606674A
CN104606674A CN201410487401.4A CN201410487401A CN104606674A CN 104606674 A CN104606674 A CN 104606674A CN 201410487401 A CN201410487401 A CN 201410487401A CN 104606674 A CN104606674 A CN 104606674A
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polysaccharide
protein
rotavirus
vaccine
carrier
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CN104606674B (en
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刘昊智
王文灏
程超
吴克
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Bravovax Co ltd
SHANGHAI BOWO BIOTECHNOLOGY CO Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6075—Viral proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12341—Use of virus, viral particle or viral elements as a vector

Abstract

The invention discloses a rotavirus polysaccharide-protein conjugate vaccine, which is a conjugated vaccine formed by polyvalent pneumococcal polysaccharide and two or more carrier proteins through a connecting body. Specifically, the connecting body is a magnetic nano-microsphere, and one of the carrier proteins is rotavirus protein. According to a preparation method of the rotavirus polysaccharide-protein conjugate vaccine, the polyvalent pneumococcal polysaccharide and two or more carrier proteins (with one of the proteins being rotavirus carrier protein) are respectively coupled with the magnetic nano-microsphere so as to obtain the rotavirus polysaccharide-protein conjugate vaccine. The preparation process for the rotavirus polysaccharide-protein conjugate vaccine provided by the invention is simple, the rotavirus polysaccharide-protein conjugate vaccine adopting the nano-microsphere as the connecting substance can enhance the Th1 type immune response of mice, and the immunologic persistence, specificity and affinity of polysaccharide specific antibodies, also can induce mice to generate rotavirus antibodies, and has the prevention effects of two vaccines, thus having very broad application prospects.

Description

Rotavirus polysaccharide-protein combined vaccine and preparation method thereof
Technical field
The present invention relates to a kind of vaccine, specifically, relate to rotavirus polysaccharide-protein combined vaccine vaccine and preparation method thereof, belong to biological technical field.
Background technology
One, microorganism is to the harm of human body and counter-measure
Microorganism typically refers to the biocenose that those body volume diameters are generally less than 1mm, their structures are simple, unicellular mostly, also some even cellularity do not have yet, we do not also exist microorganism at one's side all the time, usually just can see their Morphology and structure clearly by microscope or ultramicroscope; Wherein, pathogenic microorganism refers to the disease that can cause the mankind, animal and plant, has pathogenic microorganism.A kind of pathogen pathogenic depends on its attack to host and breeds and resist host resistance in vivo and not by ability that it is eliminated.Microorganism is pathogenic generic character, and the degree of pathogenecity power is called virulence.The establishment of infectious disease, not by the virulence unilateral decision of microorganism, also will look health condition and the immune functional state of host.Generally speaking; the not immune body crossed of the infected by microbes that virulence is strong; pathological lesion can be caused to occur apparent infection etc.; and normal body can resist the infringement of many low toxicity microorganisms (as conditioned pathogen), but then can cause a disease to these microorganism susceptibles when host resistance reduces.The trial of strength between the two of the virulence of pathogen and host resistance, draw the generation of infectious disease, develop, lapse to and prognosis, because adaptedness between pathogen and host is different, the final result that both sides contend with is different, produce various different pattern of infection, i.e. the different manifestations of course of infection.Pathogenic is for specific host, and only having the mankind of having is pathogenic, have only to some animal, the then genus infecting both domestic animals and human microorganism had.And now host can only carry out the infection of combating microorganisms usually by the immune system of self, mortality rate is very high, causes each researcher to investigated vaccine to resist the infringement of invasive organism to human body.
And vaccine is divided into therapeutic and preventative two kinds, by therapeutic vaccine disease therapy, and by preventative vaccine protection human body not by the infringement of invasive organism.By inoculating against property vaccine prevent disease be the mankind in the clinical practice in century more than one, prove effective means.Through effort for many years, medical circle has developed various different vaccine in order to prevention, and such as antibacterial, virus and fungus etc. infect the various diseases caused, drastically increase the health level of the mankind.The development of biotechnology, facilitates the variation of vaccine kind.Today, the infectious disease caused in order to pre-anti-virus has inactivation of viruses technological development vaccine out, as Vaccinum Encephalitidis Epidemicae, poliomyelitis vaccine, influenza vaccines etc.; With attenuated virus technological development attenuated live vaccine out, as Rotavirus Vaccine, oral polio virus vaccine, measles virus vaccines, mumps virus vaccine, rubella virus vaccine and chickenpox vaccine etc.The biological macromolecule purifying technological development antibacterial class vaccines out such as the useful proteins of prevention bacterial infectious disease and polysaccharide, as tetanus toxoid, diphtheria toxoid, DT-Pa and subcellular components thereof, epidemic cerebrospinal meningitis Streptococcus polysaccharides and 23 valency pneumococal polysaccharides etc.With gene recombinant protein technological development vaccine out, as hepatitis B surface antigen (prevention hepatitis B), human nipple shape viruslike particle virus (prevention cervical cancer) etc.The prevention meningitis developed with half chemical bonding techniques and the bacterial vaccine of pneumonia, as popular influenzae type polysaccharide-protein combined vaccine, 7 valencys or 10 valency pneumococcal polysaccharide-protein combined vaccines and 4 valencys meningococcal polysacharide-protein conjugate vaccines.As can be seen here, the development of medical biotechnology is the motive power of vaccine product development, by updating biotechnology, can develop more novelvaccine product deals with the challenge of different infectious disease to human health.
Two, combined vaccine general introduction
Polysaccharide is the important immune effective ingredient of the one in pathogen, have mycelial polysaccharides (OPS) and capsular polysaccharide (CPS) point.After pathogen invades body, they are as immunogen, and body can be stimulated to produce protective immune response.But, polysaccharide molecule belongs to T cell not dependence antigen (Ti-Ag), less immunogenic, after inoculation infant, immune effect is especially undesirable, and infantile hemorrhage diarrheal dehydration of causing of meningitis, E.Coli O 157:H7 that the serious disease of current much harm causes as hemophilus influenza (Hib) etc. is occurred frequently and clinical nothing effectively treats way in infant, high (the Wang Yan etc. of case fatality rate, combined vaccine general introduction [J], microbiology immunology is in progress, 2000,28 (1): 60-63).In order to improve the immunogenicity of polysaccharide vaccine, the external chemical bond vaccine having risen polysaccharide and albumen at the beginning of 1920's, i.e. conjugate vaccines, adopt chemical method to be combined on protein carrier by polysaccharide covalent in conjunction with (taking albumen as the bacterial polysaccharides class of carrier) vaccine and be prepared into polysaccharide-protein combined vaccine, for improving the immunogenicity of bacterial vaccine polysaccharide antigen, as b type hemophilus influenza combined vaccine, meningococcus combined vaccine and pneumococcal conjugated vaccine etc., short decades, its development was quite rapid, had obtained remarkable achievement.
Three, pneumococcal harm and epidemiological study thereof
Infection caused by streptococcus pneumoniae (lung chain) is the main cause of whole world M & M.Pneumonia, heat generation bacteremia and meningitis are the most common manifestation forms of invasive pneumococcal disease, and the antibacterial diffusion in respiratory tract can cause middle ear infection, sinusitis or recurrent bronchitis.Compared with invasive disease, the form of expression of Noninvasive is usually so not serious, but more common.Due to the diffusion of antibiotics resistance sexually transmitted disease, and pneumococcal pneumonia occurs after influenza infection of being everlasting, and the probability that pneumococcal disease shows effect during influenza increases further.
The disease caused by streptococcus pneumoniae has become the important public health problem in one, the whole world.Streptococcus pneumoniae has become the number one killer of global child.The case fatality rate of China's pneumonia is 16.4%, and wherein more than 50 years old middle-aged and elderly people and 1 years old Infants Below are respectively up to 28.6% and 22.0%.The carrying rate of China streptococcus pneumoniae in healthy children is higher, and statistics show, and the carrying rate in northern area healthy children is 24.2%, and southern area is 31.3%.And described disease is the major reason causing less than 5 years old death of child.Main cause is that the growth of infant immunisation system is not perfect, and immunity is more weak.And the baby that the age is less, immunity is more weak.The nearly 90 kinds of serotypes (bacterial strain) of streptococcus pneumoniae, before the statistics display pneumococcal infection bacterial strain of China, several serotype is followed successively by: 5,6,19,23,14,2,4 types.The research display of 860 strain streptococcus pneumoniae separated strains is have collected according to one, 109 strains (12.7%) are had to show as sero-group 6, reach 100% in the erythromycin resistance of Chinese Pneumococcus serotypes 6,6A, 6B and 6C type is wherein respectively 62 strains (56.9%), 38 strains (34.9%) and 9 strains (8.2%).
Chemically in structure, above pathogen has a cell surface capsular polysaccharide (Capsular polysaccharide, CPS) or lipopolysaccharide (Lipopolysaccharide, LPS) shell, or both have concurrently, its function helps pathogenic infection host.Capsular polysaccharide can shield bacterial cell surface functional component and avoid by host immune system identification, and prevent complement system from being engulfed by bacterial surface protein activation and immunocyte, if antibacterial is engulfed, capsular polysaccharide can prevent antibacterial to be killed.In most of pathogen, different bacterial strains expresses capsular polysaccharide and the lipopolysaccharide of different structure, produces multiple different serological type strain.Pneumonia caused by streptococcus pneumoniae and meningitis are caused by a big chunk strain infection in known 90 kinds of serotype.
As can be seen here, most of bacterial polysaccharides class vaccine must containing multiple different types of bacterial polysaccharides to improve pathogenic strain coverage rate, optimize and selection to comprise which kind of antibacterial or serotype polysaccharide be a very complicated epidemiology problem in vaccine.Once which kind of polysaccharide antibody clear and definite has protective effect, then can produce vaccine with this polysaccharide as immunogen.
The 23 valency Pnu-Imune 23s that Chengdu Inst. of Biological Products of Chinese biological technology group produces have chosen 23 kinds of modal pathogenic bacterium (1,2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F), respectively fermentation culture various polysaccharide on separating-purifying pneumococcal capsule, is mixed and made into vaccine by equal proportion.The polysaccharide of antibacterial is a kind of thymus independent antigen, and the main distinction of this antigen and thymus dependent antigen is that the former does not need lymphocytic the assisting of T to produce antibody.Clinical practice proves, the vaccine that capsular polysaccharide makes is clearly effective, and multiple countrywidely use.But there is following problem in this kind of polysaccharide vaccine: (1) can only produce faint immunoreation in brood or infants, does not even produce immunoreation, and immunoreation strengthens with the growth at age; (2) antibody of low-affinity is produced; (3) only produce of short duration immunoreation, do not possess immunological memory when repeatedly inoculating and immune-enhancing effect; (4) easily immunologic tolerance is produced; (5) common adjuvant not easily plays the effect of immunostimulant to this antigen, the protective rate that 23 valency polysaccharide vaccines infect for intrusion type lung chain is 50-70%, and crowd's inoculation of more than 2 years old can only be used for, and the peak age of onset of pneumonia is the 6-12 monthly age; (6) polysaccharide with repetitive structure is T cell independentthe immunogen of type 2 class, does not have the participation of T cell, and they are cannot inducing immunological memory effect, stimulates antibody mainly IgM and IgG2 that body produces, can not activating complement system effectively.
