CN107137703B - A kind of rabies immune originality conjugate and vaccine and preparation method thereof - Google Patents
A kind of rabies immune originality conjugate and vaccine and preparation method thereof Download PDFInfo
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- CN107137703B CN107137703B CN201710381237.2A CN201710381237A CN107137703B CN 107137703 B CN107137703 B CN 107137703B CN 201710381237 A CN201710381237 A CN 201710381237A CN 107137703 B CN107137703 B CN 107137703B
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- rabies
- carrier protein
- rabies virus
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Classifications
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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
- A61K39/12—Viral antigens
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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
- A61K39/385—Haptens or antigens, bound to carriers
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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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5258—Virus-like particles
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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
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
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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
- 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]
-
- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/20011—Rhabdoviridae
- C12N2760/20111—Lyssavirus, e.g. rabies virus
- C12N2760/20134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Virology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to a kind of new rabies immune originality conjugates（Rabies Immunogenic Conjugate RIC）And preparation method thereof, which includes Rabies Virus Antigen（RabAg）And carrier protein（Carrier Protein CP）The Rabies Virus Antigen is keyed with carrier protein through chemistry, the carrier protein is keyed with Rabies Virus Antigen through chemistry, and the chemical bond connects Rabies Virus Antigen to form Rabies Virus Antigen carrier protein immunogenic conjugates with carrier protein.The invention further relates to a kind of new rabies combined vaccines prepared according to above-mentioned rabies immune originality conjugate（Rabies Conjugate Vaccine RCV）And preparation method thereof.Preliminary proof, the present invention overcomes the defects of the prior art, have preferable effect.
Description
Technical field
The present invention relates to medical fields, are specifically related to the preparation of vaccine and vaccine, and especially a kind of rabies immune is former
Property conjugate and preparation method thereof and a kind of rabies combined vaccine and preparation method thereof.
Background technology
Rabies (Rabies) are the acute infectious diseases caused by hydrophobin (Rabies Virus) infects, and are a kind of
The impatient sexually transmitted disease of infecting both domestic animals and human of Natur al foca.Hydrophobin is mainly in direct contact brokenly by being infected the saliva of animal
The skin or mucous membrane of damage infect people.For rabies once falling ill, case fatality rate is almost 100%.Dog is that hydrophobin is main
Host, up to 99% human rabies are infected through dog.Bat is the main source of America human rabies death, is occurred recently
Bat rabies in Australia and West Europe threaten as publilc health.There is rabies hair in all continents in addition to the Antarctic Continent
Raw, the WHO estimations whole world is every year because the number of rabies death is up to 59,000, wherein being more than 95% human rabies case hair
Life is in Asia and Africa (http://www.who.int/mediacentre/factsheets/fs099/en/).The Chinese human world
Rabies are fallen ill at epidemic situation peak in 2007, and annual report case load is up to 3300,2004-2014, rabies death toll
It is in first 3 (Chinese Center for Disease Control and Prevention rabies prophylaxis control technique guidelines of China's Death of Infectious Diseases number always
.2016)。
Hydrophobin belongs to Rhabdoviridae (Rhabdovividae) lyssavirus (Lyssaviruses).It is mad
Dog disease Tobamovirus include 12 kinds, 4 serotypes, 7 genotype, hydrophobin is to cause human infection's rabies main
Kind, belong to I type serotype, I type genotype.The typical form of hydrophobin particle is the slightly concave shape of one end flat, the other end
It is semi-circular, exactly like the therefore named rhabdovirus of bullet (Rhabdoyivus).Viral size is 75~80 × 170~180nm, viral base
Because group is sub-thread strand RNA, 11162 nucleotide encode 5 kinds of structural proteins, respectively nucleoprotein (Nucleoprotein,
N), phosphoprotein (Phosphoprotein, P), stromatin (Matrixprotein, M), glycoprotein (Glycoprotein, G)
With the RNA polymerases (RNA dependent RNA polymerase or Large protein, L) of dependenc RNA.Rabies
Viral genetic is relatively stablized, and the rabies viruses detached from world wide can be neutralized by monospecific antiserum.
It there is no whether detection means diagnosis has infected hydrophobin before clinical symptoms appearance at present.So far
Rabies are still the disease that the mankind can not cure, because there is no medicine at present, once its case fatality rate of falling ill is almost
100%.It is only capable of extending the course of disease using the special rabies immune globulin of antiviral drug, interferon and large dosage, still not
It is avoided that death.Unlike many other infectious diseases, rabies be by the preventable disease of vaccine inoculation, and
Even if timely vaccine inoculation and being used in combination with rabies immune globulin can effectively prevent dead after being exposed to hydrophobin
Die (Rabies vaccines:WHO position paper 2010).
Rabic incubation period is typically several weeks to the several months, can also be as short as a couple of days and grow to several years (ACIP.Use of a
Reduced(4-Dose)Vaccine Schedule for Postexposure Prophylaxis to Prevent Human
Rabies.2010).The incubation period of few new report 249 cases of Guilin City's rabies of Huang is most only 1 day, up to 3650 days short
(the few new occupations of Huang and health .2013,29 (17):2123-2126).Liu Fuqiang etc. reports 1059 cases of Hunan Province's rabies
Incubation period it is most short be 2 days, up to 4636 days (such as Liu Fuqiang China Natural medicine magazine .2008,10 (4):263-268).
Cao Minghua etc. reports that the incubation period of 416 cases of Anhui Province's rabies is most only 2 days short, up to 5606 days (the peaces such as Cao Minghua
Emblem preventive medicine magazine .2013,19 (1):1-4).Yu Chun etc. reports that the incubation period of 2065 cases of Guizhou Province's rabies is most short only
It it is 2 days, (the medical faunaes such as Yu Chun prevent .2016 (2) to longest within 13 years:145-147).
Rabies vacciness be largely for exposure after immune (Post Exposure Prophylaxis PEP), lead to
Passive immunity or active immunity effect are crossed in hydrophobin intrusion central nervous system (Central Nervous System
CNS it is stopped before), to play effect (Wilde H.Vaccine.2007,25 (44) of PEP:7605-9).Since there are mad
Dog disease incubation period is only the case of a couple of days, and vaccine is needed to induce immune response rapidly in human body, generates early immune and protects
Shield, and lasting immune protection effectiveness is provided.However, for incubation period shorter case, hydrophobin can invade rapidly god
Through system, PEP measures are unable to blocking virus duplication, cannot prevent hydrophobin from infecting (Terryn S, et to brain
al.PLOS Neglected Tropical Diseases.2016,8,2:1-15)。
Since Louis Pasteur invention Antirabic Vaccine in 1885, rabies vacciness experienced the animal god of early stage
Through tissue vaccine, avian embryo vaccine, cell culture crude vaccine, develop to current through primary hamster kidney cell, primary chicken embryo
Cell, primary duck embryo cells, African green monkey kidney cell (Vero cells), rhesus macaque fetus diploid cell, human diploid cell
The purified vaccine of culture.No matter early stage crude vaccine or present purified vaccine, rabies vacciness antigen is equal since 130 years
It is the whole virus particles (Virus Particle) of inactivation.The whole virus particles boosting vaccine body of inactivation generates hydrophobin
Specific C D4+T cells, B cell and antibody (Faber M et al.Proc Natl Acad Sci USA.2009,106
(27):11300-11305).The rabies whole virus particles antigen of these inactivations is mainly offered by MHC class Ⅱmolecules in vivo,
Induce CD4+T cells, generate antibody, belong to based on humoral immunity immune response mechanism (Li Mao light Vaccinum Encephalitis Bs and
The cellular immunity research Master degree candidates academic dissertation .2008 of rabies vacciness).The rabies inactivated in Mice Body are entirely sick
Malicious particle vaccines are to induce Th2 to generate the humoral immunity of IgG1 antibody as main immune response (Ertl H.Plos Neglected
Tropical Diseases,2009,3(9):1-9).The U.S. is immunized rabies Working Committee of Advisory Board and analyzes 1976-
The whole virus particles vaccine of the vaccine clinical research that 12 are delivered between 2008, existing rabies inactivation all generates in human body
Time of neutralizing antibody needs 14 days or more (Rupprecht CE, et al.Vaccine.2009.27:7141-8).It learns in China
The 7th day neutralizing antibody GMT is only 0.24-0.27IU/ml, Conversion rate after person reports the inoculation of Vero cell rabies head needles
12.2-28.9%, the 14th day neutralizing antibody GMT reach 2.52-7.29IU/ml, (the Chinese beast such as Xiao Qiyou of Conversion rate 100%
Illness journal .2009,25 (5) altogether:493-494；The disease surveillance .2009,24 such as Yang Shuhong (6):409-411).
In the case of no immunoglobulin joint injection, it is that rabies cannot be prevented dead only to depend on the PEP of vaccine
(Servat A,et al.Vaccine.2003.22(2):244-9).Pasteur Institut, Wuhan Biological Products Inst.
Experiment is first subcutaneous after independently being exposed three times using small white mouse and Beagle dogs with Military Veterinary Institute, Academy of Military Medical Sciences
Or intramuscular infection rabies street strain virus, rabies vaccine for immunization is then used again, and it is anti-to be as a result shown in no use in conjunction
Under serum profiles, commercially available Antirabic Vaccine's protective rate is only 0~30% (the auspicious equal China experiment of vast stretch of wooded country and clinic both at home and abroad
Journal of Virology .2016.30 (1):37-40).People is less able to pass through after infecting hydrophobin if do not received to be immunized after exposing
It induces the immune system of itself to generate protective effect and removes virus.The whole virus particles vaccine of inactivation is not due to can induce inflammation
Reaction is to cannot effectively activate Th1 lymphocytes and bone-marrow-derived lymphocyte response, poor (the Marciani DJ.Drug of immunogenicity
DiscovToday.2003.8(20):934-943).Researches show that the rabies whole virus particles of inoculation inactivation are vaccine-induced
The hydrophobin that humoral immunity or the passive immunity preparation of injection can only remove periphery prevents it from invading nerve endings, carefully
The effect that born of the same parents are immunized is limited, and protection (Hooper DC, et can not be provided if cell entry CNS
al.J.Virol.1998.72(5):3711-3719；Lafon M.Human rabies vaccines.2nd ed.San
Diego:Academic Press.2007.p489-504).This is because neutralizing antibody passes through blood-brain barrier (Blood Brain
Barrier BBB) very limited (the Hooper DC et al.Future Virol.2011.6 (3) of ability:387-397).
It can be seen that the whole virus particles vaccine of existing inactivation based on its do not generate cellular immunity or cellular immunity it is faint with
And the slow-footed critical defect of humoral immunity is generated, antibody generation obviously lags behind rabies viruses and breeds and invade in local muscle
The speed of nerve ending and time, especially for serious, the position highly dangerous that bites, incubation period, short resurrectionist cannot generate
Effect protection (Wang Shu sound application preventive medicine .2010.16 (1):1-4).
In view of the foregoing, it needs the rabies whole virus particles vaccine progress to existing inactivation to be transformed revolutionaryly, makes
Novel vaccine faster generates immune response, the higher immune level of induction in human body, provides more longlasting immunoprotection effect
Power could meet people's antirabic urgent needs after exposure.
Carrier protein and Rabies Virus Antigen are formed rabies immune originality conjugate by the present invention through chemistry key connection,
Specifically by carrier protein and rabies whole virus particles antigen through chemistry key connection or by carrier protein and hydrophobin
Sample particle (Virus Like Particle VLP) antigen is through chemistry key connection or by carrier protein and rabies totivirus
Outer virionic membrane segment (Fragment of Virus Envelope) antigen after grain cracking is through chemistry key connection or by carrier egg
White and rabies virus glucoprotein (G) antigen forms a kind of new rabies immune originality conjugate through chemistry key connection.Mad dog
Carrier protein induction body in sick immunogenic conjugates generates the cell immune response to Rabies Virus Antigen, and makes
Cell immune response and humoral immune reaction reach collaboration in vivo, so that new rabies immune originality conjugate is in people
Early immune responsing reaction is faster generated in body, the higher immune level of induction, provides more longlasting immune protection effectiveness.
In recent years it has been reported that rabies vacciness has certain control for malignant mela noma, cervical carcinoma, glioblastoma
Therapeutic effect, it has further been found that there is anti-phosphatidylinositols in the blood of all malignant tumor patients
(phosphatidylinositol PI) antibody, and phosphatidylinositols is the important component of high-content in hydrophobin coating,
It is considered that the rabies vacciness by large dosage influences cancer cell by exciting the activity of lymphocyte to a greater extent, and proposes
Rabies vacciness can be as the effect (Faiderbe of a kind of method of new treating cancer and the existing cancer immunotherapy of promotion
S,et al.C R Acad Sci III.1989；310(3):49-52；Altinoz MA,et al.Clin Transl
Oncol.Published online:16January 2017；Bongiorno E,et al.J Immunol.2013；190
(1Supplement):126-5.).And a kind of new rabies immune originality conjugate of the present invention can stimulate body to generate
Cellullar immunologic response, and cooperateed with humoral immunity realization, and then generate stronger immune response effect.Hence, it is believed that this
The rabies immune originality conjugate of invention can be also used for preventing or treating or auxiliary treatment includes malignant mela noma, uterine neck
Cancer including cancer, glioblastoma.
In existing rabies vacciness and preparation method thereof described in published patent text containing tetanus toxoid and
The vaccine of Rabies Virus Antigen and the patent of preparation method include CN200910177427.8, CN201010607735.2,
CN201110458972.1、CN201210137897.3、US9,296,796B2、US9,220,770B2、US9,200,042B2、
US9,056,901B2, US8,956,812B2, US7,090,853B2, CN201210137897.3, however the technology of the above patent
Take precautions against the technological deficiency and deficiency more than all having.
In conclusion the rabies immune originality conjugate emphasis of the present invention solves existing rabies whole virus particles epidemic disease
The defect that seedling does not generate cellular immunity or cellular immunity is faint and generation antibody is slow is improving the same of existing vaccine Vaccine effectiveness
When greatly accelerate the vaccine early immune responsing reaction time.