The method of half chemical bonding techniques (also claiming combination technology) vaccine development is the technology of the exploitation bacterial vaccine from the appearance eighties in 20th century.John Robbins passes through popular influenzae type (Haemophilusinfluenzaetype b; Hib) capsular polysaccharide (Polyribosylribitolphosphate; PRP); be connected to protein carrier (tetanus toxoid) by the form of covalent bond and synthesize popular influenzae type polysaccharide PRP-tetanus toxoid conjugate (PRP-TT); after immune animal; the protection antibody of sterilization can be produced, thus started bacterial vaccine development technique of new generation.Especially meaningfully; be less than to the infant of 2 years old the age; due to developing immune system imperfection; polysaccharide is belong to a kind of T-independent antigen for infant, can not stimulate body as adult, produce the antibacterial specificity protection IgG antibody of polysaccharide origin as described in long-acting being directed to.Therefore, polysaccharide is a kind of hapten for the infant being less than 2 years old, can not inoculate the child being less than 2 years old as vaccine.When bacterial polysaccharides is connected to protein carrier with the form of covalent bond; as tetanus toxoid (Tetanus Toxoid; TT); because protein is a kind of T cell dependence antigen; the polysaccharide that covalent bond connects can be transformed into T cell dependence antigen; thus stimulating body to produce the specific IgG antibodies being directed to described polysaccharide, protection body is not by the infection of antibacterial.The success of popular influenzae type polysaccharide-tetanus toxoid (PRP-TT) combined vaccine exploitation, start the technology platform of an exploitation bacterial vaccine, by bacterial polysaccharides, as capsular polysaccharide, O-specific polysaccharide (O-specificpolysaccharide) or oligosaccharide (Oligosaccharide), be connected to the combined vaccine that protein carrier is made with covalent bond.Based on the success of this concept, the combined vaccine of medical biotechnology research circle by adopting same chemical synthesis process to have developed different bacterium; The combined vaccine of same antibacterial is equally also developed by different synthetic technologys.
Capsular polysaccharide can shield bacterial cell surface functional component, makes it avoid by host immune system identification, prevents complement system from being engulfed by the protein activation of bacterium surface and immunocyte.If antibacterial is engulfed by immunocyte, capsular polysaccharide also can avoid antibacterial to be killed.Capsular polysaccharide is one of major antigen composition of meninges Neisseria, has certain protection as vaccine to larger child.But capsular polysaccharide is to the community immunity weak effect of 2 years old Infants Below, old people and B cell immunodeficiency, and Inoculant can not reach antibody level of protection, and antibody disappears very soon.Described polysaccharide vaccine is the same with other polysaccharide vaccine, belongs to T cell independent antigen, has age relevant immunogenicity, and does not induce the dependent booster response of T cell.By by polysaccharide and certain protein covalent bond, polysaccharide conversion can be made to become T cell dependence antigen, thus stimulate the synthesis of the T cell dependency antibody of infant, and can booster response be produced, the antibody ratios of immunoglobulin (IgG) can also be improved simultaneously.This polysaccharide conjugate vaccine can not only protect infant (less than 2 years old child), and the resistance of poor patient to bacteriological infection of can also building up resistance widely, therefore has very wide application prospect.1980, JohnRobbins Bromine cyanide. activates Hib capsular polysaccharide at random, then, by adipyl dihydrazide (Adipic Dihydrazide, ADH) be added on the polysaccharide of activation as junctional complex (linker), finally with EDC method, the polysaccharide covalent key after derivation is connected on carrier protein tetanus toxoid, has synthesized popular influenzae type polysaccharide-tetanus toxoid conjugate (PRP-TT).Owing to there being multiple activation point on each polysaccharide chain, same on protein carrier have multiple junction point, and the conjugate of formation is a kind of polysaccharide and the cross-coupled macromole of albumen, and mean molecule quantity is about 5 × 106Da.1980, Harold Jennings was in United States Patent (USP) 4356170, and set forth with bovine serum albumin (Bovine serum albumin, BSA) is carrier, by meningococcal A, C group polysaccharide by reduction amine method altogetherbe connected on BSA valence link, synthesized epidemic cerebrospinal meningitis polysaccharide-BAS combined vaccine.1987 and nineteen ninety, Porter Anderson is respectively in United States Patent (USP) 4673574 and 4902506, describe with variation avirulent strain diphtheria toxin, diphtherotoxin 197 (Cross reaction material sub197, CRM197) as carrier protein, popular influenzae type oligosaccharide-variation avirulent strain diphtheria toxin, diphtherotoxin 197 (HbOC-CRM197) combined vaccine has been synthesized to reduce amine method.Concrete method is that producing at two ends is the oligosaccharide of aldehyde radical, by reducing agent sodium cyanoborohydride (sodiumcyanoborohydride), is connected on protein carrier by oligosaccharide covalent bond with sodium periodate oxidation Hib capsular polysaccharide.Form molecular weight and be about the combined with lipopolysaccharide thing of 90kDa, made containing 30% polysaccharide and each albumen on the combined vaccine of 6 glycan molecules.Subsequently, Merck (Merck, Sharpe and Dohme) with mercapto chemistry, with scorching B group's bacterial strain (Neisseria meningitidis groups B) the bacterial surface protein complex of neisseria meningitis (the outer membrane protein complex of purification, OMP) as protein carrier, popular influenzae type polysaccharide-bacterial surface protein complex (PRP-OMP) combined vaccine is synthesized.Use these synthetic technologys, successively have developed the popular influenzae type combined vaccine that three kinds have now been widely used in clinical inoculation, i.e. PRP-TT, PRP-HbOC and PRP-OMP.The success of Hib combined vaccine provides theory and technology basis for developing other antibacterial combined vaccine, exploitation subsequently enters the technology more complicated multivalence combined vaccine stage, its reason is some infectious disease, as the pneumonia that streptococcus pneumoniae causes, meningitis etc. caused by epidemic cerebrospinal meningitis coccus, can caused by multiple different serotype or strain infection, and due to the difference of the chemical constitution of bacterial surface polysaccharides between each serotype or bacterial strain, its antibody does not have cross-immune reaction, therefore, inoculate single serotype or bacterial strain combined vaccine, the infection being vaccinated human body and avoiding other serotype or bacterial strain cannot be protected.For this reason, the protection coverage rate that synthesis and preparation multivalence combined vaccine expand vaccine becomes the main target of exploitation.
By effort for many years, have developed the wide multivalence combined vaccine of multiple coverage rate with combination technology.Bacterial eapsular polysaccharide-protein conjugate vaccines appears at the thirties in 20th century the earliest; 3 type pneumococal polysaccharides are connected on horse serum globulin by Goebel and Avery; the conjugate produced can produce the single-minded antibody of polysaccharide in animals, provides corresponding immunoprotection simultaneously.1987, first GL-PP combined vaccine in the world, Type B hemophilus influenza (HiB) polysaccharide-tetanus toxoid (TT) combined vaccine was come into the market by U.S. FDA approval.Merck & Co., Inc., Pfizer and Novartis Co., Ltd develop HiB polysaccharide-tetanus toxoid conjugate and epidemic cerebrospinal meningitis polysaccharide-tetanus toxoid conjugate in succession, and successfully go on the market.2000, U.S. Hui Shi (Wyeth) company successfully develops and the 7 valency pneumococal polysaccharide-CRM197 combined vaccines that gone on the market, be by 7 different Pneumococcus serotypes polysaccharide respectively covalent bond be connected on CRM197 protein carrier, a kind of polyvalent vaccine of mixed preparing, in order to preventing child pneumonia, described 7 Pneumococcal serotype cover the North America streptococcus pneumoniae different serotypes bacterial strain popular with Europe more than 90%.2006, Sanofi Pasteur have developed 4 valency meningococcal polysacharide-tetanus toxoid conjugates, for preventing 4 kinds of epidemic cerebrospinal meningitis coccus groups, i.e. A, C, Y, W135, caused meningitis.2009, GlaxoSmithKline (GSK) have also been developed a kind of 10 valency pneumococcal polysaccharide-protein combined vaccines, in order to the pneumonia of preventing 10 kinds of Pneumococcus serotypes to cause.Described vaccine has used three kinds of protein as protein carrier, and wherein topmost carrier is albumen-D (Protein D, PD), has 8 serotype polysaccharide to be as carrier with this albumen.Albumen-D be with the gene recombination method of the popular haemophilus of non-separable express without esterification surface albumen; body can be stimulated to produce protection antibody; there is the acute otitis media caused by popular hemophilus infection of the non-separable of potential prevention; other also have tetanus toxoid and diphtheria endotoxin as carrier, are used separately as the protein carrier of the combined vaccine of serotype 18C and 19F.From the design of above combined vaccine product, can find out, the exploitation of combined vaccine is transitioned into technically more complicated polyvalent vaccine from univalent vaccine, improves the coverage rate of bacterial vaccine.
GL-PP conjugate vaccines (combined vaccine) is current state-of-the-art vaccine technologies, and specific antigen adds protein carrier, can increase its immunogenicity.Protein carrier has T cell dependency characteristic, and the polysaccharide antigen of non-T cell pauper character can be changed into the antigen of T cell pauper character by GL-PP conjugate vaccines, and the t helper cell of excitating organism produces a series of immune-enhancing effect.Capsular polysaccharide conjugate vaccine, polysaccharide adds protein carrier, becomes T cell dependence antigen from T-independent antigen, increases its immunogenicity.The antibody produced after combined vaccine inoculation quality and be quantitatively generation vaccine 400-1000 doubly, produce immunoprotection wider stronger, guard time more for a long time, reaches efficient protection.2000, beautiful pfizer of statecompany's first 7 valent pneumococcal conjugate vaccine listing; Beautiful pfizer of statecompany's exploitation infant more targetedly 7 (4,6B, 9V, 14,18C, 19F and 23F) valency pneumoprotein vaccine, also effective to less than 5 years old child.Capsular polysaccharide protein conjugate vaccines, polysaccharide adds protein carrier, becomes T cell dependence antigen from T-independent antigen, can increase its immunogenicity, can be used for the child in more than 6 week age.Current Pfizer China's registration 13 valency combined vaccines, add 6 serotypes (1,3,5,7F, 6A, 19A).But the data of China display pneumococcal infection bacterial strain serotype is followed successively by: 5,6,1,19,23,14,2,4, Pfizer 7 valent pneumococcal conjugate vaccine only has about 50% to China's common causative bacterial type coverage rate, and the coverage rate of 13 valency combined vaccines also only has 70%.Hui Shi 7 valent pneumococcal conjugate vaccine needs inoculation 4 injection, 860 yuan, every pin, expensive, is unfavorable for promoting.13 valency vaccines estimate that its price will be more expensive.Therefore beautiful pfizer of state7 and 13 valency combined vaccines of company are not too applicable to the large-scale children Streptococcus prevention of China.
Although 13 valent pneumococcal conjugate vaccines go on the market, wherein there is the immune effect of several serotype lower relative to other serotypes, as 3 types.And along with the increase of different serotypes and reusing of carrier protein of the same race, make carrier protein consumption increase, its immunogenicity reduces on the contrary.GlaxoSmithKline is in the design of exploitation 10 valency pneumococcal Polysaccharide Conjugate Vaccine, and have selected albumen-D is carrier, and its reason is the consideration based on two aspects.One, be in order to avoid reuse as the tetanus toxoid of DPT vaccine component and diphtheria toxoid as carrier.Clinical trial shows, when multivalence pneumonia combined vaccine and other contain with the univalent vaccine of protein carrier same composition or multiple vaccines Simultaneous vaccination time, such as Hib-TT, whooping cough-Hib polysaccharide conjugate vaccine-IPV (PKV)-Hepatitis B virus vaccine (being called for short 6 vaccines), the immunogenicity of the most of serotype polysaccharide in multivalent pneumococcal combined vaccine can be suppressed, particularly on especially obvious as the serotype polysaccharide immunogenic impact of carrier with tetanus toxoid.Reason be in multivalent pneumococcal combined vaccine as the tetanus toxoid of carrier and diphtheria toxoid total concentration too high, when inoculation contains the combined vaccine of whooping cough component at the same time, as 6 vaccines, so-called vector receptors competitive inhibition effect can be caused, reduce the immunogenicity of combined vaccine saccharide portion.7 valency pneumococal polysaccharide-OMP combined vaccine (PCV-OMP) of the 11 valency pneumococal polysaccharide-TT of SanofiPasteur and DT mixed carrier protein conjugate vaccines and Merck, be all that clinical test results is good and cause the example of product development failure, reason is just therewith.Its two, be give protein carrier with real protective immunity originality function.Clinical trial proves that albumen-D can stimulate body to produce protection antibody, has the acute otitis media that the popular hemophilus infection of the non-separable of potential prevention causes.The 10 valency pneumococcal Polysaccharide Conjugate Vaccines of GlaxoSmithKline select albumen-D as carrier, and the antibody that protein carrier is produced 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 subject to the low restriction of described bacterial infection rate, and it is lower that it infects the acute otitis media sickness rate caused.But these combined vaccines have a common ground shortcoming; be exactly that protein carrier does not give immunogenic defencive function; that is; although combined vaccine carrier can stimulate body to produce antibody; but; the protectiveness that the designer of vaccine does not utilize its antibody to have keeps off infection, and meanwhile, does not also determine whether its antibody produced reaches protectiveness titre levels.The traditional protein such as the nontoxic variant toxin of tetanus toxoid, diphtheria toxoid and diphtheria are selected as the main cause of carrier; be not because the antibody of their generation has protectiveness, but consider from its safety and the immunogenicity that can strengthen polysaccharide conjugate.Obviously, tetanus toxoid and diphtheria toxoid have been two components of PertussisDiphtheriaTetanus triple vaccine, by traditional vaccination; So; it is unimportant whether the tetanus toxoid in combined vaccine and diphtheria toxoid carrier can stimulate body generation to reach protection antibody titre; contrary, the antibody titer of the saccharide portion in combined vaccine is only the subject matter that vaccine design person needs to pay close attention to.In addition, the carrier that some combined vaccine products just under development are used, the Recombinant pseudomonas aeruginosa exotoxin A (rEPA) of the deletion mutant detoxification expressed by E.coli, the recombinant cholera toxin etc. of the deletion mutant detoxification expressed by E.coli is also all consider based on identical.
The kind of new polysaccharide conjugate vaccine and type are increasing year by year, and alternative carrier protein kind is less, and it is more that carrier protein reused by different vaccine.The different combined vaccines of inoculation same vehicle albumen may produce immunosuppressive effect, cause influencing each other of immune effect between different vaccine.And, the main carriers albumen of polysaccharide conjugate is as TT etc., itself is as vaccine ring vaccination infant, and in people colony, original high titre specific antibody for carrier protein may suppress body to the specific immune response of polysaccharide in combined vaccine.
Chinese patent (ZL02159032.X) discloses a kind of preparation method of polysaccharide-protein combined vaccine, is also to prepare one of technology that polysaccharide conjugate vaccine the most often uses at present.In described technology, using adipic dihydrazide (ADH) as bridging agent in conjunction with polysaccharide and albumen.First this combination needs by polysaccharide through cyanogen bromide-activated, namely acts on the hydroxyl on polysaccharide molecule in the basic conditions with Bromine cyanide., forms cyanate, then reacts with ADH; A C―O bond cleavage in cyanate, with the amino generation additive reaction of ADH one end, thus imports polysaccharide molecule by ester hydrazides (AH) group, forms polysaccharide-AH derivatives; Polysaccharide-AH derivatives forms stable conjugate with carrier protein under the mediation of carbodiimide (EDAC).It is sterically hindered that such combination can reduce that polysaccharide is combined with carrier protein, remains the epitope of polysaccharide, avoid the dissolubility of polysaccharide itself simultaneously, reduces the side effect that polysaccharide and antiserum react.
But above-mentioned traditional polysaccharide-protein combination technology also exists following weak point: (1) polysaccharide-AH derivatives can 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 is while mediation ADH derivation polysaccharide is combined with carrier protein, easily causes the self-crosslinking of polysaccharide and carrier protein, thus reduces the joint efficiency of polysaccharide-protein; (3) polysaccharide and carrier protein are macro-organism molecule, middle by only there being the ADH of 6 carbon atom length to be connected, the structure of polysaccharide and protein will certainly influence each other, make some important epitopes of polysaccharide easily shield by protein, and then reduce the immunogenicity of polysaccharide.Therefore, the immunogenicity of polysaccharide-protein combined vaccine and antibody lasting effect still need to be improved further, as polysaccharide conjugate vaccine needs immunity three ability to produce immune effect.These weak points limit further developing of polysaccharide conjugate vaccine.
Four, people takes turnsthe harm of shape virus and epidemiological study thereof
Worldwide, rotavirus (Rotavirus) is the main pathogens causing child's seriousness to be suffered from diarrhoea, in prosperity country, because of in the child that acute diarrhea is in hospital, the recall rate of rotavirus accounts for 35 ~ 52%.In the U.S., estimate have 300 ten thousand children to suffer from rotavirus diarrhea every year, cause 82000 people's hospitalization, 150 people are dead.In development country, rotavirus is also cause 2 years old modal pathogen of Infants Below severe gastro-enteritis, and estimating has the child more than 1.25 100000000 below 5 years old to suffer from Diarrhea Caused by Porcine rotavirus every year, and wherein 18,000,000 children suffer from mild diarrhea, and 87 die ten thousand deaths dies.In China, Child birth rat about 17,000,000, estimates that nearly 350,000 children cause death owing to suffering from rotavirus diarrhea every year, row second place of the world.The sickness rate of suffering from diarrhoea to 2 years old Infants Below due to the infection of described virus and the increase of fatality rate have remarkable effect, exploitation prevention rotavirus infection, effectively and the vaccine of safety is the task of top priority.
Rotavirus Reoviridae (Reoviridae genus) is the pathogen causing the mankind and numerous animal diarrhea.Totivirus diameter 70nm, has three special shell structures, and the nucleocapsid structure of innermost layer is nucleocapsid protein, is enclosed with viral gene.The gene of virus is made up of 11 stagewise fragments of AMPLIGEN, is 6 structural protein and 5 non-structural protein codings.Nucleocapsid protein is made up of VP1, VP2 and VP3 tri-virus proteins; Middle case albumen is VP6, and coat protein is made up of VP4 and VP7.Gene due to rotavirus is bifilar RNA, multiple clips structure, and the gene of this structure can carry out intergenic reconfiguring to a certain degree, i.e. the reprovision (reassortment) of rotavirus gene.After two or multiple virus infect a host cell simultaneously, at the packing stage of reovirion, the genetic fragment of each virus will reconfigure in cell, cause reprovision gene to occur.The ability of this reprovision of rotavirus gene result in human body to its immunoreactive multiformity, too increases the difficulty making specificity Rotavirus Vaccine.
Rotavirus can be divided into different types according to the difference of virus antigenicity, hypotype and serotype.Had now found that 7 serotypes (A type ~ G type), most of human pathogen belongs to A, B and C type.Epidemiological study finds in the rotavirus causing human and animal ill the most common with A type, is the main target of vaccine development.A type rotavirus, according to the difference of its VP6 antigenic characteristic, is categorized as hypotype further, and most Strain belongs to the one in hypotype I or II.Different with epitope cluster in different rotavirus surface glutelin VP7 and VP4, can independentthe neutrality antibody that ground induction is respective, the serotype of virus can be determined by the specificity of VP4 and VP7 antigen.A type rotavirus can be categorized into G serotype and P serotype further according to the difference of VP7 and VP4.Because rotavirus gene is made up of 11 fragments, the encoding gene of VP7 and VP4 can independentbe separated combination, create 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, and nucleotide sequence specific with it, namely genotype (genotype) is corresponding; That is, the genotype that each G serotype has it special, therefore, usual G serotype and G genotype can be general.In human disease's rotavirus strain that global authorization goes out, more than 90% is G1, G2, G3, G4, and G9 strain, have 10 serotypes from human body separate (see table 1).VP4 a kind ofly can be cut into two different virus proteins by trypsin, i.e. VP8 and VP5 virus protein, the serotype determined by its antigenic difference is P serotype, and some P serotype can be further divided into 2 sub-serotypes.Owing to lacking serum or the monoclonal antibody of the different VP4 Serotypes of difference, hinder the Serotypes of VP4.The application of RT-PCR just makes the genotyping of VP4 become possibility, and applies to the Epidemiological study of sample.Therefore, VP4 classifies according to gene order, finds 14 P serotypes and at least 26 P genotype (being marked in bracket) altogether at present.P serotype and P genotype may not be corresponding, need to mark simultaneously, and such as, Rotavirus Wa strain strain is denoted as P1A [8] G1 virus.In the strain of human disease, P and the G serotype of G1, G3, G4 and G9 combines normally P1A [8], and G2 normally P1B [4].As can be seen here, worldwide popular rotavirus enjoys epitope cluster (epitopes) the P1 serotype of identical cross-neutralization, has at least 7 VP4 serotypes to be found in human rotavirus.The G serotype 1,3 of significant mankind's strain on epidemiology, and 4 is the sub-serotype of 1A belonging to P serotype, and G serotype 2 is the sub-serotype of 1B of P serotype.
Because natural rotavirus infection can be protected by induction of immunity well, at least to the infection of severe rotavirus, therefore, the effort of most of vaccine development is placed on attenuated live vaccine.Initial research concentrates on and uses animal rotavirus strain, and by being referred to as Jennerian method to carry out, reason is the animal strain of nature attenuation is safe at human body, and main generation is mixed type immunoprotection.