Invention content
In order to overcome the shortcoming of existing rabies whole virus particles vaccine, the purpose of the present invention is to provide a kind of new
Rabies immune originality conjugate and preparation method thereof, and prepared according to above-mentioned immunogenic conjugates a kind of new mad
Dog disease combined vaccine and preparation method thereof.Its difference and advantageous effect are compared with the existing technology：
(1) rabies immune originality conjugate of the present invention and the mad dog prepared according to this rabies immune originality conjugate
Antigenic structure is different from the prior art and document contained by virus combined vaccine.In existing literature report and above-mentioned patent text institute
In disclosed scheme, Rabies Virus Antigen and be to exist in hybrid form using tetanus toxoid as the albumen of representative is that is, mad
Without chemistry key connection between dog disease viral antigen and tetanus toxoid, Rabies Virus Antigen and tetanus toxoid do not have yet
There are the connection on recurring structure, Rabies Virus Antigen and tetanus toxoid to exist in the form of completely independent from one another.And at this
The rabies immune originality conjugate of invention and the hydrophobin combination prepared according to this rabies immune originality conjugate
In vaccine, between Rabies Virus Antigen and carrier protein including tetanus toxoid it is keyed through chemistry, mad dog
Sick viral antigen and carrier protein form the connection in structure.
(2) rabies immune originality conjugate of the present invention and the mad dog prepared according to this rabies immune originality conjugate
Carrier protein function is different from the prior art and document contained by virus combined vaccine.Existing literature report and it is foregoing
It is more the function of playing the part of adjuvant by the albumen of representative of tetanus toxoid, to reach in scheme disclosed in patent text
For the purpose of the immunogenicity for enhancing Rabies Virus Antigen.And in the rabies immune originality conjugate of the present invention and according to this
In hydrophobin combined vaccine prepared by rabies immune originality conjugate, the function of carrier protein is that stimulation body generates needle
To the cellular immunity of rabies antigen, humoral immunity was not only generated but also had generated cellular immunity, immune response is faster generated to reach
Reaction, enhancing immunogenicity, the purpose for promoting immune persistence.
(3) rabies immune originality conjugate of the present invention and the mad dog prepared according to this rabies immune originality conjugate
Virus combined vaccine preparation method is different from the prior art and document.Disclosed in existing literature report and above-mentioned patent text
Scheme in, using tetanus toxoid as the albumen of representative be added to when vaccine semi-finished product stoste is prepared hydrophobin original
In liquid, chemical bonds do not occur between tetanus toxoid and Rabies Virus Antigen, there is semi-finished product stoste independently of one another
And in finished product.And in the rabies immune originality conjugate of the present invention and according to the preparation of this rabies immune originality conjugate
In hydrophobin combined vaccine, carrier protein forms rabies immune originality with Rabies Virus Antigen by chemistry key connection
Conjugate, then it is configured to rabies combined vaccine using rabies immune originality conjugate as stoste, the method includes following
Step：
(a) purified that Rabies Virus Antigen stoste is made；
(b) Rabies Virus Antigen stoste prepared by step (a) is subjected to chemical key connection with carrier protein and purified, made
At Rabies Virus Antigen-carrier protein immunogenic conjugates.
(c) Rabies Virus Antigen-carrier protein immunogenic conjugates prepared by step (b) are diluted, are prepared,
Packing becomes finished product vaccine.
(4) rabies immune originality conjugate of the present invention and the mad dog prepared according to this rabies immune originality conjugate
Virus combined vaccine preparation method is different from the prior art and document.It needs that people is added in rabies vacciness in the prior art
Albumin is as protective agent or stabilizer.In the present invention, carrier protein of the Rabies Virus Antigen through chemical bonds is complete
Instead of needing to be added function of the human albumin as protective agent or stabilizer in existing rabies vacciness, thus it is of the invention mad
Dog disease combined vaccine does not include human albumin.
The present invention provides a kind of rabies immune originality conjugate (Rabies Immunogenic Conjugate,
RIC), including Rabies Virus Antigen (RabAg) and carrier protein (Carrier Protein, CP), which is characterized in that described
Rabies Virus Antigen includes at least the Rabies Virus Antigen being keyed through chemistry with carrier protein, and the carrier protein is at least
Include with carrier protein of the Rabies Virus Antigen through chemistry key connection, the chemical bond is by Rabies Virus Antigen and carrier egg
White connection forms Rabies Virus Antigen-carrier protein immunogenic conjugates.Carrier protein and Rabies Virus Antigen are passed through
Chemistry key connection forms rabies immune originality conjugate, first, can promote Th1 cell recognition carrier proteins, stimulate B cell
Stronger immune response is generated to Rabies Virus Antigen-carrier protein immunogenic conjugates, and it is cell-mediated to can induce Th1
Immune anamnestic reaction, promote more B cells to generate specific antibodies；Secondly, Rabies Virus Antigen-carrier protein is immune
Originality conjugate increases relative to Rabies Virus Antigen molecular weight, advantageously anti-with the immune response in quick inductor
It answers；It is prepared in the way of amalgamation and expression in addition, being connected directly chemical bond Rabies Virus Antigen with carrier protein
Rabies Virus Antigen-carrier protein immunogenic conjugates are compared, have the function of following and technique effect and overcome with
Lower defect：1) due to Rabies Virus Antigen with carrier protein by being connected between chemical bond, remain Rabies Virus Antigen
With the respective protein structure of carrier protein and effective action site, the immunogenicity of Rabies Virus Antigen is maintained；Gram 2)
Taken larger fusion protein be difficult to express, purify and purification process in be easy to the defect of inactivation；3) Rabies Virus Antigen
It is more easy to obtain with carrier protein, has saved production cost and time.Carrier protein and Rabies Virus Antigen are connected through chemical bond
It connects to form rabies immune originality conjugate, so that cell immune response and humoral immune reaction is reached collaboration in vivo, and then make
The rabies immune originality conjugate obtained newly faster generates early immune responsing reaction, the higher immune water of induction in human body
It puts down, more longlasting immune protection effectiveness is provided.
Preferably, the Rabies Virus Antigen and Rabies Virus Antigen of the carrier protein through chemistry key connection, institute
State the carrier protein of carrier protein and Rabies Virus Antigen through chemistry key connection, the chemical bond by Rabies Virus Antigen and
Carrier protein connects to form Rabies Virus Antigen-carrier protein immunogenic conjugates.
Any of the above-described is preferably, and the Rabies Virus Antigen includes rabies whole virus particles, through recombinant expression
Hydrophobin sample particle, rabies whole virus particles prepared after cracking hydrophobin outer membrane segment, from rabies
The rabies virus glucoprotein of virus isolation, the nucleoprotein isolated and purified from hydrophobin are detached from hydrophobin
The phosphoprotein of purifying, the stromatin isolated and purified from hydrophobin, the polymerase isolated and purified from hydrophobin, through weight
The rabies virus glucoprotein of group expression, the rabies virus nucleoprotein through recombinant expression, the hydrophobin through recombinant expression
At least one in phosphoprotein, the hydrophobin stromatin through recombinant expression and the hydrophobin polymerase through recombinant expression
Kind.
Any of the above-described is preferably, and the rabies whole virus particles are hydrophobins through cell culture, inactivation, pure
The Rabies Virus Antigen of change.
Any of the above-described is preferably, and the hydrophobin sample particle is that the hydrophobin prepared through recombinant expression resists
It is former.
Any of the above-described is preferably, and the hydrophobin outer membrane segment is that rabies whole virus particles are prepared through cracking
The Rabies Virus Antigen containing furcella and stromatin.
Any of the above-described is preferably, and the rabies virus glucoprotein is isolated and purified or passed through from hydrophobin
The Rabies Virus Antigen containing glycoprotein of recombinant expression.
Any of the above-described is preferably, and the carrier protein includes tetanus toxoid (TT), diphtheria toxoid (DT), white
Larynx toxin non-toxic variant (CRM197), B group meningitis coccis outer membrane protein (OMP), Pneumococal surface protein A (PspA),
PsaA (PsaA), pneumolysin (Ply), haemophilus influenzae D albumen (PD), pertussis poison
Plain (PT), pertussis filamentous hemagglutinin (FHA), pertussis adhesin (PRN), cholera toxin (CT), muramyl dipeptide (MDP),
E.coli LT, Escherichia coli ST, purified protein derivative of tuberculin (PPD), Pseudomonas aeruginosa exotoxin A (PEA), albumen
Albumen, keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) (BSA), hepatitis b virus s antigen (HBsAg), hepatitis B
At least one of virus core antigen (HBcAg), Tetanus Toxin Fragment C (TTC).
Any of the above-described is preferably, and the Rabies Virus Antigen is with Rabies Virus Antigen-carrier protein conjugate
Form be present in the rabies immune originality conjugate, the Rabies Virus Antigen-carrier protein conjugate includes
Single self-existent and/or 2 or more Rabies Virus Antigen-carrier protein conjugates, the Rabies Virus Antigen-
Contain single self-existent and/or 2 or more rabies virus antigens, i.e. Rabies Virus Antigen in carrier protein conjugate
With RabAg-CP, RabAg-CP-RabAg, RabAg-CP-RabAg-CP-RabAg, RabAg-CP-RabAg-CP-RabAg-CP-
RabAg and (RabAg-CP)n- RabAg-RabAg at least one forms are present in rabies immune originality conjugate, wherein
" RabAg " is that Rabies Virus Antigen, " CP " represent carrier protein, "-" as connection Rabies Virus Antigen and carrier protein
Chemical bond, the single self-existent Rabies Virus Antigen not by between carrier protein chemical bond and other lists
A self-existent Rabies Virus Antigen establishes the connection in structure, n >=1, and described 2 or more Rabies Virus Antigens lead to
The chemical bond crossed between carrier protein establishes the connection in structure each other.Single self-existent and/or 2 or more mad dogs
Sick viral antigen-carrier protein conjugate effectively increases the immunogenicity of obtained vaccine, can promote Th1 cell recognitions
Carrier protein, stimulation B cell generate stronger immune response to Rabies Virus Antigen-carrier protein immunogenic conjugates,
And the cell-mediated immune anamnestic reactions of Th1 are can induce, promote more B cells to generate specific antibody, keeps cellular immunity anti-
Collaboration should be reached in vivo with humoral immune reaction, so that new rabies immune originality conjugate faster produces in human body
Raw early immune responsing reaction, provides more longlasting immune protection effectiveness at the higher immune level of induction.
Any of the above-described is preferably, and the Rabies Virus Antigen is with Rabies Virus Antigen-carrier protein conjugate
Form be present in the rabies immune originality conjugate, the Rabies Virus Antigen-carrier protein conjugate is single
A self-existent Rabies Virus Antigen-carrier protein conjugate (RabAg-CP), the Rabies Virus Antigen-carrier egg
Single Rabies Virus Antigen is contained only in white conjugate, i.e. Rabies Virus Antigen is present in mad dog in the form of RabAg-CP
In sick immunogenic conjugates, carrier protein, "-" are represented to connect mad dog wherein " RabAg " is Rabies Virus Antigen, " CP "
The chemical bond of sick viral antigen and carrier protein, single self-existent Rabies Virus Antigen not by with carrier protein it
Between chemical bond establish the connection in structure with other single self-existent Rabies Virus Antigens.It is single self-existent mad
Dog disease viral antigen-carrier protein conjugate effectively increases the immunogenicity of obtained vaccine, and Th1 cells can be promoted to know
Other carrier protein, stimulation B cell generate stronger be immunized to Rabies Virus Antigen-carrier protein immunogenic conjugates and answer
It answers, and can induce the cell-mediated immune anamnestic reactions of Th1, promote more B cells to generate specific antibody, make cellular immunity
Reaction and humoral immune reaction reach collaboration in vivo, so that new rabies immune originality conjugate is in human body faster
It generates early immune responsing reaction, the higher immune level of induction, provide more longlasting immune protection effectiveness.
Any of the above-described is preferably, and the Rabies Virus Antigen is with Rabies Virus Antigen-carrier protein conjugate
Form be present in the rabies immune originality conjugate, the Rabies Virus Antigen-carrier protein conjugate be 2
Above Rabies Virus Antigen-carrier protein conjugate ((RabAg-CP)n- RabAg, n >=1), the hydrophobin is anti-
Containing 2 or more rabies virus antigens in original-carrier protein conjugate, i.e., Rabies Virus Antigen is with RabAg-CP-
RabAg、RabAg-CP-RabAg-CP-RabAg、RabAg-CP-RabAg-CP-RabAg-CP-RabAg、(RabAg-CP)n-
RabAg at least one forms are present in rabies immune originality conjugate, wherein " RabAg " be Rabies Virus Antigen,
" CP " represent carrier protein, "-" as connect Rabies Virus Antigen and carrier protein chemical bond, n >=1, described 2 or more
Rabies Virus Antigen establishes the connection in structure by the chemical bond between carrier protein each other.2 or more rabies
Viral antigen-carrier protein conjugate effectively increases the immunogenicity of obtained vaccine, and Th1 cell recognitions can be promoted to carry
Body protein, stimulation B cell generate stronger immune response to Rabies Virus Antigen-carrier protein immunogenic conjugates, and
The cell-mediated immune anamnestic reactions of Th1 are can induce, promotes more B cells to generate specific antibody, makes cell immune response
Reach collaboration in vivo with humoral immune reaction, so that new rabies immune originality conjugate faster generates in human body
Early immune responsing reaction, provides more longlasting immune protection effectiveness at the higher immune level of induction.
Any of the above-described is preferably, and the Rabies Virus Antigen contains one or more to carry out with carrier protein
The functional groups of chemistry key connection.
Any of the above-described is preferably, and the Rabies Virus Antigen contains one or more to carry out with carrier protein
Chemistry key connection functional groups include hydroxyl (- OH), carboxyl (- COOH), aldehyde radical (- CHO) and amino (- NH2) at least
It is a kind of.
Any of the above-described is preferably, and the carrier protein contains one or more to carry out with Rabies Virus Antigen
The functional groups of chemistry key connection.
Any of the above-described is preferably, and the carrier protein contains one or more to carry out with Rabies Virus Antigen
Chemistry key connection functional groups include hydroxyl (- OH), carboxyl (- COOH), aldehyde radical (- CHO), amino (- NH2) at least one
Kind.
Any of the above-described is preferably, the Rabies Virus Antigen be according to selected from PAS plants, PV plants, PM plants, CVS plants,
Nishigara plants, Flury plants, Vnukovo-32 plants, CTN-1V plants, at least one of aGV plants hydrophobin fixed virus
The Rabies Virus Antigen of preparation, or according to selected from PAS plants, PV plants, PM plants, CVS plants, Nishigara plants, Flury plants,
The mad dog of Vnukovo-32 plants, CTN-1V plants, at least one of aGV plants hydrophobin fixed virus DNA recombinant expression preparations
Sick viral antigen.
Any of the above-described is preferably, the chemical bond connection method packet of the Rabies Virus Antigen and the carrier protein
Include carbodlimide method (EDC), mixed anhydride method (chloromethyl isobutyl ester process), N- hydroxyl succinimides ester process, glutaraldehyde method, diazonium
Change method, succinic anhydride method, carbonyl dimidazoles method, maleimide method, disulfide bond method, periodate oxidation method, carboxymethyl hydroxylamine assay
At least one.
Any of the above-described is preferably, and the chemical bond bridging agent of the Rabies Virus Antigen and the carrier protein includes
1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC), 1- cyclohexyl -3- (2-N- morpholinyl ethyls) carbon two
Imines (CMC), dicyclohexylcarbodiimide (DCC), N, N'- diisopropylcarbodiimide (DIC), the different evil of 2- ethyl -5- phenyl
Azoles -3'- sulfonate (Woodward ' s reagent K), N, N'- carbonyl dimidazoles (CDI), schiff bases generate and reduction amination
At least one of the reagent of reaction such as sodium cyanoborohydride (NaBH3CN), sodium borohydride (NaBH4).
Any of the above-described is preferably, and the chemistry key connection of the Rabies Virus Antigen and the carrier protein is direct
Connection, it is described to be directly connected at least not include linking arm (Linker Arm), spacerarm (Spacer Arm) and bridging molecules
(Bridging Molecule), the Rabies Virus Antigen and carrier protein are that zero-length connects (Zero-Length
Linking) or zero-length is crosslinked (Zero-Length Crosslinking) or zero-length bridging (Zero-Length
Bridging), the chemical bond of Rabies Virus Antigen and carrier protein is connected in the Rabies Virus Antigen and carrier protein
Between do not include new adatom or molecule.
Any of the above-described is preferably, and the chemistry key connection of the Rabies Virus Antigen and the carrier protein is to pass through
The connection that linking arm, spacerarm or at least one mode of bridging molecules carry out, the linking arm or spacerarm or bridging newly created point
Rabies Virus Antigen and carrier protein are formed Rabies Virus Antigen-carrier protein immunogenicity knot by son through chemistry key connection
Object is closed, it includes new between Rabies Virus Antigen and carrier protein to connect the chemical bond of Rabies Virus Antigen and carrier protein
Adatom or molecule.
Any of the above-described is preferably, and the linking arm or spacerarm or bridging molecules include that (succinyl is sub- for two thiobis
Amidos propionic acid ester) (DSP), two thiobis (sulfosuccinimide base propionic ester) (DTSSP), disuccinimidyl suberate
(DSS), bis- (thiosuccimide base) suberate sodium salts (BS3), two succinimide of tartaric acid (DST), tartaric acid two
Sulfosuccinimide ester (sulfo-DST), bis- (2- (succinimidyloxycarbonyl oxygen) ethyl) sulfones (BSOCOES), bis- (2-
(sulfosuccinimide oxygen carbonyl oxygen) ethyl) sulfone (sulfo-BSOCOES), bis- (the succinimide ester succinic acid of ethylene glycol-
Ester) (EGS), ethylene glycol-bis- (sulfosuccinimide ester succinate) (sulfo-EGS), double amber imide glutarates
(DSG), oneself two sub- carboxylic acid amide esters (DMA), heptanedioyl imidic acid diformazan of bis- succinimidyl carbonate of N, N'- (DSC), dimethyl
Ester (DMP), dimethyl-octa dinitrate (DMS), bis- thiobis the third imido dimethyl phthalates (DTBP) of 3,3'-, bis- (3'- of 1,4-
(2'- disulfide groups pyridine) propionic acid acylamino-) butane (DPDPB), dimaleimide base hexane (BMH), difluorodinitrobenzene
(DFDNB), difluorodinitrobenzene base sulfone (DFDNPS), curing two (β-(4- nitrine salicyloyls amino) ethyl) (BASED), first
Aldehyde, glutaraldehyde, 1,4- butanediols glycidol ether, adipic acid dihydrazide (ADH), carbohydrazide, diamino dimethyl diphenyl, to diamino
Base biphenyl, nitrogen-amber star argon ammonia -3 (2- pyridines two are thio)-acid esters (SPDP), long-chain-nitrogen--3 (2- pyridines two of amber star argon ammonia
It is thio)-acid esters (LC-SPDP), -3 (2- pyridines two are thio)-acid esters (sulfo-LC- of sulfo group long-chain-nitrogen-amber star argon ammonia
SPDP), succinimido oxo carbonyl-methyl-(2- pyridylthios) benzene (SMPT), sulfo group long-chain-succinimido
Oxo carbonyl-methyl-(2- pyridylthios) benzene (sulfo-LC-SMPT), 4- (N- maleimidomethyls) hexamethylene carboxylic
Sour N-hydroxy-succinamide ester (SMCC), the thio-N- succinyls of 4- (N- maleimidomethyls) hexamethylene -1- carboxylic acids 3-
Imines ester sodium salt (sulfo-SMCC), maleimide yl benzoic acid succinimide ester (MBS), M- maleimidobenzoyls
Succinimide ester (sulfo-MBS), N- succinimides (4- iodoacteyls) aminobenzoic acid (SIAB), sulfo group-N- ambers
Amber acid imide (4- iodoacteyls) aminobenzoic acid (sulfo-SIAB), 4- (4- maleimidophenyls) butyric acid succinyl
Imines ester (SMPB), sulfosuccinimide base -4- (P- maleimidophenyls) butyrate (sulfo-SMPB), the Malaysias 4-
Imide butyric acid-N- succinimide esters (GMBS), sulfo group maleimidobutyric acid-N- succinimide esters (sulfo-
GMBS), succinimido -6- ((iodoacetyl) amino) capronate (SIAX), succinimido -6- (6- (((4- iodos
Acetyl group) amino) hexanoyl) amino) caproic acid (SIAXX), succinimido -4- (((iodoacteyl) amino) methyl) hexamethylene
Alkane -1- carboxylic acids (SIAC), succinimido -6- ((((4 (iodoacteyl) amino) methyl) hexamethylene-is -1- carbonyls) ammonia
Base) caproic acid (SIACX), 4- nitro phenyl ester iodoacetic acid (NPIA), 4- (4-N- maleimide benzene methanamines ester) butyric acid hydrazides
(MPBH), 4-N- maleimidomehyls hexamethylene -1- carboxyl hydrazides (M2C2H), 3- (2- pyridyl groups two are thio) propionyl hydrazides
(PDPH), n-hydroxysuccinimide -4- azidos salicylic acid (NHS-ASA), n-Hydroxysulfosuccinimide -4- azidos
Salicylic acid (sulfo-NHS-ASA), sulfosuccinimide -4- nitrine salicyl aminocaproic acids (sulfo-NHS-LC-ASA),
Sulfosuccinimide base -2- (P- azidos-salicyloyl amino) ethyl -1,3'- disulfide groups propionic ester (SASD), succinyl are sub-
Amido -4- triazobenzenes formic acid esters (HSAB), sulfosuccinimide base -4- triazobenzenes formic acid esters (sulfo-HSAB), N-
Succinimido -6- (4'- azido -2'- nitro-phenylaminos) capronate (SANPAH), sulfosuccinimide base -6-
(4'- azido -2'- nitro-phenylaminos) capronate (sulfo-SANPAH), 5- azido -2- nitrobenzoic acid-N- ambers
Imide ester (ANB-NOS), sulfosuccinimide -2- (M- nitroazides base-benzamido)-ethyl -1,3'- two are thio
Dipropionate (SAND), N- succinimidos-(4- azidos phenyl) -1,3'- dithiopropionic acids ester (SADP), N- sulfo group ambers
Amber imide-(4- azidos phenyl) -1,3'- dithiopropionic acids ester (sulfo-SADP), sulfosuccinimide base -4-
(P- azidophenyls) butyric acid (Sulfo-SAPB), sulfosuccinimide -2- (7- nitrine -4- methyl cumarin -3- acetyl
Amine) ethyl -1,3'- dithiopropionic acids ester (SAED), sulfosuccinimide -7- azido -4- methylcoumarin -3- acetic acid esters
(Sulfo-SAMCA), p-nitrophenyl diazonium pyruvic acid (pNPDP), p-nitrophenyl -2- diazonium 3,3,3- trifluoroacetic acids
(PNP-DTP), 1- (p- nitrine salicyloyls amino) -4- (iodacetyl amido) butane (ASIB), N- (4- (p- azidosalicylamides
Base) butyl)-3'- (two sulphur of 2'- pyridyl groups) propionamide (APDP), UVINUL MS 40-iodoacetamide, UVINUL MS 40-Malaysia acyl
Imines, p- azido benzoyls hydrazine, 4- (P- nitrine salicyloyls amino)-butylamine (ASBA), P- azidophenyls glyoxal (APG), 4-
Nitrine -2- nitrobenzophenone biotin -4- nitrobenzenes base esters (ABNP), sulfosuccinimide base -2- (6- (biotin amides
Base) -2- (p- azido benzoyls amino) acylamino-s) ethyl -1,3- disulfide groups propionic ester (sulfo-SBED), the thio sulphur of methane
Four fluoro- long-chain-biotin (MTS-ATF-biotin) of sour azido, methane thiosulfonic acid azido tetrafluoro biotin (MTS-
ATF-LC-biotin), at least one in three (hydroxymethyl) hydrogen phosphide (THP), three (hydroxymethyl) phosphorus base propionic acid (THPP)
Kind.
Any of the above-described is preferably, and is connected again by chemical bond with carrier protein after the Rabies Virus Antigen is activated
It connects.
Any of the above-described is preferably, and is connected again by chemical bond with Rabies Virus Antigen after the carrier protein is activated
It connects.
Any of the above-described is preferably, Rabies Virus Antigen and carrier protein distinguish it is activated after connected again by chemical bond
It connects.
Any of the above-described is preferably, and the rabies immune originality conjugate diagnoses for rabies, prevents and control
It treats.
Any of the above-described is preferably, and the rabies immune originality conjugate is used to prevent, treat or auxiliary treatment
Cancer.
The present invention also provides a kind of methods preparing rabies immune originality conjugate, and the method includes following steps
Suddenly：
(a) purified that Rabies Virus Antigen stoste is made；
(b) the Rabies Virus Antigen stoste for preparing step (a) carries out chemical key connection with carrier protein, then through pure
Rabies Virus Antigen-carrier protein immunogenic conjugates are made in change.
Carrier protein and Rabies Virus Antigen are formed into rabies immune originality conjugate through chemistry key connection, first,
It can promote Th1 cell recognition carrier proteins, stimulation B cell is to Rabies Virus Antigen-carrier protein immunogenic conjugates
Stronger immune response is generated, and can induce the cell-mediated immune anamnestic reactions of Th1, promotes more B cells to generate special
Property antibody；Secondly, Rabies Virus Antigen-carrier protein immunogenic conjugates increase relative to Rabies Virus Antigen molecular weight
Add, advantageously with the immune response in quick inductor；In addition, Rabies Virus Antigen and carrier protein are passed through
Chemical bond is connected directly and is prepared Rabies Virus Antigen-carrier protein immunogenic conjugates phase in the way of amalgamation and expression
Than having the function of following and technique effect and overcoming following defect：1) since Rabies Virus Antigen and carrier protein are logical
It crosses between chemical bond and is connected, remain Rabies Virus Antigen and the respective protein structure of carrier protein and effectively act on position
Point maintains the immunogenicity of Rabies Virus Antigen；2) larger fusion protein is overcome to be difficult to express, purify and purify
It is easy to the defect of inactivation in the process；3) Rabies Virus Antigen and carrier protein are more easy to obtain, and have saved production cost and time.
Carrier protein and Rabies Virus Antigen are formed into rabies immune originality conjugate through chemistry key connection, make cell immune response
Reach collaboration in vivo with humoral immune reaction, so that new rabies immune originality conjugate faster generates in human body
Early immune responsing reaction, provides more longlasting immune protection effectiveness at the higher immune level of induction.
Preferably, the Rabies Virus Antigen includes rabies whole virus particles, the rabies disease through recombinant expression
Hydrophobin outer membrane segment that malicious sample particle, rabies whole virus particles are prepared after cracking, detached from hydrophobin it is pure
The dog disease viral glycoprotein of change, the nucleoprotein isolated and purified from hydrophobin, the phosphoprotein isolated and purified from hydrophobin,
The stromatin that is isolated and purified from hydrophobin, the polymerase isolated and purified from hydrophobin, the mad dog through recombinant expression
Sick viral glycoprotein, the rabies virus nucleoprotein through recombinant expression, the hydrophobin phosphoprotein through recombinant expression, through recombination
At least one of the hydrophobin stromatin of expression, hydrophobin polymerase through recombinant expression.
Any of the above-described is preferably, and in step (a), the Rabies Virus Antigen stoste is to fix hydrophobin
Rabies whole virus particles stoste is made in seed culture of viruses inoculating cell, culture, harvest virus liquid, inactivation, purifying.
Any of the above-described is preferably, and in step (a), the Rabies Virus Antigen stoste is through recombinantly expressing, purifying
Hydrophobin sample particle stoste is made.
Any of the above-described is preferably, and in step (a), the Rabies Virus Antigen stoste is to obtain in accordance with the following steps
Hydrophobin outer membrane segment stoste, hydrophobin fixes seed culture of viruses inoculating cell, culture, harvest virus liquid, inactivation, purifying
And manufactured rabies whole virus particles stoste；The rabies whole virus particles stoste prepared in above-mentioned steps is through cracking, purifying
Hydrophobin outer membrane segment stoste is made.
Any of the above-described is preferably, and in step (a), rabies are made to be purified in the Rabies Virus Antigen stoste
Viral glycoprotein stoste.