Finding that rotavirus is the pathogen after 10 years causing child's seriousness to be suffered from diarrhoea; in nineteen eighty-three; start with porcine rotavirus strain RIT4237 (G6P [1] type); first the rotavirus attenuated live vaccine made has carried out clinical trial; in the result display of Finland's test; described vaccine is that safety is with effective; by the effect of mixed type human rotavirus (heterotypichuman rotaviruses), the protective rate of prevention severe rotavirus diarrhoea reaches 80%.But, at subsequently other countrythe clinical trial result carried out is disappointing, shows low or does not have protective effect, and described test ends in failure.In 1987, monkey rotavirus RRV strain was used to develop attenuated live vaccine, was another in the Rotavirus Vaccine of first exploitation.Clinical trial shows, although described vaccine can induce body to produce protection antibody, unstable result, trace it to its cause and be, the G serotype of RRV strain is G3P [3], when the rotavirus of human infection is the G serotype of homotype, i.e. G3, the Be very effective of vaccine; If when veriform G serotype infects, then poor effect.Subsequently, the VP7 gene in mankind's strain is incorporated in described strain by the method for gene resortment by RRV strain, makes other three kinds of G serotype G1, G2 and G4 common in human rotavirus also can at RRV recombined strain upper tablereach, which results in succeeding in developing of 4 valency Rotashield vaccines.Described vaccine obtained FDA in 1998 and ratifies list marketing, but owing to having found minority after large-scale inoculation, but the generation of intussusception side effect case that quantity obviously raises, in 1999 by production company under city.In 1988, start by another colyliform sick poison poisonstrain, i.e. WC3 pig strain (G6P [5] type), when carrying out clinical trial, starting result proves effectively, but display does not have significant protectiveness in a subsequent experiment, and described vaccine also stopped and continues test.To nineteen ninety, in order to make the antigenic structure of WC3 strain closer to human rotavirus, by the method for gene resortment (gene reassortment), be incorporated into WC3 recombined strain by the gene for VP4 and VP7 encoding histone from human rotavirus, this method is referred to as the Jennerian method improved.Described strain and method are exactly in order to develop the development approach of 5 valencys (pentavalent) vaccine of RotaTeq by Merck.2006, the WC3-gene resortment vaccine RotaTeq producing 5 valencys by Merck also went through listing, described vaccine contain these two mankind's rotavirus protein substituent groups of VP7 and VP4 because of, i.e. G1, G2, G3, corresponding VP7 protein gene in G4, and the corresponding VP4 of P [8].Clinical trial shows, described vaccine does not have intussusception side effect, is 74%, reaches 98% to the protective rate of severe gastro-enteritis to the protective rate of the gastroenteritis caused by G1-G4 rotavirus, is 94.5% to the protective rate of being in hospital and emergency case is accessed.In the same year, human rotavirus's attenuated live vaccine Rotarix that GSK produces also obtains listing approval, and this vaccine is the mankind's strain 89-12 based on attenuation, and serotype is G1P [8] strain, is most common serotype in world wide.Described virus suffers from the patient clinical samples of rotavirus gastroenteritis from one separating, and obtain after histiocyte repeatedly goes down to posterity variation attenuation.Clinical trial shows, after injection two dosage, reaches more than 87%, reach 96% to the protective rate of serious gastroenteritis to the protective rate of all rotavirus infections, reaches 100% to needing the protective rate of gastroenteritis case in hospital.Further test shows, the scope protection of described vaccine not only comprises the gastroenteritis caused by G1P [8] strain, and includes the G3P [8] relevant with VP4, the gastroenteritis that G4 [8] and G9P [8] strain cause.To effectiveness and G1 identical of G3, G4 and G9 rotavirus infection protection, exceeded 95%, and be 75% to the effectiveness of G2 strain, causing to all rotavirus the protective rate that gastroenteritis is in hospital is 75%.
Statistical data shows, G1 strain is worldwide the strain the most often detected, and in Asia, North America and Europe, G1-G4 strain accounts for always infects 97.5% of rotavirus.In South America, Africa, Australia accounts for 83.5%-90.4%, meanwhile, starts to become important at these regional G5, G8 and G9 strains.From P serotype, P1A [8] strain is the most common, and ensuing is P1B [4] strain.This result is predictable, because VP7 serotype G1 is P1A [8], is the VP7 serotype the most often detected; The VP7 serotype that other two epidemiology are important, G3 and G4, also has identical VP4 serotype.The VP4 of another predominant serotypes G2 has the feature of P1B [4].Although G and P serotype or genotype can have multiple different combination, the combination of 4 kinds of P-G, i.e. P [8] G1, P [4] G2, P [8] G3, and P [8] G4 constitutes the common pathogenic strain of 88.5%.
As can be seen here, if configure vaccine with the VP7 albumen of G serotype, need to comprise multiple different G serotype strain to improve the coverage rate of vaccine, i.e. G1, G2, G3, G4, G9, G8 and G5, the rotavirus attenuated live vaccine of 7 valencys is also the striving direction of current Development of New Generation vaccine.If develop Rotavirus Vaccine from another strategy, determine that the difference of albumen VP4 prepares the vaccine of a new generation with P serotype, then can greatly reduce the strain kind that comprises in vaccine to reach identical coverage rate.From above analysis, if prepare vaccine with P [8] and P [4] two P serotype strains, the effect of immunity is by suitable with the G serotype of 7 valencys.From Rotarix and the Rotateq effect used clinically, almost as broad as long, and existing statistical data is seen, Rotarix seems more effective than Rotateq.From the composition analyzing the strain serotype decision albumen that these two vaccines comprise, Rotarix is only containing this kind of VP7 virus protein of G1; Although Rotateq contains four kinds of VP7 virus proteins of G1, G2, G3 and G4, immune effect does not strengthen; And the quantity of albumen VP4 is determined from the P serotype that both contain, it is a kind of that Rotarix contains P [8], and Rotateq contains P [8] (one of them strain) and P [5] (the original strain of WC-3 is G6P [5]) two kinds; But the content of the important antigenic component P [8] in Rotarix is by higher than the content in Rotateq.As can be seen here, assess from the angle of P serotype, the VP4 strain Rotavirus Vaccine containing P [8] has great meaning.If include the P [8] of P serotype, P [4] and P [6] three kinds of VP4 antigenic components in vaccine of new generation, the epidemic isolates of global most area can be contained.
Five, the application and future of Nano microsphere in biotechnology
Nano material refers to that crystallite dimension is less than monocrystal or the polycrystal of 100 nanometers, unique small-size effect and the effect such as surface or interface, and many excellences that made it possess or brand-new performance, it is subject to people's attention just day by day.Such as nano material, on the continuous infiltration in drug research field and impact, has caused the revolution that drug world one field depth is far away.Medicine be the mankind for resisting and prophylactic important substance, for a long time, the research and development of medicine provide many treatment meanss, for patient brings many benefits for clinical.But, existing medicine still also exists many problems, as medicine cannot be detained in blood circulation and reach valid density, specific therapeutic goal cannot be arrived, cannot by blood brain barrier, higher concentration cannot be partially formed at certain and do not produce simultaneously toxic and side effects etc. (Wu Xinrong. the application of drug-carried nanometer and progress [J]. Chinese Hospitals materia medica magazine, 2001,21 (3): 171-173.).Magnetic Nano microsphere pharmaceutical carrier is the product that nanotechnology is combined with modern medicine and pharmacology, there is due to it the advantages such as small-size effect, good targeting, biocompatibility, biological degradability and functional group, be therefore expected to overcome these defects that conventional medicament brings.Magnetic nanometer particles also can be used for the purification of protein and enzyme, recovery and enzyme immobilizatio, simple to operate, and improves the stability of enzyme.Utilize magnetic nanometer particles to carry out immunoassay, have specificity good, be separated feature that is fast, favorable reproducibility.Use Magnetic Microspheres-Carrier to carry out interventional therapy, carry out thromboembolism at magnetic control Ink vessel transfusing, then there is the advantages such as magnetic control guiding, target position thromboembolism.
The application of magnetic Nano microsphere in biomedicine going deep into day by day extensively with research, specifically has:
1, immobilized enzyme
Biopolymer such as enzyme molecule etc. all has a lot of functional group, by modes such as physical absorption, crosslinked, covalent couplings, they can be fixed on the surface of magnetic particle.By the advantage of magnetic Nano microsphere immobilized enzyme be: be easy to enzyme-to-substrate and product separation; Improve biocompatibility and the immunocompetence of enzyme; Improve the stability of enzyme, and simple to operately to reduce costs.Bendkiene etc. have prepared chitosan magnetic microsphere, as fixation support.After enzyme is fixed on this carrier, can easily by magnetic devices Separation and Recovery from the mixed liquor of reaction.Domestic researcher has also done exploration to this respect, Ding little Bin etc. adopt dispersion copolymerization method, synthesize Fe3O4/ (St-MPEO) (St-styrene, MPEO-poly(ethylene oxide) polymeric monomer) microsphere, described microsphere has amphiphilic structure, in most of polarity, in apolar medium, all there is good swelling behavior, the compound making microsphere immobilized all has higher activity (Ding XB in medium, Wei L, Zhao HZ.Synthesis and characterization of aliphatic polycarbonatediols [J] .Applied Polymer Science, 2001, 79 (3): 1847-1851).Described magnetic Nano microsphere carrier is expected for the purification of protein and enzyme, recovery and the field such as enzyme immobilizatio, cell separation.
2, targeted drug
The medicament carrier microspheres with targeting refers to that medicine carrying microballoons energy height is selectively distributed in effective object, thus heightens the effect of a treatment, reduces side effect.Initial target medicine carrier microsphere is according to clinical needs, by selecting, medicine carrying microballoons is made to the various tissue of body or the different carrier of diseased region affinity, or monoclonal antibody is combined with carrier, the specific part for the treatment of and expecting to reach is transported to enable medicine.Along with the requirement of people to treatment is more and more higher, targeting location also can not be entirely satisfactory because of the restriction by substrate, has therefore just occurred magnetic Nano microsphere drug-loading system.This system is under the effect of externally-applied magnetic field, by dynamic (quiet) arteries and veins note people to pathological tissues, carrier is directed to diseased region (target position), contained drug is made to obtain location release, focus on diseased region to have an effect (A Paul, Alivisatos.Ultrasensitive magnetic biosensor for homogeneous immunoassay [J] .Science, 2001,12 (5): 53-60).The Lubbe etc. of Germany completes the clinical experiment of first case applied magnetic drug targeting treatment in the world.In the magnetic target therapy to 14 patients with advanced solid tumors, find that the toleration of patient to magnetic targeted drug is fine.Lexion etc. also applied magnetic microsphere treat scale cancer (the Lexion C of rabbit as pharmaceutical carrier, Amold W, Klein RJ, et al.Locotegional cancer treatment with magnetic drug targeting [J] .Cancer Res, 2000, 60 (23): 6641-6648), the adriamycin magnetic albumin microspheres treatment Mus implanted gastric tumor (Tao Kaixiong such as domestic Tao Kaixiong, Sun Hongwu, Chen Daoda, Deng. Targeting Treatment with Adriamycin Magnetic Albumin Microspheres in Human Mus implanted gastric tumor [J]. Chinese experimental surgery magazine, 2000, 17 (1): 63-64)), Bleomycin A5 magnetic microsphere treatment top collar portion, oral cavity cavernous hemangioma 25 example such as Guo Jun.In addition, high-intensity magnetic field also have cancer suppressing action (Guo Jun, Li Cheng, Wu Hanjiang. the Bleomycin A5 magnetic microsphere targeted therapy oromaxillo-facial region routine clinical report of cavernous hemangioma 25 [J]. Nanjing Railway College of Medicine's journal, 2000,19 (2): 112-114)).The glucose magnetic microsphere immobilized L-Radix Asparagi phthalein amine enzyme such as Xu Huixian treats acute lymphatic leukaemia, also achieves good therapeutic effect.These results all show, the magnetic and medicated gathering at tumor locus of the raising with magnetic field intensity increases, and utilizes the magnetic steering effect of externally-applied magnetic field, makes medicine fix a point in target site, play concentrated, efficient antitumor action.Just because of the targeting of magnetic Nano microsphere and the idiosyncratic carrier of surface combination, make people be expected to utilize it to follow the trail of and eliminate the cancerous cell shifted, thus become " biological missile " of eliminating cancerous cell in human body.But, the tumor multidigit of current applied magnetic Drug therapy is in body surface or table is comparatively near in vitro, and therefore the intensity of externally-applied magnetic field can be more weak, and be easy to control, if the tumor for the treatment of deep organ or tissue, then need to be optimized magnetic field intensity, location and Pharmaceutical carrier particles size etc.The intensity of magnetization improving magnetic microsphere then has certain difficulty, because the clad on magnetic particle surface can reduce its magnetic property greatly, in addition, the controlled of granular size is also have a problem to be solved.
3, cell separation and immunoassay
If magnetic particle surface draws to connect have bioactive specific antibodies, under the effect of externally-applied magnetic field, utilize the specific binding of antibody and cell, just can obtain immune magnetic microsphere (Immunomagnetic microspheres, or immune magnetic pearl (Immunomagnetic beads IMMS), IMBS), utilize them can fast and effeciently by cell separation or carry out immunoassay.When especially adopting IMBS to be separated antigenic substance specific to cell, organelle surface, have easy fast, separation purity is high, retain the features such as target species activity.Mccole etc. are separated the T lymphocyte in the Adult Bovine peripheral blood infected by Liver fluke with IMBS, the cell separated is pure, respond well for Liver fluke infection mechanism.The John IMMS being connected with monoclonal antibody detects Salmonella, and whole testing process only needs 2-3h, and sensitivity is 103-104 thalline/ml, and the existence of a certain amount of blood and feces is noiseless to analysis, and compare with immunofluorescence with agglutination, sensitivity improves 103 times.Glenn is separated to breathe with IMBS and closes inclusion virus, desmoenzyme linked immune analysis, and reduce the diffusion in conventional tube and micropore analysis and non-specific adsorption, the formation of complex only needs 7min, and conventional microporous rule needs 120min.The isolation technics of cell also can be used for the treatment of cancer.The superfine physical absorption of Kang Ji is in conjunction with the covalently bound method of chemical bond, anti-human wing moon bright carcinoma monoclonal antibody is connected to the surface of previously prepared Magnetic Polystyrene Microsphere carrier, constructs and can be combined with target cell specifically and give it with the immune magnetic microsphere of magnetic responsiveness.Result shows, constructed IMMS can combine with target cell effectively, and the preliminary experiment being separated cancerous cell with IMMS from animal bone marrow shows, IMM effectively can remove cancerous cell, and medullary cell only has the loss of seldom amount.Immunoassay is a kind of important method in modern biotechnology analytical technology, and it plays huge effect to the quantitative analysis of protein, antigen, antibody and cell.Utilize the carrier-bound antigen of magnetic nanometer particles or antibody to carry out immunoassay, there is the features such as specificity is high, separation is fast, favorable reproducibility.Jing Xiaoyan etc. adopt agalactosis polymerization, in alcohol one aqueous systems, take potassium persulfate as initiator, formed at Fe3O4 magnetic fluid particle surface and cause point, with acrylic acid (AA) for stabilizing agent, by styrene (ST) and propylene phthalein amine copolymer, prepare monodispersed amido magnetic microsphere, described microsphere can be direct, fast and antibody (antigen) protein cross, protein need be adopted when avoiding other functional group microsphere binding immunoassay reagent to be the shortcoming (Jing Xiaoyan of cross-linking agent, Wang Jun, Li Rumin, Deng. the preparation research [J] of magnetic function polymer microsphere. applicating technology, 2000, 27 (1): 16-17)).
4, magnetic control inspection plug
In common Jie's human therapy process, the phenomenons such as dystopy thromboembolism and infarction can be there is, and cause serious complication, this is the thorny problem being badly in need of clinically solving, and use Jie's human therapy of Magnetic Microspheres-Carrier, carrying out thromboembolism at magnetic control Ink vessel transfusing and then there is the advantages such as magnetic control guiding, target position thromboembolism, providing approach for solving an above difficult problem.Scholars focus on the aspects such as magnetic spherolite footpath, magnetic control time, magnetic field intensity, magnetic microsphere lapping (coarse, carry positive charge, have hydrophobic property) and have done careful research.The therapy that Minalnimura etc. use thermotherapy and arterial thrombosis to combine is for the research of Mus liver cancer model.They have developed DM-MS ductus arteriosus topical, the magnetic field of additional 500kHz.After treatment 3d, tumor rate of increase (thromboembolism-thermotherapy group, simple embolization group and matched group) is respectively 28%, 124% and 385% (Minalnimura T, Sato H, Kasaoka S, et al.Tumor regression by inductive hyperthermia combined with hepatic embolization using dextran magnetite incorporated microspheres in rats [J] .Int J Oncol, 2000,16 (6): 1153-1160).This research display DM-MS thermotherapy and the thromboembolism therapy that combines are a kind of antitumor feasibility therapies, have wide investigation and application prospect.To amycin magnetic microsphere hepatic artery embolism and drug targeting, the toxicity to antitumor therapy is studied (Goodwin SC to Goodwin etc., Bittner CA, Peterson CL, et al.Single-dose toxicity study of hepatic intra-arterial infusion of doxorubicin coupled to a novel magnetically targeted drug carrier [J] .Toxical, 2001,60 (1): 117-183).The Hepar Sus domestica cancer model result display amycin magnetic microsphere low dosage that they set up has no side effect.Only have when just there is good effect content >=75mg (contain or the do not contain amycin) target area of magnetic microsphere, the degree of necrosis of hepatoma carcinoma cell is directly proportional to thromboembolism degree, and amycin can not freely circulate at whole body and successfully be controlled in target area.The homemade poly-methyl methacrylate vinegar magnetic microsphere such as Hui Xuhui is inquired into Endovascular Embolization, experiment shows, the PMMA magnetic microsphere of 30-50um has that magnetic response ability is strong, magnetic control effect of embolization is good, in high Hemodynamic environment situation, still can realize the advantages such as target position thromboembolism, a kind of magnetic control Endovascular Embolization material (Hui Xuhui preferably, high Rieter, He Nengqian. polymethyl methacrylate magnetic microsphere Endovascular Embolization experimentation [J]. Sichuan medical science, 2001,22 (10): 928-929)).In magnetic control thromboembolism, the size of Magnetic Microspheres-Carrier affects the most important factor of Target localization.If particle diameter is less, then magnetic responsiveness is weak, and magnetic control degree is poor, can not be used for high Hemodynamic environment or compared with the endovascular magnetic control thromboembolism of Large Diameter Pipeline.Therefore, different with magnetic microsphere application in other respects, in magnetic control thromboembolism Jie human therapy, the magnetic microsphere that general employing particle diameter is larger.
Also have report to point out, Nano microsphere can be used as carrier protein and the adjuvant of DNA vaccination, but is applied to preventative polysaccharide and/or protide vaccine has no report.
Summary of the invention
For the problems referred to above that prior art exists, the object of this invention is to provide the rotavirus polysaccharide-protein combined vaccine that a kind of immunogenicity is stronger.
For achieving the above object, the technical solution used in the present invention is as follows:
Rotavirus polysaccharide-protein combined vaccine, it is multivalent pneumococcal polysaccharide and two or more the carrier protein conjugate vaccines through connector, and wherein, connector is magnetic Nano microsphere, and one of carrier protein is rotavirus protein.
As a kind of preferred version, the inside that the magnetic particle of described magnetic Nano microsphere is positioned at magnetic Nano microsphere is core, and macromolecular material is wrapped in the outside of magnetic particle.
As further preferred version, magnetic particle is Fe 3o 4.
As further preferred version, magnetic Nano microsphere particle diameter is 0.1-10 μm, is preferably 0.1-5 μm.
As another kind of preferred version, macromolecular material is bioabsorbable polymer material, is selected from one or more the mixture in chitosan, Polyethylene Glycol, Poly(D,L-lactide-co-glycolide (PLGA), PLA-PEG copolymer (PELA); The best is PLGA or PELA.
As another kind of preferred version, multivalent pneumococcal polysaccharide is multiple pneumococcal capsular polysaccharide, be preferably the capsular polysaccharide on separating-purifying Pneumococcal serotype pod membrane, the serotype of described Pneumococcal serotype comprise 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 version, the mass ratio of multivalent pneumococcal polysaccharide and two kinds of carrier proteins is (0.5 ~ 2): 1, and be preferably (0.5 ~ 1): 1, the mass ratio wherein between each carrier protein is preferably 1:1.
As further preferred version, described rotavirus protein is restructuring people takes turnsshape virus protein.
As the preferred preferred version of one, described carrier protein is selected from diphtheria toxoid, tetanus toxoid, carrier protein CRM197, bloodthirsty hemophilus influenza surface protein HiD, pertussis Prn surface protein, pertussis Fha antigen and/or Pneumococal surface protein A (rPspA).
As further preferred version, having a kind of in described carrier protein is bloodthirsty hemophilus influenza surface protein HiD or Pneumococal surface protein A (rPspA).
As further preferred version, restructuring people takes turnsshape virus protein is partial amino-acid series or the complete sequence of P genotype rotavirus protein, is preferably P genotype colyliform sick poison poisonone in strain P [8], P [4], P [6] or P [11]; Wherein, P genotype colyliform is sick poison poisonthe one of selecting good strains in the field for seed in P [8] G1, P [4] G2, P [8] G3, P [8] G4, P [8] G9, P [8] G5 or P [6] G8.
As further preferred version, the colyliform of P genotype P [8] is sick poison poisonthe one of selecting good strains in the field for seed in Wa, Ku, P, YO, MO, VA70, D, AU32, CH-32, CH-55, CHW2, CH927A, W161, F45, Ai-75, Hochi, Hosokawa, BR1054, WT78 or WI79 strain.
As further preferred version, the colyliform of P genotype P [4] is sick poison poisonthe one of selecting good strains in the field for seed in DS-1, RV-5, S2, L26, KUN, E210, CHW17, AU64,107E18, MW333 or TB-Chen strain.
As further preferred version, the colyliform of P genotype P [6] is sick poison poisonselect good strains in the field for seed from M37,1076, one in RV-3, ST3, SC2, BrB, McN13, US1205, MW023, US585 or AU19 strain.
As the further preferred version of another kind, restructuring people takes turnsshape virus protein is selected from the one in VP8, VP4, VP8 polypeptide chain fragment, core VP8, VP4 polypeptide chain fragment, VP8 specific antigen bunch peptide chain or VP4 specific antigen bunch peptide chain.
As the further preferred version of another kind, recombinant rotavirus albumen is partial amino-acid series or the complete sequence of the VP7 albumen of G serotype rotavirus.
As the further preferred version of another kind, recombinant rotavirus albumen is partial amino-acid series or the complete sequence of the VP7 albumen of G serotype rotavirus.
As further preferred version, G serotype colyliform is sick poison poisonthe one of selecting good strains in the field for seed in G1, G2, G3, G4, G9, G5, G8, G10 or G11 serotype.
As further preferred version, described G serotype colyliform is sick poison poisonthe one of selecting good strains in the field for seed in P [8] G1, P [4] G2, P [8] G3, P [8] G4, P [8] G9, P [8] G5 or P [6] G8 serotype.