Any of the above-described is preferably, and the carrier protein is selected from tetanus toxoid (TT), diphtheria toxoid (DT), white
Larynx toxin non-toxic variant (CRM197), B group meningitis coccis outer membrane protein (OMP), Pneumococal surface protein A (PspA),
PsaA (PsaA), pneumolysin (Ply), haemophilus influenzae D albumen (PD), pertussis poison
Plain (PT), pertussis filamentous hemagglutinin (FHA), pertussis adhesin (PRN), cholera toxin (CT), muramyl dipeptide (MDP),
E.coli LT, Escherichia coli ST, purified protein derivative of tuberculin (PPD), Pseudomonas aeruginosa exotoxin A (PEA), albumen
Albumen, keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) (BSA), hepatitis b virus s antigen (HBsAg), hepatitis B
At least one of virus core antigen (HBcAg), Tetanus Toxin Fragment C (TTC).
Any of the above-described is preferably, the Rabies Virus Antigen be according to selected from PAS plants, PV plants, PM plants, CVS plants,
Nishigara plants, Flury plants, Vnukovo-32 plants, CTN-1V plants, at least one of aGV plants hydrophobin fixed virus
The Rabies Virus Antigen of preparation, or according to selected from PAS plants, PV plants, PM plants, CVS plants, Nishigara plants, Flury plants,
The mad dog of Vnukovo-32 plants, CTN-1V plants, at least one of aGV plants hydrophobin fixed virus DNA recombinant expression preparations
Sick viral antigen.
Any of the above-described is preferably, and the Rabies Virus Antigen contains one or more to carry out with carrier protein
The functional groups of chemistry key connection.
Any of the above-described is preferably, and the Rabies Virus Antigen contains one or more to carry out with carrier protein
Chemistry key connection functional groups include hydroxyl (- OH), carboxyl (- COOH), aldehyde radical (- CHO) and amino (- NH2) at least
It is a kind of.
Any of the above-described is preferably, and the carrier protein contains one or more to carry out with Rabies Virus Antigen
The functional groups of chemistry key connection.
Any of the above-described is preferably, and the carrier protein contains one or more to carry out with Rabies Virus Antigen
Chemistry key connection functional groups include hydroxyl (- OH), carboxyl (- COOH), aldehyde radical (- CHO), amino (- NH2) at least one
Kind.
Any of the above-described is preferably, the chemical bond connection method packet of the Rabies Virus Antigen and the carrier protein
Include carbodlimide method (EDC), mixed anhydride method (chloromethyl isobutyl ester process), N- hydroxyl succinimides ester process, glutaraldehyde method, diazonium
Change method, succinic anhydride method, carbonyl dimidazoles method, maleimide method, disulfide bond method, periodate oxidation method, carboxymethyl hydroxylamine assay
At least one of.
Any of the above-described is preferably, and the chemical bond bridging agent of the Rabies Virus Antigen and the carrier protein includes
1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC), 1- cyclohexyl -3- (2-N- morpholinyl ethyls) carbon two
Imines (CMC), dicyclohexylcarbodiimide (DCC), N, N'- diisopropylcarbodiimide (DIC), the different evil of 2- ethyl -5- phenyl
Azoles -3'- sulfonate (Woodward ' s reagent K), N, N'- carbonyl dimidazoles (CDI), schiff bases generate and reduction amination
At least one of the reagent of reaction such as sodium cyanoborohydride (NaBH3CN) or sodium borohydride (NaBH4).
Any of the above-described is preferably, and the chemistry key connection of the Rabies Virus Antigen and the carrier protein is direct
Connection, it is described to be directly connected at least not include linking arm (Linker Arm), spacerarm (Spacer Arm) and bridging molecules
(Bridging Molecule), Rabies Virus Antigen and carrier protein are that zero-length connects (Zero-Length Linking)
Or zero-length is crosslinked (Zero-Length Crosslinking) or zero-length bridging (Zero-Length Bridging), connection
The chemical bond of Rabies Virus Antigen and carrier protein is between Rabies Virus Antigen and carrier protein and not comprising newly-increased original
Son or molecule.
Any of the above-described is preferably, and the chemistry key connection of the Rabies Virus Antigen and the carrier protein is to pass through
The connection of linking arm, spacerarm or bridging molecules, the linking arm newly created, spacerarm or bridging molecules are by Rabies Virus Antigen
With carrier protein Rabies Virus Antigen-carrier protein immunogenic conjugates, connection rabies disease are formed through chemistry key connection
The chemical bond of malicious antigen and carrier protein includes new adatom or molecule between Rabies Virus Antigen and carrier protein.
Any of the above-described is preferably, and the linking arm or spacerarm or bridging molecules include that (succinyl is sub- for two thiobis
Amidos propionic acid ester) (DSP), two thiobis (sulfosuccinimide base propionic ester) (DTSSP), disuccinimidyl suberate
(DSS), bis- (thiosuccimide base) suberate sodium salts (BS3), two succinimide of tartaric acid (DST), tartaric acid two
Sulfosuccinimide ester (sulfo-DST), bis- (2- (succinimidyloxycarbonyl oxygen) ethyl) sulfones (BSOCOES), bis- (2-
(sulfosuccinimide oxygen carbonyl oxygen) ethyl) sulfone (sulfo-BSOCOES), bis- (the succinimide ester succinic acid of ethylene glycol-
Ester) (EGS), ethylene glycol-bis- (sulfosuccinimide ester succinate) (sulfo-EGS), double amber imide glutarates
(DSG), oneself two sub- carboxylic acid amide esters (DMA), heptanedioyl imidic acid diformazan of bis- succinimidyl carbonate of N, N'- (DSC), dimethyl
Ester (DMP), dimethyl-octa dinitrate (DMS), bis- thiobis the third imido dimethyl phthalates (DTBP) of 3,3'-, bis- (3'- of 1,4-
(2'- disulfide groups pyridine) propionic acid acylamino-) butane (DPDPB), dimaleimide base hexane (BMH), difluorodinitrobenzene
(DFDNB), difluorodinitrobenzene base sulfone (DFDNPS), curing two (β-(4- nitrine salicyloyls amino) ethyl) (BASED), first
Aldehyde, glutaraldehyde, 1,4- butanediols glycidol ether, adipic acid dihydrazide (ADH), carbohydrazide, diamino dimethyl diphenyl, to diamino
Base biphenyl, nitrogen-amber star argon ammonia -3 (2- pyridines two are thio)-acid esters (SPDP), long-chain-nitrogen--3 (2- pyridines two of amber star argon ammonia
It is thio)-acid esters (LC-SPDP), -3 (2- pyridines two are thio)-acid esters (sulfo-LC- of sulfo group long-chain-nitrogen-amber star argon ammonia
SPDP), succinimido oxo carbonyl-methyl-(2- pyridylthios) benzene (SMPT), sulfo group long-chain-succinimido
Oxo carbonyl-methyl-(2- pyridylthios) benzene (sulfo-LC-SMPT), 4- (N- maleimidomethyls) hexamethylene carboxylic
Sour N-hydroxy-succinamide ester (SMCC), the thio-N- succinyls of 4- (N- maleimidomethyls) hexamethylene -1- carboxylic acids 3-
Imines ester sodium salt (sulfo-SMCC), maleimide yl benzoic acid succinimide ester (MBS), M- maleimidobenzoyls
Succinimide ester (sulfo-MBS), N- succinimides (4- iodoacteyls) aminobenzoic acid (SIAB), sulfo group-N- ambers
Amber acid imide (4- iodoacteyls) aminobenzoic acid (sulfo-SIAB), 4- (4- maleimidophenyls) butyric acid succinyl
Imines ester (SMPB), sulfosuccinimide base -4- (P- maleimidophenyls) butyrate (sulfo-SMPB), the Malaysias 4-
Imide butyric acid-N- succinimide esters (GMBS), sulfo group maleimidobutyric acid-N- succinimide esters (sulfo-
GMBS), succinimido -6- ((iodoacetyl) amino) capronate (SIAX), succinimido -6- (6- (((4- iodos
Acetyl group) amino) hexanoyl) amino) caproic acid (SIAXX), succinimido -4- (((iodoacteyl) amino) methyl) hexamethylene
Alkane -1- carboxylic acids (SIAC), succinimido -6- ((((4 (iodoacteyl) amino) methyl) hexamethylene-is -1- carbonyls) ammonia
Base) caproic acid (SIACX), 4- nitro phenyl ester iodoacetic acid (NPIA), 4- (4-N- maleimide benzene methanamines ester) butyric acid hydrazides
(MPBH), 4-N- maleimidomehyls hexamethylene -1- carboxyl hydrazides (M2C2H), 3- (2- pyridyl groups two are thio) propionyl hydrazides
(PDPH), n-hydroxysuccinimide -4- azidos salicylic acid (NHS-ASA), n-Hydroxysulfosuccinimide -4- azidos
Salicylic acid (sulfo-NHS-ASA), sulfosuccinimide -4- nitrine salicyl aminocaproic acids (sulfo-NHS-LC-ASA),
Sulfosuccinimide base -2- (P- azidos-salicyloyl amino) ethyl -1,3'- disulfide groups propionic ester (SASD), succinyl are sub-
Amido -4- triazobenzenes formic acid esters (HSAB), sulfosuccinimide base -4- triazobenzenes formic acid esters (sulfo-HSAB), N-
Succinimido -6- (4'- azido -2'- nitro-phenylaminos) capronate (SANPAH), sulfosuccinimide base -6-
(4'- azido -2'- nitro-phenylaminos) capronate (sulfo-SANPAH), 5- azido -2- nitrobenzoic acid-N- ambers
Imide ester (ANB-NOS), sulfosuccinimide -2- (M- nitroazides base-benzamido)-ethyl -1,3'- two are thio
Dipropionate (SAND), N- succinimidos-(4- azidos phenyl) -1,3'- dithiopropionic acids ester (SADP), N- sulfo group ambers
Amber imide-(4- azidos phenyl) -1,3'- dithiopropionic acids ester (sulfo-SADP), sulfosuccinimide base -4-
(P- azidophenyls) butyric acid (Sulfo-SAPB), sulfosuccinimide -2- (7- nitrine -4- methyl cumarin -3- acetyl
Amine) ethyl -1,3'- dithiopropionic acids ester (SAED), sulfosuccinimide -7- azido -4- methylcoumarin -3- acetic acid esters
(Sulfo-SAMCA), p-nitrophenyl diazonium pyruvic acid (pNPDP), p-nitrophenyl -2- diazonium 3,3,3- trifluoroacetic acids
(PNP-DTP), 1- (p- nitrine salicyloyls amino) -4- (iodacetyl amido) butane (ASIB), N- (4- (p- azidosalicylamides
Base) butyl)-3'- (two sulphur of 2'- pyridyl groups) propionamide (APDP), UVINUL MS 40-iodoacetamide, UVINUL MS 40-Malaysia acyl
Imines, p- azido benzoyls hydrazine, 4- (P- nitrine salicyloyls amino)-butylamine (ASBA), P- azidophenyls glyoxal (APG), 4-
Nitrine -2- nitrobenzophenone biotin -4- nitrobenzenes base esters (ABNP), sulfosuccinimide base -2- (6- (biotin amides
Base) -2- (p- azido benzoyls amino) acylamino-s) ethyl -1,3- disulfide groups propionic ester (sulfo-SBED), the thio sulphur of methane
Four fluoro- long-chain-biotin (MTS-ATF-biotin) of sour azido, methane thiosulfonic acid azido tetrafluoro biotin (MTS-
ATF-LC-biotin), at least one in three (hydroxymethyl) hydrogen phosphide (THP), three (hydroxymethyl) phosphorus base propionic acid (THPP)
Kind.
Any of the above-described is preferably, and is connected again by chemical bond with carrier protein after the Rabies Virus Antigen is activated
It connects.
Any of the above-described is preferably, and passes through chemistry with the Rabies Virus Antigen again after the carrier protein is activated
Key connection.
Any of the above-described is preferably, and is passed through again after the Rabies Virus Antigen and the carrier protein are activated respectively
Chemistry key connection.
Any of the above-described is preferably, and the rabies immune originality conjugate being prepared is for rabies diagnosis, prevention
And treatment.
Any of the above-described is preferably, and the rabies immune originality conjugate being prepared is for preventing, treating or assisting
Treating cancer.
The present invention also provides a kind of rabies combined vaccine (Rabies Conjugate Vaccine RCV), including it is mad
Dog disease immunogenic conjugates, the rabies immune originality conjugate include Rabies Virus Antigen and carrier protein, described
Rabies Virus Antigen includes at least the Rabies Virus Antigen being keyed through chemistry with carrier protein, and the carrier protein is at least
Include with carrier protein of the Rabies Virus Antigen through chemistry key connection, the chemical bond is by Rabies Virus Antigen and carrier egg
White connection forms Rabies Virus Antigen-carrier protein immunogenic conjugates.Carrier protein and Rabies Virus Antigen are passed through
Chemistry key connection forms rabies immune originality conjugate, first, can promote Th1 cell recognition carrier proteins, stimulate B cell
Stronger immune response is generated to Rabies Virus Antigen-carrier protein immunogenic conjugates, and it is cell-mediated to can induce Th1
Immune anamnestic reaction, promote more B cells to generate specific antibodies；Secondly, Rabies Virus Antigen-carrier protein is immune
Originality conjugate increases relative to Rabies Virus Antigen molecular weight, advantageously anti-with the immune response in quick inductor
It answers；It is prepared in the way of amalgamation and expression in addition, being connected directly chemical bond Rabies Virus Antigen with carrier protein
Rabies Virus Antigen-carrier protein immunogenic conjugates are compared, have the function of following and technique effect and overcome with
Lower defect：1) due to Rabies Virus Antigen with carrier protein by being connected between chemical bond, remain Rabies Virus Antigen
With the respective protein structure of carrier protein and effective action site, the immunogenicity of Rabies Virus Antigen is maintained；Gram 2)
Taken larger fusion protein be difficult to express, purify and purification process in be easy to the defect of inactivation；3) Rabies Virus Antigen
It is more easy to obtain with carrier protein, has saved production cost and time.Carrier protein and Rabies Virus Antigen are connected through chemical bond
It connects to form rabies immune originality conjugate, so that cell immune response and humoral immune reaction is reached collaboration in vivo, and then make
The rabies immune originality conjugate obtained newly faster generates early immune responsing reaction, the higher immune water of induction in human body
It puts down, more longlasting immune protection effectiveness is provided.