Another object of the present invention is to provide the preparation method of this rotavirus polysaccharide-protein combined vaccine, is specifically formed through coupling by the carrier protein of multivalent pneumococcal polysaccharide and two kinds or more.
As a kind of preferred version, the preparation method of described conjugate vaccines, specifically comprises the following steps:
A) capsular polysaccharide on difference separating-purifying various serotype pneumococcal capsule;
B) carrier protein also selected by separating-purifying is prepared respectively;
C) respectively various capsular polysaccharide is become polysaccharide-magnetic Nano microsphere couplet with magnetic Nano microsphere coupling;
D) again polysaccharide-magnetic Nano microsphere couplet is combined with variety carrier albumen coupling respectively;
E) purification procedures d) gained couplet becomes described conjugate vaccines stock solution.
As further preferred version, the preparation method of described conjugate vaccines, specifically comprises the following steps:
A) capsular polysaccharide on difference separating-purifying various serotype pneumococcal capsule;
B) the variety carrier albumen respectively selected by separating-purifying;
C) respectively various capsular polysaccharide is become polysaccharide-magnetic Nano microsphere couplet with magnetic Nano microsphere coupling, then through chemical modification, thus-the OH that described polysaccharide-magnetic Nano microsphere couplet surface is not reacted with polysaccharide is modified as-CHO;
D) again polysaccharide-magnetic Nano microsphere couplet is combined with variety carrier albumen coupling respectively;
E) purification procedures d) gained couplet becomes described conjugate vaccines stock solution.
As further preferred version, described preparation method also comprises and prepares magnetic Nano microsphere, comprises the process first prepared magnetic nanoparticle and prepare magnetic Nano microsphere again; Wherein magnetic nanoparticle is prepared by chemical coprecipitation, hydro-thermal method or sol method pyrolysismethod, magnetic Nano microsphere, by the preparation of investment, monomer polymerization method or in-situ method, is preferably and adopts chemical coprecipitation to prepare magnetic nanoparticle again magnetic nanoparticle and macromolecular material to be prepared magnetic Nano microsphere through fast film emulsifying in conjunction with solvent extraction and/or investment or monomer polymerization method; Most preferably be the magnetic Nano microsphere adopting chemical coprecipitation to prepare magnetic nanoparticle again magnetic nanoparticle and macromolecular material to be prepared uniform particle diameter in conjunction with solvent extraction and/or investment through fast film emulsifying; Wherein magnetic particle is preferably Fe 3o 4.
As further preferred version, the preparation method of above-mentioned nano-magnetic ion can refer to Fe in prior art 3o 4the method preparation of magnetic Nano microsphere, specifically can see following detailed description of the invention, not as the whether attainable key factor of the present invention.
As further preferred version, in step a, the separation purifying technique of polysaccharide can operate according to prior art according to required polysaccharide and protein classes.
As further preferred version, the preparation of the albumen in step b and separation purifying technique can operate according to prior art according to required polysaccharide and protein classes.
As further preferred version, the concrete operations of step c are: in coupling medium reaction buffer, under room temperature, under pH4.0-9.0, step a gained polysaccharide and magnetic Nano microsphere coreaction are obtained polysaccharide-magnetic Nano microsphere couplet in 6-24 hour, by 25% glutaraldehyde, further modification is carried out to it again, make-the OH that polysaccharide-magnetic Nano microsphere couplet surface is not reacted with polysaccharide be modified as-CHO; Wherein coupling medium is preferably PB, PBS or TBS, and the best is 0.1M TBS solution; The Optimal pH of reaction buffer is 6; Optimum reacting time is 12-16 hour.
As further preferred version, the concrete operations of steps d are: in coupling medium reaction buffer, 4 DEG C, under pH4.0-9.0, by step c gained through the polysaccharide-magnetic Nano microsphere couplet of chemical modification and carrier protein coreaction 12-24 hour; Wherein coupling medium is preferably PB, PBS or TBS, and the best is 0.1M TBS solution; The Optimal pH of reaction buffer is 6; Optimum reacting time is 24 hours.
As further preferred version, the separation and purification of step e is by coupling conjugate and unreacted polysaccharide and carrier protein separation and purification, and purification process can by chromatography or ultrafiltration; Can carry out separation and purification according to the molecular size of the conjugate vaccines obtained, chromatography preferably adopts Superdex200 solvent resistant column, Sepharose CL-4B or Sepharose CL-6B to carry out; And ultrafiltration adopts different retention molecular weight film to come separating and combining thing and unreacted reactant.
Described conjugate vaccines preparation can adopt water preparation or lyophilized preparation.In order to strengthen its immunogenicity, can add adjuvant, conventional adjuvant has aluminium adjuvant, as aluminium hydroxide, aluminum phosphate, and prioritizing selection aluminum phosphate of the present invention.The solvent of conjugate can be 0.2 sodium chloride solution, 1 × PBS buffer or other can stablize the buffer of polysaccharide or conjugate.The preparation method of each preparation of conjugate vaccines of the present invention adopts the conventional means of the art to prepare.Wherein, preferably under sugar (such as sucrose or lactose) exists, lyophilizing is carried out.
Conjugate vaccines provided by the invention can carry out immunity with any existing approach, comprises skin corium or the form such as percutaneous drug delivery, intramuscular delivery.Wherein, the amount given is that those skilled in the art are confirmable according to general knowledge.
Term definition
Core VP8: be that in rotavirus protein VP8 one section has the polypeptide chain containing sialic acid (sialic acid) adhesive function with cell surface, usually containing 160 amino acid residues.
VP8 polypeptide chain fragment: for any molecular weight is lower than the polypeptide chain of total length VP8.
VP4 polypeptide chain fragment: for any molecular weight is lower than the polypeptide chain of total length VP4.
VP8 specific antigen bunch chain: full VP8 polypeptide chain contains multiple antigenic determinant, is sheared by the method for gene recombinaton and goes not containing important aminoacid, and retains the polypeptide chain containing specific antigen bunch, and molecular weight is less than total length VP8 polypeptide chain usually.
VP4 specific antigen bunch chain: full VP4 polypeptide chain contains multiple antigenic determinant, is sheared by the method for gene recombinaton and goes not containing important aminoacid, and retains the polypeptide chain containing specific antigen bunch, and molecular weight is less than total length VP4 polypeptide chain usually.
VP8 fusion rotein: with gene recombination method, by VP8 albumen and other soluble protein polypeptide chain amalgamation and expression, to improve VP8 solubility in aqueous; Or strengthen the immunogenicity of VP8.
NSP4 albumen: be non-structural protein, has enterotoxin characteristic, and molecular weight is 28kDa, containing 175 aminoacid.
VP8-NSP4 fusion rotein: by the method for gene recombinaton, by VP8 and NSP4 polypeptide chain amalgamation and expression, to improve VP8 solubility in aqueous, makes fusion rotein have the protection antibody stimulating body to produce anti-NSP4 simultaneously.
Capsular polysaccharide fragment: by physics (as ultrasound wave, particle spray), the chemistry method such as (as acid, alkali, enzymic digestion) by the polysaccharide (claiming full polysaccharide or original polysaccharide) degraded (depolymerization) by purification in inoculum the polysaccharide fragment that obtains, molecular weight is usually less than original polysaccharide.Pod membrane oligosaccharide: by physics (as ultrasound wave, particle spray), the chemistry method such as (as acid, alkali, enzymic digestion) by the polysaccharide (claiming full polysaccharide or original polysaccharide) degraded (depolymerization) by purification in inoculum the polysaccharide fragment that obtains, the monosaccharide residue in molecular structure is usually less than 10.But the definition of monosaccharide residue quantity is variant, and the polysaccharide chain being less than 20 monosaccharide residues more than 10 is also become oligosaccharide by some document.
Compared with prior art, the present invention has following advantage:
1. conjugate vaccines described in is the immunoconjugates containing two or more different carriers albumen, compared with existing pneumococcal conjugated vaccine, its immunogenicity is stronger, the polysaccharide antibody level of bringing out is higher than single carrier conjugates, immunne response can be caused, especially infant in more wide crowd; Carrier epi-position is avoided to transship by reducing each carrier dosage; T-helper cell activity is strengthened by two kinds of carriers;
2. due to there is in two kinds of carrier proteins protectiveness Protein Epitopes also can induction ratio two kinds of albumen hybrid injections time higher immunoreation, mutual synergism, enhances the immunogenicity of carrier protein further, adds the immunoreation of body to polysaccharide;
3. the employing magnetic Nano microsphere initiated of the present invention is as the connector of polysaccharide and multichip carrier albumen, effectively avoids the autoimmunity syndrome in preparation process between capsular polysaccharide and protein, can improve the yield in conjunction with product, and be beneficial to the quality control of product; Effectively can extend the space length between capsular polysaccharide and carrier protein, reduce the spatial masking effect of carrier protein to capsular polysaccharide antigen epi-position, be conducive to the immunogenicity improving capsular polysaccharide;
4. pioneering in the preparation process of conjugate vaccines described in the-OH on magnetic Nano microsphere surface is first carried out coupling reaction with polysaccharide, then by couplet through chemical modification unreacted-OH is modified as-CHO with-the NH in carrier protein 2carry out coupling combination, associated methods more of the prior art is more stable;
5. its preparation method is simple, is applicable to the needs that large-scale industrial is produced, and does not significantly change the architectural feature of capsular polysaccharide and carrier protein.
To sum up state, conjugate vaccines preparation technology provided by the invention is simple, employing magnetic Nano microsphere is that the conjugate vaccines of junctional complex can strengthen mice Th1 type immunne response, and the immune lasting effect of polysaccharide specificity antibody, specificity and affinity, rotavirus antibody can be produced by inducing mouse in addition; Possesses the preventive effect of two kinds of vaccines; Therefore there is very wide application prospect.
Accompanying drawing explanation
fig. 1conjugate vaccines obtained by the embodiment of the present invention 1 1h-NMR composes figure;
fig. 2for the immunne response experimental result of the polysaccharide specificity antibody of conjugate vaccines provided by the invention is illustrated figure;
fig. 3for the immune lasting effect experimental result signal of the polysaccharide specificity antibody of conjugate vaccines provided by the invention figure;
fig. 4for the rotavirus neutralization test result of conjugate vaccines provided by the invention is illustrated figure.
Detailed description of the invention
Do to illustrate in detail, intactly further to the present invention below in conjunction with embodiment.Following agents useful for same or equipment are commercially available kind, if no special instructions, operate all to specifications, do not repeat at this.
Be below that the present invention is further illustrated in conjunction with specific embodiments, but should not be considered as limitation of the invention.