Preferably, the Rabies Virus Antigen and Rabies Virus Antigen of the carrier protein through chemistry key connection, institute
State the carrier protein of carrier protein and Rabies Virus Antigen through chemistry key connection, the chemical bond by Rabies Virus Antigen and
Carrier protein connects to form Rabies Virus Antigen-carrier protein immunogenic conjugates.
Any of the above-described is preferably, and the Rabies Virus Antigen includes rabies whole virus particles, through recombinant expression
Hydrophobin sample particle, rabies whole virus particles prepared after cracking hydrophobin outer membrane segment, from rabies
The rabies virus glucoprotein of virus isolation, the nucleoprotein isolated and purified from hydrophobin are detached from hydrophobin
The phosphoprotein of purifying, the stromatin isolated and purified from hydrophobin, the polymerase isolated and purified from hydrophobin, through weight
The rabies virus glucoprotein of group expression, the rabies virus nucleoprotein through recombinant expression, the hydrophobin through recombinant expression
At least one in phosphoprotein, the hydrophobin stromatin through recombinant expression and the hydrophobin polymerase through recombinant expression
Kind.
Any of the above-described is preferably, and the rabies whole virus particles are hydrophobins through cell culture, inactivation, pure
The Rabies Virus Antigen of change.
Any of the above-described is preferably, and the hydrophobin sample particle is that the hydrophobin prepared through recombinant expression resists
It is former.
Any of the above-described is preferably, and the hydrophobin outer membrane segment is that rabies whole virus particles are prepared through cracking
The Rabies Virus Antigen containing furcella and stromatin.
Any of the above-described is preferably, and the rabies virus glucoprotein is isolated and purified or passed through from hydrophobin
The Rabies Virus Antigen containing glycoprotein of recombinant expression.
Any of the above-described is preferably, and the carrier protein includes tetanus toxoid (TT), diphtheria toxoid (DT), white
Larynx toxin non-toxic variant (CRM197), B group meningitis coccis outer membrane protein (OMP), Pneumococal surface protein A (PspA),
PsaA (PsaA), pneumolysin (Ply), haemophilus influenzae D albumen (PD), pertussis poison
Plain (PT), pertussis filamentous hemagglutinin (FHA), pertussis adhesin (PRN), cholera toxin (CT), muramyl dipeptide (MDP),
E.coli LT, Escherichia coli ST, purified protein derivative of tuberculin (PPD), Pseudomonas aeruginosa exotoxin A (PEA), albumen
Albumen, keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) (BSA), hepatitis b virus s antigen (HBsAg), hepatitis B
At least one of virus core antigen (HBcAg), Tetanus Toxin Fragment C (TTC).
Any of the above-described is preferably, and the rabies combined vaccine includes adjuvant.
Any of the above-described is preferably, and the adjuvant includes aluminium adjuvant, calcium phosphate adjuvant, cholera toxin (CT), cholera poison
Plain B subunits (CTB), pertussis toxin (PT), pertussis toxin B subunits (PTB), pertussis filamentous hemagglutinin (FHA), hundred
Day cough adhesin (PRN), Soap chitins QS-21, alpha-tocopherol, squalene, lipoid, liposome (liposomes), monophosphate class
Fat A (MPL-A), MF59, viruslike particle proteoliposome (Virosomes), polyglycolide (PLA) microballoon, polylactic acid-glycollic acid
(PLGA) microballoon, lipid-cholesterol (DC-Chol), dimethyl double octadecyl quaternary ammonium bromides (DDA), immunostimulating complexes
(ISCOM)、Montanide ISA50、Montanide ISA51、Montanide ISA206、Montanide ISA720、
Montanide ISA series of adjuvants, AS01, AS02, AS03, AS04, AS series of adjuvants, muramyl dipeptide (MDP), bacterium fat are more
Sugared (OM-174), e. coli heat-labile toxin (LT), IL-1, IL-2, IL-6, IL-12, IL-15, IL-18, IFN-γ, GM-
CSF, CpG ODN, trehalose dimycolate (TDM), containing poly- inosinic acid (Poly I) and/or poly- born of the same parents it is phonetic
At least one of the substance of the pick up adjuvant of pyridine nucleotide (Poly C).
Any of the above-described is preferably, and the Rabies Virus Antigen is with Rabies Virus Antigen-carrier protein conjugate
Form be present in the rabies immune originality conjugate, the Rabies Virus Antigen-carrier protein conjugate includes
Single self-existent and/or 2 or more Rabies Virus Antigen-carrier protein conjugates, the Rabies Virus Antigen-
Contain single self-existent and/or 2 or more rabies virus antigens, i.e. Rabies Virus Antigen in carrier protein conjugate
With RabAg-CP, RabAg-CP-RabAg, RabAg-CP-RabAg-CP-RabAg, RabAg-CP-RabAg-CP-RabAg-CP-
RabAg and (RabAg-CP)n- RabAg at least one forms are present in rabies immune originality conjugate, wherein " RabAg "
For Rabies Virus Antigen, " CP " represent carrier protein, "-" as connect Rabies Virus Antigen and carrier protein chemical bond,
The single self-existent Rabies Virus Antigen not by between carrier protein chemical bond and other single independences
Existing Rabies Virus Antigen establishes the connection in structure, n >=1, described 2 or more Rabies Virus Antigens by with load
Chemical bond between body protein establishes the connection in structure each other.Single self-existent and/or 2 or more hydrophobins
Antigen-carrier protein conjugates effectively increase the immunogenicity of obtained vaccine, can promote Th1 cell recognition carrier eggs
In vain, stimulation B cell generates stronger immune response to Rabies Virus Antigen-carrier protein immunogenic conjugates, and can lure
The cell-mediated immune anamnestic reactions of Th1 are led, promotes more B cells to generate specific antibody, makes cell immune response and body
Liquid immune response reaches collaboration in vivo, so that new rabies immune originality conjugate faster generates early stage in human body
Immune response, provides more longlasting immune protection effectiveness at the higher immune level of induction.
Any of the above-described is preferably, and the Rabies Virus Antigen is with Rabies Virus Antigen-carrier protein conjugate
Form be present in the rabies immune originality conjugate, the Rabies Virus Antigen-carrier protein conjugate is single
A self-existent Rabies Virus Antigen-carrier protein conjugate (RabAg-CP), the Rabies Virus Antigen-carrier egg
Single Rabies Virus Antigen is contained only in white conjugate, i.e. Rabies Virus Antigen is present in mad dog in the form of RabAg-CP
In sick immunogenic conjugates, carrier protein, "-" are represented to connect mad dog wherein " RabAg " is Rabies Virus Antigen, " CP "
The chemical bond of sick viral antigen and carrier protein, single self-existent Rabies Virus Antigen not by with carrier protein it
Between chemical bond establish the connection in structure with other single self-existent Rabies Virus Antigens.It is single self-existent mad
Dog disease viral antigen-carrier protein conjugate effectively increases the immunogenicity of obtained vaccine, and Th1 cells can be promoted to know
Other carrier protein, stimulation B cell generate stronger be immunized to Rabies Virus Antigen-carrier protein immunogenic conjugates and answer
It answers, and can induce the cell-mediated immune anamnestic reactions of Th1, promote more B cells to generate specific antibody, make cellular immunity
Reaction and humoral immune reaction reach collaboration in vivo, so that new rabies immune originality conjugate is in human body faster
It generates early immune responsing reaction, the higher immune level of induction, provide more longlasting immune protection effectiveness.
Any of the above-described is preferably, and the Rabies Virus Antigen is with Rabies Virus Antigen-carrier protein conjugate
Form be present in the rabies immune originality conjugate, the Rabies Virus Antigen-carrier protein conjugate be 2
Above Rabies Virus Antigen-carrier protein conjugate ((RabAg-CP)n- RabAg, n >=1), the hydrophobin is anti-
Containing 2 or more rabies virus antigens in original-carrier protein conjugate, i.e., Rabies Virus Antigen is with RabAg-CP-
RabAg、RabAg-CP-RabAg-CP-RabAg、RabAg-CP-RabAg-CP-RabAg-CP-RabAg、(RabAg-CP)n-
RabAg at least one forms are present in rabies immune originality conjugate, wherein " RabAg " be Rabies Virus Antigen,
" CP " represent carrier protein, "-" as connect Rabies Virus Antigen and carrier protein chemical bond, n >=1, described 2 or more
Rabies Virus Antigen establishes the connection in structure by the chemical bond between carrier protein each other.2 or more rabies
Viral antigen-carrier protein conjugate effectively increases the immunogenicity of obtained vaccine, and Th1 cell recognitions can be promoted to carry
Body protein, stimulation B cell generate stronger immune response to Rabies Virus Antigen-carrier protein immunogenic conjugates, and
The cell-mediated immune anamnestic reactions of Th1 are can induce, promotes more B cells to generate specific antibody, makes cell immune response
Reach collaboration in vivo with humoral immune reaction, so that new rabies immune originality conjugate faster generates in human body
Early immune responsing reaction, provides more longlasting immune protection effectiveness at the higher immune level of induction.
Any of the above-described is preferably, and the Rabies Virus Antigen contains one or more to carry out with carrier protein
The functional groups of chemistry key connection.
Any of the above-described is preferably, and the Rabies Virus Antigen carries out the functionality of chemical key connection with carrier protein
Group includes at least one of hydroxyl (- OH), carboxyl (- COOH), aldehyde radical (- CHO) amino (- NH2).
Any of the above-described is preferably, and the carrier protein contains one or more to carry out with Rabies Virus Antigen
The functional groups of chemistry key connection.
Any of the above-described is preferably, and the carrier protein carries out the functionality of chemical key connection with Rabies Virus Antigen
Group includes at least one of hydroxyl (- OH), carboxyl (- COOH), aldehyde radical (- CHO), amino (- NH2).
Any of the above-described is preferably, the Rabies Virus Antigen be according to selected from PAS plants, PV plants, PM plants, CVS plants,
Nishigara plants, Flury plants, Vnukovo-32 plants, CTN-1V plants, at least one of aGV plants hydrophobin fixed virus
The Rabies Virus Antigen of preparation, or according to selected from PAS plants, PV plants, PM plants, CVS plants, Nishigara plants, Flury plants,
The mad dog of Vnukovo-32 plants, CTN-1V plants, at least one of aGV plants hydrophobin fixed virus DNA recombinant expression preparations
Sick viral antigen.
Any of the above-described is preferably, the chemical bond connection method packet of the Rabies Virus Antigen and the carrier protein
Include carbodlimide method (EDC), mixed anhydride method (chloromethyl isobutyl ester process), N- hydroxyl succinimides ester process, glutaraldehyde method, diazonium
Change method, succinic anhydride method, carbonyl dimidazoles method, maleimide method, disulfide bond method, periodate oxidation method, carboxymethyl hydroxylamine assay
At least one.
Any of the above-described is preferably, and the chemical bond bridging agent of the Rabies Virus Antigen and the carrier protein includes
1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC), 1- cyclohexyl -3- (2-N- morpholinyl ethyls) carbon two
Imines (CMC), dicyclohexylcarbodiimide (DCC), N, N'- diisopropylcarbodiimide (DIC), the different evil of 2- ethyl -5- phenyl
Azoles -3'- sulfonate (Woodward ' s reagent K), N, N'- carbonyl dimidazoles (CDI), schiff bases generate and reduction amination
At least one of the reagent of reaction such as sodium cyanoborohydride (NaBH3CN), sodium borohydride (NaBH4).
Any of the above-described is preferably, and the chemistry key connection of the Rabies Virus Antigen and carrier protein is directly to connect
It connects, it is described to be directly connected at least not include linking arm (Linker Arm), spacerarm (Spacer Arm) and bridging molecules
(Bridging Molecule), the Rabies Virus Antigen and carrier protein are that zero-length connects (Zero-Length
Linking) or zero-length is crosslinked (Zero-Length Crosslinking) or zero-length bridging (Zero-Length
Bridging), the chemical bond of Rabies Virus Antigen and carrier protein is connected in the Rabies Virus Antigen and carrier protein
Between do not include new adatom or molecule.
Any of the above-described is preferably, and the chemistry key connection of the Rabies Virus Antigen and carrier protein is to pass through connection
The connection that arm, spacerarm or at least one mode of bridging molecules carry out, the linking arm or spacerarm or bridging molecules newly created will
Rabies Virus Antigen and carrier protein form Rabies Virus Antigen-carrier protein immunogenicity through chemistry key connection and combine
Object, it includes newly-increased between Rabies Virus Antigen and carrier protein to connect the chemical bond of Rabies Virus Antigen and carrier protein
Atom or molecule.
Any of the above-described is preferably, and the linking arm or spacerarm or bridging molecules include that (succinyl is sub- for two thiobis
Amidos propionic acid ester) (DSP), two thiobis (sulfosuccinimide base propionic ester) (DTSSP), disuccinimidyl suberate
(DSS), bis- (thiosuccimide base) suberate sodium salts (BS3), two succinimide of tartaric acid (DST), tartaric acid two
Sulfosuccinimide ester (sulfo-DST), bis- (2- (succinimidyloxycarbonyl oxygen) ethyl) sulfones (BSOCOES), bis- (2-
(sulfosuccinimide oxygen carbonyl oxygen) ethyl) sulfone (sulfo-BSOCOES), bis- (the succinimide ester succinic acid of ethylene glycol-
Ester) (EGS), ethylene glycol-bis- (sulfosuccinimide ester succinate) (sulfo-EGS), double amber imide glutarates
(DSG), oneself two sub- carboxylic acid amide esters (DMA), heptanedioyl imidic acid diformazan of bis- succinimidyl carbonate of N, N'- (DSC), dimethyl
Ester (DMP), dimethyl-octa dinitrate (DMS), bis- thiobis the third imido dimethyl phthalates (DTBP) of 3,3'-, bis- (3'- of 1,4-
(2'- disulfide groups pyridine) propionic acid acylamino-) butane (DPDPB), dimaleimide base hexane (BMH), difluorodinitrobenzene
(DFDNB), difluorodinitrobenzene base sulfone (DFDNPS), curing two (β-(4- nitrine salicyloyls amino) ethyl) (BASED), first
Aldehyde, glutaraldehyde, 1,4- butanediols glycidol ether, adipic acid dihydrazide (ADH), carbohydrazide, diamino dimethyl diphenyl, to diamino
Base biphenyl, nitrogen-amber star argon ammonia -3 (2- pyridines two are thio)-acid esters (SPDP), long-chain-nitrogen--3 (2- pyridines two of amber star argon ammonia
It is thio)-acid esters (LC-SPDP), -3 (2- pyridines two are thio)-acid esters (sulfo-LC- of sulfo group long-chain-nitrogen-amber star argon ammonia
SPDP), succinimido oxo carbonyl-methyl-(2- pyridylthios) benzene (SMPT), sulfo group long-chain-succinimido
Oxo carbonyl-methyl-(2- pyridylthios) benzene (sulfo-LC-SMPT), 4- (N- maleimidomethyls) hexamethylene carboxylic
Sour N-hydroxy-succinamide ester (SMCC), the thio-N- succinyls of 4- (N- maleimidomethyls) hexamethylene -1- carboxylic acids 3-
Imines ester sodium salt (sulfo-SMCC), maleimide yl benzoic acid succinimide ester (MBS), M- maleimidobenzoyls
Succinimide ester (sulfo-MBS), N- succinimides (4- iodoacteyls) aminobenzoic acid (SIAB), sulfo group-N- ambers
Amber acid imide (4- iodoacteyls) aminobenzoic acid (sulfo-SIAB), 4- (4- maleimidophenyls) butyric acid succinyl
Imines ester (SMPB), sulfosuccinimide base -4- (P- maleimidophenyls) butyrate (sulfo-SMPB), the Malaysias 4-
Imide butyric acid-N- succinimide esters (GMBS), sulfo group maleimidobutyric acid-N- succinimide esters (sulfo-
GMBS), succinimido -6- ((iodoacetyl) amino) capronate (SIAX), succinimido -6- (6- (((4- iodos
Acetyl group) amino) hexanoyl) amino) caproic acid (SIAXX), succinimido -4- (((iodoacteyl) amino) methyl) hexamethylene
Alkane -1- carboxylic acids (SIAC), succinimido -6- ((((4 (iodoacteyl) amino) methyl) hexamethylene-is -1- carbonyls) ammonia
Base) caproic acid (SIACX), 4- nitro phenyl ester iodoacetic acid (NPIA), 4- (4-N- maleimide benzene methanamines ester) butyric acid hydrazides
(MPBH), 4-N- maleimidomehyls hexamethylene -1- carboxyl hydrazides (M2C2H), 3- (2- pyridyl groups two are thio) propionyl hydrazides
(PDPH), n-hydroxysuccinimide -4- azidos salicylic acid (NHS-ASA), n-Hydroxysulfosuccinimide -4- azidos
Salicylic acid (sulfo-NHS-ASA), sulfosuccinimide -4- nitrine salicyl aminocaproic acids (sulfo-NHS-LC-ASA),
Sulfosuccinimide base -2- (P- azidos-salicyloyl amino) ethyl -1,3'- disulfide groups propionic ester (SASD), succinyl are sub-
Amido -4- triazobenzenes formic acid esters (HSAB), sulfosuccinimide base -4- triazobenzenes formic acid esters (sulfo-HSAB), N-
Succinimido -6- (4'- azido -2'- nitro-phenylaminos) capronate (SANPAH), sulfosuccinimide base -6-
(4'- azido -2'- nitro-phenylaminos) capronate (sulfo-SANPAH), 5- azido -2- nitrobenzoic acid-N- ambers
Imide ester (ANB-NOS), sulfosuccinimide -2- (M- nitroazides base-benzamido)-ethyl -1,3'- two are thio
Dipropionate (SAND), N- succinimidos-(4- azidos phenyl) -1,3'- dithiopropionic acids ester (SADP), N- sulfo group ambers
Amber imide-(4- azidos phenyl) -1,3'- dithiopropionic acids ester (sulfo-SADP), sulfosuccinimide base -4-
(P- azidophenyls) butyric acid (Sulfo-SAPB), sulfosuccinimide -2- (7- nitrine -4- methyl cumarin -3- acetyl
Amine) ethyl -1,3'- dithiopropionic acids ester (SAED), sulfosuccinimide -7- azido -4- methylcoumarin -3- acetic acid esters
(Sulfo-SAMCA), p-nitrophenyl diazonium pyruvic acid (pNPDP), p-nitrophenyl -2- diazonium 3,3,3- trifluoroacetic acids
(PNP-DTP), 1- (p- nitrine salicyloyls amino) -4- (iodacetyl amido) butane (ASIB), N- (4- (p- azidosalicylamides
Base) butyl)-3'- (two sulphur of 2'- pyridyl groups) propionamide (APDP), UVINUL MS 40-iodoacetamide, UVINUL MS 40-Malaysia acyl
Imines, p- azido benzoyls hydrazine, 4- (P- nitrine salicyloyls amino)-butylamine (ASBA), P- azidophenyls glyoxal (APG), 4-
Nitrine -2- nitrobenzophenone biotin -4- nitrobenzenes base esters (ABNP), sulfosuccinimide base -2- (6- (biotin amides
Base) -2- (p- azido benzoyls amino) acylamino-s) ethyl -1,3- disulfide groups propionic ester (sulfo-SBED), the thio sulphur of methane
Four fluoro- long-chain-biotin (MTS-ATF-biotin) of sour azido, methane thiosulfonic acid azido tetrafluoro biotin (MTS-
ATF-LC-biotin), at least one in three (hydroxymethyl) hydrogen phosphide (THP), three (hydroxymethyl) phosphorus base propionic acid (THPP)
Kind.
Any of the above-described is preferably, and is connected again by chemical bond with carrier protein after the Rabies Virus Antigen is activated
It connects.
Any of the above-described is preferably, and is connected again by chemical bond with Rabies Virus Antigen after the carrier protein is activated
It connects.
Any of the above-described is preferably, Rabies Virus Antigen and carrier protein distinguish it is activated after connected again by chemical bond
It connects.
Any of the above-described is preferably, and the potency unit of the rabies combined vaccine is denoted as >=2.5IU/ agent.
Any of the above-described is preferably, and the rabies combined vaccine includes protective agent and/or stabilizer.
Any of the above-described is preferably, and the protective agent and/or stabilizer of the rabies combined vaccine include human albumin.
Any of the above-described is preferably, and the rabies combined vaccine does not include protective agent and/or stabilizer.
Any of the above-described is preferably, and the rabies combined vaccine does not include human albumin.
Any of the above-described is preferably, and the rabies combined vaccine does not include any preservative.
Any of the above-described is preferably, and the rabies combined vaccine includes preservative.
Any of the above-described is preferably, and the preservative includes at least one in thimerosal, 2- Phenoxyethanols, benzyl alcohol
Kind.
Any of the above-described is preferably, and rabies combined vaccine is for rabies diagnosis, prevention and treatment.
Any of the above-described is preferably, and rabies combined vaccine is used to prevent, treat or auxiliary for treating cancer.
The present invention also provides a kind of methods preparing rabies combined vaccine, the described method comprises the following steps：
(a) purified that Rabies Virus Antigen stoste is made；
(b) Rabies Virus Antigen stoste prepared by step (a) is subjected to chemical key connection with carrier protein and purified, made
At Rabies Virus Antigen-carrier protein immunogenic conjugates.
(c) Rabies Virus Antigen-carrier protein immunogenic conjugates prepared by step (b) are diluted, are prepared,
Packing becomes finished product vaccine.
Carrier protein and Rabies Virus Antigen are formed into rabies immune originality conjugate through chemistry key connection, first,
It can promote Th1 cell recognition carrier proteins, stimulation B cell is to Rabies Virus Antigen-carrier protein immunogenic conjugates
Stronger immune response is generated, and can induce the cell-mediated immune anamnestic reactions of Th1, promotes more B cells to generate special
Property antibody；Secondly, Rabies Virus Antigen-carrier protein immunogenic conjugates increase relative to Rabies Virus Antigen molecular weight
Add, advantageously with the immune response in quick inductor；In addition, Rabies Virus Antigen and carrier protein are passed through
Chemical bond is connected directly and is prepared Rabies Virus Antigen-carrier protein immunogenic conjugates phase in the way of amalgamation and expression
Than having the function of following and technique effect and overcoming following defect：1) since Rabies Virus Antigen and carrier protein are logical
It crosses between chemical bond and is connected, remain Rabies Virus Antigen and the respective protein structure of carrier protein and effectively act on position
Point maintains the immunogenicity of Rabies Virus Antigen；2) larger fusion protein is overcome to be difficult to express, purify and purify
It is easy to the defect of inactivation in the process；3) Rabies Virus Antigen and carrier protein are more easy to obtain, and have saved production cost and time.
Carrier protein and Rabies Virus Antigen are formed into rabies immune originality conjugate through chemistry key connection, make cell immune response
Reach collaboration in vivo with humoral immune reaction, so that new rabies immune originality conjugate faster generates in human body
Early immune responsing reaction, provides more longlasting immune protection effectiveness at the higher immune level of induction.
Preferably, the Rabies Virus Antigen includes rabies whole virus particles, the rabies disease through recombinant expression
Hydrophobin outer membrane segment that malicious sample particle, rabies whole virus particles are prepared after cracking, detached from hydrophobin it is pure
The rabies virus glucoprotein of change, the nucleoprotein isolated and purified from hydrophobin, the phosphorus egg isolated and purified from hydrophobin
In vain, the stromatin that is isolated and purified from hydrophobin, the polymerase isolated and purified from hydrophobin, through the mad of recombinant expression
Dog disease viral glycoprotein, the rabies virus nucleoprotein through recombinant expression, the hydrophobin phosphoprotein through recombinant expression, through weight
Hydrophobin stromatin, at least one of the hydrophobin polymerase through recombinant expression of group expression.
Any of the above-described is preferably, and the Rabies Virus Antigen stoste is that hydrophobin is fixed seed culture of viruses inoculation carefully
Rabies whole virus particles stoste is made in born of the same parents, culture, harvest virus liquid, inactivation, purifying.
Any of the above-described is preferably, and in step (a), the Rabies Virus Antigen stoste is through recombinantly expressing, purifying
Hydrophobin sample particle stoste is made.
Any of the above-described is preferably, and in step (a), the Rabies Virus Antigen stoste is to obtain in accordance with the following steps
Hydrophobin outer membrane segment stoste, hydrophobin fixes seed culture of viruses inoculating cell, culture, harvest virus liquid, inactivation, purifying
And manufactured rabies whole virus particles stoste；The rabies whole virus particles stoste prepared in above-mentioned steps is through cracking, purifying
Hydrophobin outer membrane segment stoste is made.
Any of the above-described is preferably, and in step (a), rabies are made to be purified in the Rabies Virus Antigen stoste
Viral glycoprotein stoste.
Any of the above-described is preferably, and the carrier protein is selected from tetanus toxoid (TT), diphtheria toxoid (DT), white
Larynx toxin non-toxic variant (CRM197), B group meningitis coccis outer membrane protein (OMP), Pneumococal surface protein A (PspA),
PsaA (PsaA), pneumolysin (Ply), haemophilus influenzae D albumen (PD), pertussis poison
Plain (PT), pertussis filamentous hemagglutinin (FHA), pertussis adhesin (PRN), cholera toxin (CT), muramyl dipeptide (MDP),
E.coli LT, Escherichia coli ST, purified protein derivative of tuberculin (PPD), Pseudomonas aeruginosa exotoxin A (PEA), albumen
Albumen, keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) (BSA), hepatitis b virus s antigen (HBsAg), hepatitis B
At least one of virus core antigen (HBcAg), Tetanus Toxin Fragment C (TTC).
Any of the above-described is preferably, and the rabies combined vaccine includes adjuvant.
Any of the above-described is preferably, and the adjuvant includes aluminium adjuvant, calcium phosphate adjuvant, cholera toxin (CT), cholera poison
Plain B subunits (CTB), pertussis toxin (PT), pertussis toxin B subunits (PTB), pertussis filamentous hemagglutinin (FHA), hundred
Day cough adhesin (PRN), Soap chitins QS-21, alpha-tocopherol, squalene, lipoid, liposome (liposomes), monophosphate class
Fat A (MPL-A), MF59, viruslike particle proteoliposome (Virosomes), polyglycolide (PLA) microballoon, polylactic acid-glycollic acid
(PLGA) microballoon, lipid-cholesterol (DC-Chol), dimethyl double octadecyl quaternary ammonium bromides (DDA), immunostimulating complexes
(ISCOM)、Montanide ISA50、Montanide ISA51、Montanide ISA206、Montanide ISA720、
Montanide ISA series of adjuvants, AS01, AS02, AS03, AS04, AS series of adjuvants, muramyl dipeptide (MDP), bacterium fat are more
Sugared (OM-174), e. coli heat-labile toxin (LT), IL-1, IL-2, IL-6, IL-12, IL-15, IL-18, IFN-γ, GM-
CSF, CpG ODN, trehalose dimycolate (TDM), containing poly- inosinic acid (Poly I) and/or poly- born of the same parents it is phonetic
At least one of the substance of the pick up adjuvant of pyridine nucleotide (Poly C).
Any of the above-described is preferably, the Rabies Virus Antigen be according to selected from PAS plants, PV plants, PM plants, CVS plants,
Nishigara plants, Flury plants, Vnukovo-32 plants, CTN-1V plants, at least one of aGV plants hydrophobin fixed virus
The Rabies Virus Antigen of preparation, or according to selected from PAS plants, PV plants, PM plants, CVS plants, Nishigara plants, Flury plants,
The mad dog of Vnukovo-32 plants, CTN-1V plants, at least one of aGV plants hydrophobin fixed virus DNA recombinant expression preparations
Sick viral antigen.
Any of the above-described is preferably, and the Rabies Virus Antigen contains one or more to carry out with carrier protein
The functional groups of chemistry key connection.