Embodiment 1
One, magnetic Nano microsphere is prepared
1. get 2.24gFeSO 4-7H 2o and 3.24gFeCl 3-6H 2o is dissolved in the dddH that 10mL and 15mL filters deoxygenation respectively 2in O, after dissolving, mixing all hooks, and adds the dddH that 100mL filters deoxygenation 2o;
2. at N 2protection under stir 5min, disposablely add 50mL1mol/LNaOH solution, then regulate pH value of solution to 9-10, accelerate mixing speed to 200-250r/min, continuous stirring 30min;
3. reaction vessel is transferred in 65-70 DEG C of water-bath, continues at N 2protection under stir ageing 30min;
4. reaction is complete is settled to 100mL, basis of microscopic observation magnetic particle synthesis situation;
5. 400mgPLGA to be dissolved in 10mLEA solvent as oil phase (O), to add 3mL above-mentioned magnetic particle solution as interior aqueous phase (W 1), adopt ultrasonic cell disintegration instrument (120W, 60s) in ice-water bath, carry out just emulsifying and prepare colostrum, then colostrum is poured into a certain amount of aqueous solution (outer aqueous phase, W containing 15g/LPVA and 0.9% (ω) NaCl 2), magnetic agitation (300r/min, 2min) prepares pre-double emulsion (W 1/ 0/W 2), more pre-emulsion is poured in the storage tank of fast film emulsifying, with certain N 2it is pressed through SPG film by pressure repeatedly, obtains the Nano microsphere emulsion drop of uniform particle diameter.In addition, unspent Nano microsphere can be made into lyophilized preparation and continues to employ.
Or by getting Fe 3o 4magnetic particle adds by equal-volume with 50mL and dehydrated alcohol, after ultrasonic activation 30min, puts in 60 DEG C of water-baths, drips 10mLPELA slowly and carry out-NH to magnetic Nano microsphere 2terminal-modified and PELA is wrapped in Fe 3o 4outside magnetic particle, stirring reaction l0h, makes magnetic Nano microsphere under nitrogen protection; After completion of the reaction, wash paint 3 times with 50mL dehydrated alcohol, then after washing paint three times with 0.01MPBS, be settled to 50mL, basis of microscopic observation magnetic bead modification situation, to magnetic Nano microsphere surface-NH 2end changes-OH end into.
Two, the preparation of pneumococal polysaccharide
1. choose 24 kinds of serotypes (1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20,22F, 23F, 33F) streptococcus pneumoniae cultivate;
2. the capsular polysaccharide that more than purifying respectively, in various Pneumococcal serotype, antigenicity is strong: streptococcus pneumoniae, collected by centrifugation supernatant after deactivation, through ultrafiltration and concentration, (volume fraction be 70%) pre-cooled ethanol is added in right amount respectively according to each Pneumococcus serotypes characteristic, collected by centrifugation, obtains rough polysaccharide; Rough polysaccharide is dissolved in sodium acetate solution, then mixes with cold phenol in 1:2 ratio, centrifugal segregation albumen, phenol is carried 5-6 time repeatedly, collects supernatant, with distill water dialysis, after dialysis, liquid adds 2mol/L calcium chloride solution, adds ethanol and stirs, centrifugal segregation nucleic acid, collect supernatant, add ethanol (final concentration 80% stirs), centrifugal collecting precipitation, by ethanol, washing with acetone precipitation, be multivalence after dehydrate and refine capsular polysaccharide, put-20 DEG C and save backup.
Three, the preparation of rotavirus carrier protein
The preparation of rotavirus carrier protein can see the preparation method of multiple recombiant protein of the prior art, and the present embodiment adopts the method preparation mentioned in CN 101972475, and can be reduced to following steps, design parameter does not repeat:
1, the cDNA storehouse of rotavirus is set up
The VP4 gene for Wa strain VP8 albumen selected, amplimer design is as follows: sense primer HWaVP4 ρ ET28:
5 '-TTACATATGGCTTCGCTCATTTATAG-3 ', anti-sense primer AHWaVP4 ρ ET28:
5’-CCGGATCCCTAGTCTTCATTAACTTGTGCT-3’。
2, build pET28aWaVP8 and express total length VP8 plasmid
3, restructuring VP8 albumen is expressed
1) obtained pET28aWaVP8 Plastid transformation is entered in BL21 (DE3) competent cell, inoculating cell to 50 μ g/mL kanamycin LB culture dishs, in CO at 37 DEG C 2spend the night in incubator.
2) pick out a bacterium colony, be inoculated into 10 milliliters containing amplification in the kanamycin LB culture fluid (1% tryptone, 0.5% yeast extract, 1%NaCl, pH7.5) of 50 μ g/mL, 37 DEG C of overnight incubation.
3) culture fluid is turned the 50 μ g/mL kanamycin LB culture fluid being inoculated into 100 milliliters, continue to cultivate.When the OD of absorbance 600nm arrives 1.0, culture fluid is turned and is inoculated into 6 and is raised in 50 μ g/mL kanamycin LB culture fluid, continue at 37 DEG C, shake the interior cultivation of shaking table of fast 200rpm, when the OD of absorbance 600nm arrives 0.6 ~ 0.8, the IPTG (isopropyl-β-D-thiogalactoside) adding 0.3mM induces VP8 to express.
4) under identical condition of culture, induce after 4 hours, with under 4000g, at 10 DEG C after centrifugal 20 minutes, collect thalline.
5) by the 1 × PBS solution of bacterial suspension in 20mL, after French filter pressing kettle (Frenchpress) disrupt bacteria, under 10000g, centrifugal 30 minutes at 10 DEG C, abandon supernatant, the inclusion body of collecting precipitation.Before being further purified, store inclusion body in-40 DEG C.
3, purification VP8 albumen from inclusion body
1) the VP8 inclusion body 0.5 gram (weight in wet base) of preparation is taken, with cleaning buffer solution (10mMTris, 100mM phosphate buffer, 2Murea, pH8.0) suspension inclusion body, at room temperature hatch 30 minutes, with 10,000g centrifugal 10 minutes, collect inclusion body.Repeat above step 3 secondary to the foreign protein depolluted.
2) inclusion body precipitation is dissolved in dissolving buffer (10mMTris-HCl, 100mM phosphate buffer, 8M carbamide, pH8.0), stirs on ice and hatch 1 hour.With 16,000g centrifugal 30 minutes, collect supernatant, abandon insoluble precipitate.
3) with fixing metal ions affinity chromatograph chromatography (IMAC) purification His-tagged restructuring VP8 albumen.
4) collect containing the eluent of VP8, proceed in bag filter and dialyse in the TBS (pH4.0) containing 20mM beta-mercaptoethanol 1 and 8M carbamide, and reduce the concentration (namely 8 of carbamide gradually, 6,4,2, and 1M), dialysed overnight in 4 DEG C.Then in the TBSpH5.5 solution containing 2mM beta-mercaptoethanol, dialyse twice, finally dialyse in TBS solution.Different according to the strain of restructuring VP8 dietary protein origin, come finally to dialyse.
Four, the preparation of Pneumococal surface protein A (rPspA)
PspA albumen is cloned into escherichia coli and carries out expression and separation and purification, specifically comprise:
1. the optimization of genes of interest and the structure of recombinant expression plasmid
In GenBank, obtain PspA gene order (GI:193804931) and be optimized, full genome synthesis is carried out after adding His label, by the sequence of synthesis after Sac I and Nde I double digestion, directed cloning is in the expression vector pET-30a (+) of same double digestion, transform competent E. coli BL21Star (DE3), 37 DEG C of incubated overnight, picking positive monoclonal bacterium colony, plasmid is extracted after amplification cultivation, with Nde I and the qualification of Sac I double digestion, and send order-checking, by recombinant expression plasmid called after pET-30a-rPspA correct for order-checking.
2. the abduction delivering of recombiant protein and purification
Recovery engineering bacteria, with the ratio of 1:100 inoculation 2 × LB culture medium, at 37 DEG C, amplification culture under 237r/min, when thalline value is about 12, adding IPTG to final concentration is 1mmol/L, 37 DEG C of induction 4h, sampling is carried out SDS-PAGE analysis centrifugal and is collected induction thalline, add the resuspended washing of normal saline 2 times, the resuspended thalline of buffer of 05mol/LNaCl5mmol/L imidazoles 20mmol/LPB (pH7.4) is added with the ratio of 1:10 (g/mL), ultrasonic disruption thalline, the centrifugal 40min of 8000 × g, collect supernatant, purification is carried out in nickel ion chromatographic column, by specification operation purified product and analyze e. coli bl21 Star (DE3) whole cell (contrast) that carrier pET-30a (+) is transformed with by upper step purification of samples after SDS-PAGE is separated electrotransfer on nitrocellulose filter, 2h is closed with 5% defatted milk powder shaking table slight oscillatory, add His Mus source monoclonal antibody (1: 800 dilution), 4 DEG C are spent the night, TBST cleans 3 times, adds the sheep anti-mouse igg (1: 2000 dilution) of HRP labelling, incubated at room 1h, wash 3 times, DAB develops the color.
Five, the preparation of rotavirus polysaccharide-protein combined vaccine
1. polysaccharide-magnetic Nano microsphere coupling
Under 0.1MTBS solution buffer, pH6.0 adds above-mentioned steps obtained times one or more capsular polysaccharide and magnetic Nano microsphere, adopt in the present embodiment 13 valency capsular polysaccharides (1,3,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F), the mass ratio of capsular polysaccharide and magnetic Nano microsphere is 1:(0.5-1), under room temperature, react 6-24 hour; The mass ratio wherein optimized is 1:1, aldolisation 16 hours under room temperature.After reaction terminates, use bag filter enough hemodialysis, removing unreacted magnetic Nano microsphere.
2. couplet modification
Get step 1 polysaccharide-magnetic Nano microsphere couplet 30mL, slowly drip 2mL25% 1,3-propanedicarboxylic acid under stirring condition, 200r/min stirring reaction 6 hours; After completion of the reaction, after washing three times with 0.01MPBS, be settled to 30mL, basis of microscopic observation magnetic Nano microsphere modification situation, make-the OH that polysaccharide-magnetic Nano microsphere couplet surface is not reacted with polysaccharide be modified as-CHO; N 2, 4 DEG C save backup.
3. with PspA carrier protein and rotavirus protein coupling altogether
Under 0.1MTBS solution buffer, 4 DEG C, under pH4.0-9.0, by modified polysaccharide-magnetic Nano microsphere respectively with rotavirus protein and PspA albumen coreaction 24 hours (for ensure enough carrier proteins and magnetic Nano microsphere, adopt excessive nano-carrier albumen addition, as mass ratio adopts polysaccharide: magnetic Nano microsphere: PspA: rotavirus protein=1:1:2:2).
4. the separation and purification of rotavirus polysaccharide-protein combined vaccine
Carry out separation and purification with Superdex200 solvent resistant column (2.6cm × 60cm), eluent is the phosphate buffer (pH7.4) of 20mM, and flow velocity is 3mL/min.Collect the eluting peak corresponding to polysaccharide-magnetic Nano microsphere-complex carries albumen.
The composition analysis of obtained rotavirus polysaccharide-protein combined vaccine
With 1h-NMR carries out detecting described rotavirus polysaccharide-protein combined vaccine, and testing result is shown in fig. 1. as Fig. 1shown in, compared with capsular polysaccharide molecule, there is characteristic peak at 0.4-1.4ppm place in conjugate, corresponding to the aliphatic chain amino acid residue of carrier protein.The characteristic peak corresponding to the aromatic amino acid residue of carrier protein has been there is at 7.2ppm place.This shows capsular polysaccharide molecule successfully coupling rotavirus protein and PspA carrier protein two kinds of carrier proteins.Occurred the characteristic peak corresponding to butanimide at 6.2ppm place, this shows in the cross structure of polysaccharide conjugate vaccine containing butanimide.In addition, the characteristic peak of 5.2,1.6,3.6 outlets PELA proves that magnetic Nano microsphere PELA is as junctional complex.Therefore, capsular polysaccharide is in conjunction with before and after two kinds of carrier proteins, and obvious change does not occur its structure.