Any of the above-described is preferably, and the Rabies Virus Antigen contains one or more to carry out with carrier protein
Chemistry key connection functional groups include hydroxyl (- OH), carboxyl (- COOH), aldehyde radical (- CHO) and amino (- NH2) at least
It is a kind of.
Any of the above-described is preferably, and the carrier protein contains one or more to carry out with Rabies Virus Antigen
The functional groups of chemistry key connection.
Any of the above-described is preferably, and the carrier protein contains one or more to carry out with Rabies Virus Antigen
Chemistry key connection functional groups include hydroxyl (- OH), carboxyl (- COOH), aldehyde radical (- CHO), amino (- NH2) at least one
Kind.
Any of the above-described is preferably, the chemical bond connection method packet of the Rabies Virus Antigen and the carrier protein
Include carbodlimide method (EDC), mixed anhydride method (chloromethyl isobutyl ester process), N- hydroxyl succinimides ester process, glutaraldehyde method, diazonium
Change method, succinic anhydride method, carbonyl dimidazoles method, maleimide method, disulfide bond method, periodate oxidation method, carboxymethyl hydroxylamine assay
At least one of.
Any of the above-described is preferably, and the chemical bond bridging agent of the Rabies Virus Antigen and the carrier protein includes
1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC), 1- cyclohexyl -3- (2-N- morpholinyl ethyls) carbon two
Imines (CMC), dicyclohexylcarbodiimide (DCC), N, N'- diisopropylcarbodiimide (DIC), the different evil of 2- ethyl -5- phenyl
Azoles -3'- sulfonate (Woodward ' s reagent K), N, N'- carbonyl dimidazoles (CDI), schiff bases generate and reduction amination
At least one of the reagent of reaction such as sodium cyanoborohydride (NaBH3CN) or sodium borohydride (NaBH4).
Any of the above-described is preferably, and the chemistry key connection of the Rabies Virus Antigen and the carrier protein is direct
Connection, it is described to be directly connected at least not include linking arm (Linker Arm), spacerarm (Spacer Arm) and bridging molecules
(Bridging Molecule), Rabies Virus Antigen and carrier protein are that zero-length connects (Zero-Length Linking)
Or zero-length is crosslinked (Zero-Length Crosslinking) or zero-length bridging (Zero-Length Bridging), connection
The chemical bond of Rabies Virus Antigen and carrier protein is between Rabies Virus Antigen and carrier protein and not comprising newly-increased original
Son or molecule.
Any of the above-described is preferably, and the chemistry key connection of the Rabies Virus Antigen and the carrier protein is to pass through
The connection of linking arm, spacerarm or bridging molecules, the linking arm newly created, spacerarm or bridging molecules are by Rabies Virus Antigen
With carrier protein Rabies Virus Antigen-carrier protein immunogenic conjugates, connection rabies disease are formed through chemistry key connection
The chemical bond of malicious antigen and carrier protein includes new adatom or molecule between Rabies Virus Antigen and carrier protein.
Any of the above-described is preferably, and the linking arm or spacerarm or bridging molecules include that (succinyl is sub- for two thiobis
Amidos propionic acid ester) (DSP), two thiobis (sulfosuccinimide base propionic ester) (DTSSP), disuccinimidyl suberate
(DSS), bis- (thiosuccimide base) suberate sodium salts (BS3), two succinimide of tartaric acid (DST), tartaric acid two
Sulfosuccinimide ester (sulfo-DST), bis- (2- (succinimidyloxycarbonyl oxygen) ethyl) sulfones (BSOCOES), bis- (2-
(sulfosuccinimide oxygen carbonyl oxygen) ethyl) sulfone (sulfo-BSOCOES), bis- (the succinimide ester succinic acid of ethylene glycol-
Ester) (EGS), ethylene glycol-bis- (sulfosuccinimide ester succinate) (sulfo-EGS), double amber imide glutarates
(DSG), oneself two sub- carboxylic acid amide esters (DMA), heptanedioyl imidic acid diformazan of bis- succinimidyl carbonate of N, N'- (DSC), dimethyl
Ester (DMP), dimethyl-octa dinitrate (DMS), bis- thiobis the third imido dimethyl phthalates (DTBP) of 3,3'-, bis- (3'- of 1,4-
(2'- disulfide groups pyridine) propionic acid acylamino-) butane (DPDPB), dimaleimide base hexane (BMH), difluorodinitrobenzene
(DFDNB), difluorodinitrobenzene base sulfone (DFDNPS), curing two (β-(4- nitrine salicyloyls amino) ethyl) (BASED), first
Aldehyde, glutaraldehyde, 1,4- butanediols glycidol ether, adipic acid dihydrazide (ADH), carbohydrazide, diamino dimethyl diphenyl, to diamino
Base biphenyl, nitrogen-amber star argon ammonia -3 (2- pyridines two are thio)-acid esters (SPDP), long-chain-nitrogen--3 (2- pyridines two of amber star argon ammonia
It is thio)-acid esters (LC-SPDP), -3 (2- pyridines two are thio)-acid esters (sulfo-LC- of sulfo group long-chain-nitrogen-amber star argon ammonia
SPDP), succinimido oxo carbonyl-methyl-(2- pyridylthios) benzene (SMPT), sulfo group long-chain-succinimido
Oxo carbonyl-methyl-(2- pyridylthios) benzene (sulfo-LC-SMPT), 4- (N- maleimidomethyls) hexamethylene carboxylic
Sour N-hydroxy-succinamide ester (SMCC), the thio-N- succinyls of 4- (N- maleimidomethyls) hexamethylene -1- carboxylic acids 3-
Imines ester sodium salt (sulfo-SMCC), maleimide yl benzoic acid succinimide ester (MBS), M- maleimidobenzoyls
Succinimide ester (sulfo-MBS), N- succinimides (4- iodoacteyls) aminobenzoic acid (SIAB), sulfo group-N- ambers
Amber acid imide (4- iodoacteyls) aminobenzoic acid (sulfo-SIAB), 4- (4- maleimidophenyls) butyric acid succinyl
Imines ester (SMPB), sulfosuccinimide base -4- (P- maleimidophenyls) butyrate (sulfo-SMPB), the Malaysias 4-
Imide butyric acid-N- succinimide esters (GMBS), sulfo group maleimidobutyric acid-N- succinimide esters (sulfo-
GMBS), succinimido -6- ((iodoacetyl) amino) capronate (SIAX), succinimido -6- (6- (((4- iodos
Acetyl group) amino) hexanoyl) amino) caproic acid (SIAXX), succinimido -4- (((iodoacteyl) amino) methyl) hexamethylene
Alkane -1- carboxylic acids (SIAC), succinimido -6- ((((4 (iodoacteyl) amino) methyl) hexamethylene-is -1- carbonyls) ammonia
Base) caproic acid (SIACX), 4- nitro phenyl ester iodoacetic acid (NPIA), 4- (4-N- maleimide benzene methanamines ester) butyric acid hydrazides
(MPBH), 4-N- maleimidomehyls hexamethylene -1- carboxyl hydrazides (M2C2H), 3- (2- pyridyl groups two are thio) propionyl hydrazides
(PDPH), n-hydroxysuccinimide -4- azidos salicylic acid (NHS-ASA), n-Hydroxysulfosuccinimide -4- azidos
Salicylic acid (sulfo-NHS-ASA), sulfosuccinimide -4- nitrine salicyl aminocaproic acids (sulfo-NHS-LC-ASA),
Sulfosuccinimide base -2- (P- azidos-salicyloyl amino) ethyl -1,3'- disulfide groups propionic ester (SASD), succinyl are sub-
Amido -4- triazobenzenes formic acid esters (HSAB), sulfosuccinimide base -4- triazobenzenes formic acid esters (sulfo-HSAB), N-
Succinimido -6- (4'- azido -2'- nitro-phenylaminos) capronate (SANPAH), sulfosuccinimide base -6-
(4'- azido -2'- nitro-phenylaminos) capronate (sulfo-SANPAH), 5- azido -2- nitrobenzoic acid-N- ambers
Imide ester (ANB-NOS), sulfosuccinimide -2- (M- nitroazides base-benzamido)-ethyl -1,3'- two are thio
Dipropionate (SAND), N- succinimidos-(4- azidos phenyl) -1,3'- dithiopropionic acids ester (SADP), N- sulfo group ambers
Amber imide-(4- azidos phenyl) -1,3'- dithiopropionic acids ester (sulfo-SADP), sulfosuccinimide base -4-
(P- azidophenyls) butyric acid (Sulfo-SAPB), sulfosuccinimide -2- (7- nitrine -4- methyl cumarin -3- acetyl
Amine) ethyl -1,3'- dithiopropionic acids ester (SAED), sulfosuccinimide -7- azido -4- methylcoumarin -3- acetic acid esters
(Sulfo-SAMCA), p-nitrophenyl diazonium pyruvic acid (pNPDP), p-nitrophenyl -2- diazonium 3,3,3- trifluoroacetic acids
(PNP-DTP), 1- (p- nitrine salicyloyls amino) -4- (iodacetyl amido) butane (ASIB), N- (4- (p- azidosalicylamides
Base) butyl)-3'- (two sulphur of 2'- pyridyl groups) propionamide (APDP), UVINUL MS 40-iodoacetamide, UVINUL MS 40-Malaysia acyl
Imines, p- azido benzoyls hydrazine, 4- (P- nitrine salicyloyls amino)-butylamine (ASBA), P- azidophenyls glyoxal (APG), 4-
Nitrine -2- nitrobenzophenone biotin -4- nitrobenzenes base esters (ABNP), sulfosuccinimide base -2- (6- (biotin amides
Base) -2- (p- azido benzoyls amino) acylamino-s) ethyl -1,3- disulfide groups propionic ester (sulfo-SBED), the thio sulphur of methane
Four fluoro- long-chain-biotin (MTS-ATF-biotin) of sour azido, methane thiosulfonic acid azido tetrafluoro biotin (MTS-
ATF-LC-biotin), at least one in three (hydroxymethyl) hydrogen phosphide (THP), three (hydroxymethyl) phosphorus base propionic acid (THPP)
Kind.
Any of the above-described is preferably, and is connected again by chemical bond with carrier protein after the Rabies Virus Antigen is activated
It connects.
Any of the above-described is preferably, and passes through chemistry with the Rabies Virus Antigen again after the carrier protein is activated
Key connection.
Any of the above-described is preferably, and is passed through again after the Rabies Virus Antigen and the carrier protein are activated respectively
Chemistry key connection.
Any of the above-described is preferably, and the potency unit of the rabies combined vaccine is denoted as >=2.5IU/ agent.
Any of the above-described is preferably, and the rabies combined vaccine includes protective agent and/or stabilizer.
Any of the above-described is preferably, and the protective agent and/or stabilizer of the rabies combined vaccine include human albumin.
Any of the above-described is preferably, and the rabies combined vaccine does not include protective agent and/or stabilizer.
Any of the above-described is preferably, and the rabies combined vaccine does not include human albumin.
Any of the above-described is preferably, and the rabies combined vaccine does not include any preservative.
Any of the above-described is preferably, and the rabies combined vaccine includes preservative.
Any of the above-described is preferably, and the preservative includes at least one in thimerosal, 2- Phenoxyethanols, benzyl alcohol
Kind.
Any of the above-described is preferably, and the rabies combined vaccine being prepared is for rabies diagnosis, prevention and treatment.
Any of the above-described is preferably, and the rabies combined vaccine being prepared is used to prevent, treat or auxiliary treatment cancer
Disease.
One aspect of the present invention provides a kind of rabies immune originality conjugate, and the rabies immune originality conjugate includes
Rabies Virus Antigen and carrier protein, the Rabies Virus Antigen are keyed with carrier protein through chemistry, the carrier egg
In vain with Rabies Virus Antigen through chemistry be keyed, the chemical bond Rabies Virus Antigen is connected with carrier protein to be formed it is mad
Dog disease viral antigen-carrier protein immunogenic conjugates.In other words, the present invention includes carrier protein by chemical bond by mad dog
Sick viral antigen connection is formed by Rabies Virus Antigen-carrier protein immunogenic conjugates.The present invention includes mad dog again
Carrier protein connection is formed by Rabies Virus Antigen-carrier protein immunogenicity by chemical bond and combined by sick viral antigen
Object.Carrier protein is connected with Rabies Virus Antigen the invention also includes chemical bond and is formed by Rabies Virus Antigen-load
Body protein immunogenic conjugates.Rabies Virus Antigen-carrier protein immunogenic conjugates of the present invention are used for the mad dog of people
Disease diagnosis, prevention and treatment.Rabies Virus Antigen-carrier protein immunogenic conjugates of the present invention can be also used for preventing
Or the cancer for the treatment of or auxiliary treatment including malignant mela noma, cervical carcinoma, glioblastoma.
Another aspect of the invention provides a kind of rabies immune originality conjugate, the rabies immune originality conjugate packet
Include single self-existent Rabies Virus Antigen-carrier protein, the single self-existent Rabies Virus Antigen difference
With carrier protein through chemistry be keyed, with carrier protein through chemistry key connection single self-existent Rabies Virus Antigen with
The connection in structure is not established between other single self-existent Rabies Virus Antigens, with single self-existent mad dog
Sick viral antigen-carrier protein form is present in rabies immune originality conjugate, the Rabies Virus Antigen-carrier egg
Single Rabies Virus Antigen is contained only in white conjugate, i.e., Rabies Virus Antigen is with RabAg-CP, RabAg-CP, RabAg-
CP ... form is present in rabies immune originality conjugate, wherein " RabAg " is Rabies Virus Antigen, " CP " representative load
Body protein, "-" are to connect the chemical bond of Rabies Virus Antigen and carrier protein.Therefore, the present invention includes individually being individually present
Rabies Virus Antigen connect to form rabies immune originality conjugate with carrier protein through chemical bond, it is described single independently to deposit
Rabies Virus Antigen not by between carrier protein chemical bond and other single self-existent rabies diseases
Malicious antigen is established the form connected in structure and is present in rabies immune originality conjugate.