The molecular weight distribution situation of GL-PP conjugate is detected by CL-4B and SEC-MALLS method; Albumen in different antibody serum determination GL-PP conjugates and polysaccharide kind is used by the two method expanded of immunity; The polyoses content of GL-PP conjugate is detected by anthrone method; Lowry method protein content detects the total protein content of GL-PP conjugate, then is combined than (Ratio) by the GL-PP calculating conjugate; Rotavirus protein, PspA protein concentration are detected by euzymelinked immunosorbent assay (ELISA).Result shows: in described rotavirus polysaccharide-protein combined vaccinogen liquid, the concentration ratio of each material is about: polysaccharide: magnetic Nano microsphere: PspA: rotavirus protein=1:1:1:1).
Comparative example 1
This comparative example is only with the difference of embodiment 1: in obtained vaccine, nonmagnetic nanoparticle is as connector, and by the method mentioned in prior art, complex carries albumen and polysaccharide is puted together and obtained.
Comparative example 2
This comparative example is only with the difference of embodiment 1: without PspA carrier protein in obtained vaccine, only adopt polysaccharide-magnetic nanometer particles-rotavirus carrier protein to be rotavirus polysaccharide-protein combined vaccine.
Embodiment 2
The difference of the present embodiment and embodiment 1 is only: the carrier protein of described rotavirus polysaccharide-protein combined vaccine is rotavirus carrier protein and tetanus toxoid carrier albumen.
There is the concordance within the scope of theoretical error in the composition analysis result of obtained rotavirus polysaccharide-protein combined vaccine and embodiment 1 acquired results.
Embodiment 3
The difference of the present embodiment and embodiment 1 is only: the carrier protein of described rotavirus polysaccharide-protein combined vaccine is rotavirus carrier protein and bloodthirsty hemophilus influenza surface protein HiD.
There is the concordance within the scope of theoretical error in the composition analysis result of obtained rotavirus polysaccharide-protein combined vaccine and embodiment 1 acquired results.
Embodiment 4
The difference of the present embodiment and embodiment 1 is only: connector is PLGA magnetic nanoparticle.
There is the concordance within the scope of theoretical error in the composition analysis result of obtained rotavirus polysaccharide-protein combined vaccine and embodiment 1 acquired results.
Embodiment 5
The difference of the present embodiment and embodiment 1 is only: connector is PEG magnetic nanoparticle.
There is the concordance within the scope of theoretical error in the composition analysis result of obtained rotavirus polysaccharide-protein combined vaccine and embodiment 1 acquired results.
Vaccine evaluation
The assessment experimental evaluation embodiment 1 ~ 7 below adopting the vaccine routines such as immunogenicity to tire and the immune effect of comparative example 1 ~ 2 obtained vaccine:
A. rotavirus polysaccharide-protein combined vaccine immunogenicity experiments
Choose the female Blab/C mice in 100 5 week ages, body weight is 15-22 gram.Be divided into 10 groups at random, i.e. embodiment 1 ~ 7, comparative example 1 ~ 2 and positive controls (Prevnar 13), often organize 10 mices.Lumbar injection, every per injection is 5 micrograms, injects 1 time weekly, injects 3 times altogether.Within 21 days, posterior orbit gets blood.IgG, IgG1 and the IgG2a of anti-capsular polysaccharide in mice plasma is detected by ELISA method.Experimental result is shown in table 1
table 1
as table 1shown in, in embodiment 1 ~ 7, the rotavirus polysaccharide-protein combined vaccine of gained significantly can promote antibody titer (p<0.0001**), as Fig. 2shown in, and be obviously better than comparative example 1 and comparative example 2 and positive controls; As can be seen here, adopt complex carries albumen the rotavirus polysaccharide-protein combined vaccine obtained with magnetic Nano microsphere and pneumonia polysaccharide significantly can strengthen Th1 type immunne response, and immune effect is obviously better than the single carrier protein without nano-magnetic microsphere connector and comparative example 2 of comparative example 1 and the rotavirus polysaccharide-protein combined vaccine that obtains; But also known according to the immunne response result of embodiment 1 ~ 5 gained, be that the immunne response effect of one of complex carries albumen is better by PspA or HiD, and in magnetic nano-carrier, PELA and PLGA is effective compared with PEG immunne response.
B. the immune lasting effect of polysaccharide specificity antibody, specificity and affinity experiment
Once test with embodiment 1, comparative example 1 and 2 and positive control gained rotavirus polysaccharide-protein combined vaccine respectively.
1, immune lasting effect
By measuring the titre of polysaccharide specificity antibody in 20 weeks after three pin immunity, to study the immune lasting effect of polysaccharide specificity antibody. fig. 3for described immune lasting effect signal figure, as Fig. 3shown in, its polysaccharide specificity IgG titre is lower, and increase along with inject time and reduce gradually, the 4th Zhou Houyi cannot detect.The polysaccharide specificity IgG titre that comparative example 1 and 2 produces will lower than embodiment 1 group, and higher than positive controls.Polysaccharide specificity IgG titre, at the 2nd Zhou Dafeng, declines gradually within 4-20 week, and wherein the decrease speed of positive controls is the fastest.After 18th week, the polysaccharide specificity IgG titre of embodiment 1 group, comparative example 1, comparative example 2 and positive controls is 20%, 15%, 12% and 10% of its peak value respectively.Therefore, rotavirus polysaccharide-protein combined vaccine provided by the invention can strengthen the immune lasting effect of polysaccharide specificity antibody.
2, specificity and affinity
In 200 times of the PS-TT groups of diluting, PS-PLGA-TT group and PS-PELA-TT group mice plasma, add not commensurability capsular polysaccharide, detect the antibody horizontal of anti-capsular polysaccharide in mice plasma by ELISA method, the results are shown in table 2.
table 2
as table 2shown in, along with the increase of polysaccharide addition, in polysaccharide specificity antibodies 96 orifice plate, the ability of polysaccharide reduces gradually.When the polysaccharide added reaches 20 μ g, the Disability of antibodies polysaccharide.This shows that the anti-capsular polysaccharide antibody produced by rotavirus polysaccharide-protein combined vaccine inducing mouse provided by the invention can specifically in conjunction with capsular polysaccharide.
The Antibody Avidity of anti-capsular polysaccharide is measured with ammonium thiocyanate.The polysaccharide specificity antibody index of affinity of capsular polysaccharide group (negative control) is 1.18mol/L, and the antibody index of affinity of positive controls, comparative example 1 group, comparative example 2 groups of groups and embodiment 1 group is respectively 2.65mol/L, 2.80mol/L, 3.02mol/L and 3.21mol/L.This shows that rotavirus polysaccharide-protein combined vaccine provided by the invention can significantly improve the affinity of polysaccharide specificity antibody.
C, rotavirus neutralization test result
Adopt each mice serum of each group of the mice serum (embodiment 1, comparative example 1 group and comparative example 2 groups inject a pin, two pins and three pins respectively) that method obtains respectively in embodiment 1 respectively to get 10 μ L to mix, for neutralization test Sample serum.
The neutralization test of the anti-WaVP8 antibody that injection white mice produces above carries out with BSC-1 cell at microplate (microplate).After heat inactivated serum is carried out serial dilution, sick with the Wa colyliform of 100TCID50 poison poisonafter strain mixing, cultivate 1 hour at 4 DEG C.Then BSC-1 cell is inoculated on microplate, hatches 1 hour.Add DMEM (not containing serum) and join each hole, hatch 1 hour for 37 DEG C.Finally, dilute the antiserum concentration that can prevent rotavirus cytopathic effect (CPE) from occurring be in and titre.Polysaccharide matched group serum is used as negative control.Experimental result is shown in table 3with fig. 4.
table 3: with Rotavirus Wa strain strain result of the test in rotavirus polysaccharide-protein combined vaccine injection white mice antibody
as table 3shown in, be better than comparative example 1 and comparative example 2 with rotavirus successful in rotavirus polysaccharide-protein combined vaccine, can significantly induce rotavirus antibody to produce.
Finally be necessary described herein: above embodiment is only for being described in more detail technical scheme of the present invention; can not be interpreted as limiting the scope of the invention, some nonessential improvement that those skilled in the art's foregoing according to the present invention is made and adjustment all belong to protection scope of the present invention.

Claims (10)

1. rotavirus polysaccharide-protein combined vaccine, it is characterized in that: described rotavirus polysaccharide-protein combined vaccine is multivalent pneumococcal polysaccharide and two or more the carrier protein rotavirus polysaccharide-protein combined vaccine through connector, wherein, connector is magnetic Nano microsphere, and the one in two or more carrier protein is rotavirus protein.
2. rotavirus polysaccharide-protein combined vaccine according to claim 1, is characterized in that: the inside that the micro-magnetic particle of described magnetic Nano is positioned at Nano microsphere is core, and macromolecular material is wrapped in the outside of magnetic particle.
3. rotavirus polysaccharide-protein combined vaccine according to claim 2, it is characterized in that: macromolecular material is bioabsorbable polymer material, be selected from one or more the mixture in chitosan, Polyethylene Glycol, Poly(D,L-lactide-co-glycolide (PLGA), PLA-PEG copolymer (PELA).
4. rotavirus polysaccharide-protein combined vaccine according to claim 1, is characterized in that: the mass ratio of multivalent pneumococcal polysaccharide and two kinds of carrier proteins is (0.5 ~ 2): 1.
5. rotavirus polysaccharide-protein combined vaccine according to claim 1, it is characterized in that: another carrier protein in two or more carrier protein is selected from diphtheria toxoid, tetanus toxoid, carrier protein CRM197, bloodthirsty hemophilus influenza surface protein HiD, pertussis Prn surface protein, pertussis Fha antigen and/or Pneumococal surface protein A (rPspA).
6. rotavirus polysaccharide-protein combined vaccine according to claim 1, is characterized in that: rotavirus protein is restructuring Human reoviruslike agent albumen.
7. rotavirus polysaccharide-protein combined vaccine according to claim 1, is characterized in that: recombined human rotavirus protein is partial amino-acid series or the complete sequence of P genotype rotavirus protein.
8. rotavirus polysaccharide-protein combined vaccine according to claim 1, is characterized in that: the Strain of recombined human rotavirus protein is the one in P genotype rotavirus strain P [8], P [4], P [6] or P [11].
9. the preparation method of the arbitrary described rotavirus polysaccharide-protein combined vaccine of claim 1 ~ 8, is characterized in that: formed through coupling with Nano microsphere respectively by the carrier protein of multivalent pneumococcal polysaccharide and two kinds or more.
10. the preparation method of rotavirus polysaccharide-protein combined vaccine according to claim 9, is characterized in that, specifically comprise the following steps:
A) capsular polysaccharide on difference separating-purifying various serotype pneumococcal capsule;
B) the rotavirus carrier protein also selected by separating-purifying and another carrier protein is prepared respectively;
C) respectively various capsular polysaccharide is become polysaccharide-Nano microsphere couplet with Nano microsphere coupling;
D) again polysaccharide-Nano microsphere couplet is combined with rotavirus carrier protein and another carrier protein couplet respectively;
E) purification procedures d) gained couplet becomes this rotavirus polysaccharide-protein combined vaccinogen liquid.
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