Another aspect of the invention provides a kind of rabies immune originality conjugate, the rabies immune originality conjugate packet
Include 2 or more Rabies Virus Antigen-carrier proteins, described 2 or more Rabies Virus Antigens are jointly and carrier protein
It is keyed through chemistry, carrier protein is keyed 2 or more Rabies Virus Antigens by chemistry, with carrier protein through chemical bond
The connection in structure is established between the Rabies Virus Antigen of connection and other Rabies Virus Antigens, with 2 or more mad dogs
Sick viral antigen-carrier protein form is present in rabies immune originality conjugate, the Rabies Virus Antigen-carrier egg
Containing 2 or more Rabies Virus Antigens in white conjugate, i.e., Rabies Virus Antigen with RabAg-CP-RabAg, and/or
RabAg-CP-RabAg-CP-RabAg, and/or RabAg-CP-RabAg-CP-RabAg-CP-RabAg ... form is present in mad
In dog disease immunogenic conjugates, wherein " RabAg " is Rabies Virus Antigen, " CP ", to represent carrier protein, "-" mad to connect
The chemical bond of dog disease viral antigen and carrier protein.Therefore, the present invention includes 2 or more Rabies Virus Antigens through chemical bond
Connect to form rabies immune originality conjugate with carrier protein, described 2 or more Rabies Virus Antigens by with carrier
The form that chemical bond between albumen establishes connection each other in structure is present in rabies immune originality conjugate.
Another aspect of the invention provides a kind of rabies immune originality conjugate, the rabies immune originality conjugate packet
Include single self-existent and/or 2 or more Rabies Virus Antigen-carrier proteins, it is single self-existent and/or 2
Above Rabies Virus Antigen is keyed with carrier protein through chemistry respectively and/or jointly, carrier protein not by and/or it is logical
It crosses chemistry and is keyed single self-existent and/or 2 or more Rabies Virus Antigens, be keyed through chemistry with carrier protein
Self-existent Rabies Virus Antigen and other self-existent Rabies Virus Antigens between do not establish and/or establish
Connection in structure is present in mad in the form of single and/or 2 or more self-existent Rabies Virus Antigen-carrier proteins
In dog disease immunogenic conjugates, in the Rabies Virus Antigen-carrier protein conjugate containing it is single self-existent and/
Or 2 or more Rabies Virus Antigens, i.e., Rabies Virus Antigen with RabAg-CP, and/or RabAg-CP-RabAg and/
Or RabAg-CP-RabAg-CP-RabAg, and/or RabAg-CP-RabAg-CP-RabAg-CP-RabAg ... form is present in
In rabies immune originality conjugate, carrier protein, "-" are represented as connection wherein " RabAg " is Rabies Virus Antigen, " CP "
The chemical bond of Rabies Virus Antigen and carrier protein.Therefore, the present invention includes single self-existent and/or 2 or more
Rabies Virus Antigen connect to form rabies immune originality conjugate with carrier protein through chemical bond, described to be individually individually present
And/or 2 or more self-existent Rabies Virus Antigens not by and/or by chemical bond between carrier protein
The form that connection is established in structure is present in rabies immune originality conjugate.
Another aspect of the invention provides a kind of rabies immune originality conjugate, the rabies immune originality conjugate packet
Rabies Virus Antigen and carrier protein are included, the Rabies Virus Antigen contains one or more can be with carrier protein
Carry out the functional groups of chemical key connection, the functional groups include hydroxyl (- OH), carboxyl (- COOH), aldehyde radical (-
CHO), amino (- NH2)。
Another aspect of the invention provides a kind of rabies immune originality conjugate, the rabies immune originality conjugate packet
Rabies Virus Antigen and carrier protein are included, the carrier protein contains one or more can be with Rabies Virus Antigen
Carry out the functional groups of chemical key connection, the functional groups include hydroxyl (- OH), carboxyl (- COOH), aldehyde radical (-
CHO), amino (- NH2)。
Another aspect of the invention provides a kind of rabies immune originality conjugate, the rabies immune originality conjugate packet
Rabies whole virus particles and carrier protein are included, the rabies whole virus particles are keyed with carrier protein through chemistry, described
Carrier protein and rabies whole virus particles are keyed through chemistry, and the chemical bond is by rabies whole virus particles and carrier protein
Connection forms rabies whole virus particles-carrier protein immunogenic conjugates.Therefore, the present invention includes passing through of carrier protein
It learns key and rabies whole virus particles is connected into formed rabies whole virus particles-carrier protein immunogenic conjugates.It is described
Rabies whole virus particles are Rabies Virus Antigen of the hydrophobin through cell culture, inactivation, purifying.
Another aspect of the invention provides a kind of rabies immune originality conjugate, the rabies immune originality conjugate packet
Rabies VLP and carrier protein are included, the rabies VLP is keyed with carrier protein through chemistry, the carrier protein and mad dog
Sick VLP is keyed through chemistry, and the chemical bond, which connects rabies VLP with carrier protein, to be formed rabies VLP- carrier proteins and exempt from
Epidemic focus conjugate.Therefore, the present invention includes carrier protein by chemical bond by the formed rabies VLP- of rabies VLP connections
Carrier protein immunogenic conjugates.The VLP is the Rabies Virus Antigen prepared through recombinant expression.
Another aspect of the invention provides a kind of rabies immune originality conjugate, the rabies immune originality conjugate packet
Hydrophobin outer membrane segment and carrier protein are included, the hydrophobin outer membrane segment is keyed with carrier protein through chemistry,
The carrier protein and hydrophobin outer membrane segment are keyed through chemistry, the chemical bond by hydrophobin outer membrane segment and
Carrier protein connects to form hydrophobin outer membrane segment-carrier protein immunogenic conjugates.Therefore, the present invention includes carrier
Hydrophobin outer membrane segment is connected formed hydrophobin outer membrane segment-carrier protein immunogene by albumen by chemical bond
Property conjugate.The hydrophobin outer membrane segment is that rabies whole virus particles contain furcella and stromatin through prepared by cracking
Rabies Virus Antigen.
Another aspect of the invention provides a kind of rabies immune originality conjugate, the rabies immune originality conjugate packet
Rabies virus glucoprotein (G) and carrier protein are included, the rabies virus glucoprotein is keyed with carrier protein through chemistry, institute
It states carrier protein and rabies virus glucoprotein to be keyed through chemistry, the chemical bond is by rabies virus glucoprotein and carrier egg
White connection forms rabies virus glucoprotein-carrier protein immunogenic conjugates.Therefore, the present invention includes that carrier protein passes through
Rabies virus glucoprotein is connected formed rabies virus glucoprotein-carrier protein immunogenic conjugates by chemical bond.Institute
It is the rabies disease being isolated and purified from hydrophobin or through recombinant expression containing glycoprotein to state rabies virus glucoprotein
Malicious antigen.
Another aspect of the invention provides a kind of rabies combined vaccine (Rabies Conjugate Vaccine RCV), institute
It includes rabies immune originality conjugate to state rabies combined vaccine, and the rabies immune originality conjugate includes rabies disease
Malicious antigen and carrier protein, the Rabies Virus Antigen are keyed with carrier protein through chemistry, the carrier protein and mad dog
Sick viral antigen is keyed through chemistry, and Rabies Virus Antigen is connected to form hydrophobin by the chemical bond with carrier protein
Antigen-carrier protein immunogenic conjugate.In other words, the present invention includes that carrier protein is resisted hydrophobin by chemical bond
Original connection be formed by Rabies Virus Antigen-carrier protein immunogenic conjugates it is formulated again made of rabies combination epidemic disease
Seedling.The present invention includes that carrier protein connection is formed by Rabies Virus Antigen-by Rabies Virus Antigen by chemical bond again
Rabies combined vaccine made of carrier protein immunogenic conjugates are formulated again.The invention also includes chemical bonds by carrier egg
It is white connected with Rabies Virus Antigen be formed by Rabies Virus Antigen-carrier protein immunogenic conjugates it is formulated again and
At rabies combined vaccine.The rabies combined vaccine of the present invention is prevented and treated for human rabies.
Another aspect of the invention provides a kind of rabies combined vaccine, and the rabies combined vaccine includes rabies immune
Originality conjugate, the rabies immune originality conjugate include rabies whole virus particles and carrier protein, the rabies
Whole virus particles are keyed with carrier protein through chemistry, and the carrier protein is keyed with rabies whole virus particles through chemistry,
Rabies whole virus particles are connected to form rabies whole virus particles-carrier protein immunogene with carrier protein by the chemical bond
Property conjugate.In other words, the present invention includes that the connection of rabies whole virus particles is formed mad dog by carrier protein by chemical bond
Rabies totivirus combined vaccine made of sick whole virus particles-carrier protein immunogenic conjugates are formulated again.It is described mad
Dog disease whole virus particles are Rabies Virus Antigen of the hydrophobin through cell culture, inactivation, purifying.
Another aspect of the invention provides a kind of rabies combined vaccine, and the rabies combined vaccine includes rabies immune
Originality conjugate, the rabies immune originality conjugate include rabies VLP and carrier protein, the rabies VLP with carry
Body protein is keyed through chemistry, and the carrier protein and rabies VLP are keyed through chemistry, and the chemical bond is by rabies VLP
It connects to form rabies VLP- carrier protein immunogenic conjugates with carrier protein.In other words, the present invention includes carrier protein
By chemical bond by the formed rabies VLP- carrier proteins immunogenic conjugates of rabies VLP connections it is formulated again made of
Rabies VLP combined vaccines.The VLP is the Rabies Virus Antigen prepared through recombinant expression.
Another aspect of the invention provides a kind of rabies combined vaccine, and the rabies combined vaccine includes rabies immune
Originality conjugate, the rabies immune originality conjugate include hydrophobin outer membrane segment and carrier protein, the mad dog
Sick outer virionic membrane segment is keyed with carrier protein through chemistry, and the carrier protein is with hydrophobin outer membrane segment through chemical bond
Connection, the chemical bond connect hydrophobin outer membrane segment with carrier protein to form hydrophobin outer membrane segment-carrier
Protein immunogenic conjugate.In other words, the present invention includes that carrier protein is connected hydrophobin outer membrane segment by chemical bond
Connect formed hydrophobin outer membrane segment-carrier protein immunogenic conjugates it is formulated again made of rabies cracking combine
Vaccine.The hydrophobin outer membrane segment is rabies whole virus particles prepared through cracking it is mad containing furcella and stromatin
Dog disease viral antigen.
Another aspect of the invention provides a kind of rabies combined vaccine, and the rabies combined vaccine includes rabies immune
Originality conjugate, the rabies immune originality conjugate include rabies virus glucoprotein (G) and carrier protein, the mad dog
Sick viral glycoprotein is keyed with carrier protein through chemistry, and the carrier protein connects with rabies virus glucoprotein through chemical bond
It connects, the chemical bond, which connects rabies virus glucoprotein with carrier protein, to be formed rabies virus glucoprotein-carrier protein and exempt from
Epidemic focus conjugate.In other words, the present invention includes that carrier protein is formed rabies virus glucoprotein connection by chemical bond
Rabies glycoproteins combined vaccine made of rabies virus glucoprotein-carrier protein immunogenic conjugates are formulated again.Institute
It is the rabies disease being isolated and purified from hydrophobin or through recombinant expression containing glycoprotein to state rabies virus glucoprotein
Malicious antigen.
Another aspect of the invention provides a kind of method preparing rabies immune originality conjugate, and the method includes following
Step：
(a) purified that Rabies Virus Antigen stoste is made；
(b) Rabies Virus Antigen stoste prepared by step (a) is subjected to chemical key connection with carrier protein and purified, made
At Rabies Virus Antigen-carrier protein immunogenic conjugates.
Another aspect of the invention provides a kind of method preparing rabies immune originality conjugate, and the method includes following
Step：
(a) hydrophobin fixed to seed culture of viruses inoculating cell, culture, harvest virus liquid, inactivation, that rabies are made in purifying is complete
Virion stoste；
(b) rabies whole virus particles stoste prepared by step (a) is subjected to chemical key connection with carrier protein and purified,
Rabies whole virus particles-carrier protein immunogenic conjugates are made.
Another aspect of the invention provides a kind of method preparing rabies immune originality conjugate, and the method includes following
Step：
(a) rabies VLP stostes are made through recombinantly expressing, purifying；
(b) rabies VLP stostes prepared by step (a) are subjected to chemical key connection with carrier protein and purified, is made mad
Dog disease VLP- carrier protein immunogenic conjugates.
Another aspect of the invention provides a kind of method preparing rabies immune originality conjugate, and the method includes following
Step：
(a) hydrophobin fixed to seed culture of viruses inoculating cell, culture, harvest virus liquid, inactivation, that rabies are made in purifying is complete
Virion stoste；
(b) hydrophobin outer membrane is made through cracking, purifying in rabies whole virus particles stoste prepared by step (a)
Section stoste；
(c) hydrophobin outer membrane segment stoste and the carrier protein for preparing step (b) carry out chemistry key connection and pure
Change, hydrophobin outer membrane segment-carrier protein immunogenic conjugates are made.
Another aspect of the invention provides a kind of method preparing rabies immune originality conjugate, and the method includes following
Step：
(a) purified that rabies virus glucoprotein stoste is made；
(b) rabies virus glucoprotein stoste prepared by step (a) is subjected to chemical key connection with carrier protein and purified,
Rabies virus glucoprotein-carrier protein immunogenic conjugates are made.
Another aspect of the invention provides a kind of method preparing rabies combined vaccine, the described method comprises the following steps：
(a) purified that Rabies Virus Antigen stoste is made；
(b) Rabies Virus Antigen stoste prepared by step (a) is subjected to chemical key connection with carrier protein and purified, made
At Rabies Virus Antigen-carrier protein immunogenic conjugates.
(c) Rabies Virus Antigen-carrier protein immunogenic conjugates prepared by step (b) are diluted, are prepared,
Packing becomes finished product vaccine.
Another aspect of the invention provides a kind of method preparing rabies combined vaccine, the described method comprises the following steps：
(a) hydrophobin fixed to seed culture of viruses inoculating cell, culture, harvest virus liquid, inactivation, that rabies are made in purifying is complete
Virion stoste；
(b) rabies whole virus particles stoste prepared by step (a) is subjected to chemical key connection with carrier protein and purified,
Rabies whole virus particles-carrier protein immunogenic conjugates are made.
(c) rabies whole virus particles-carrier protein immunogenic conjugates prepared by step (b) are diluted, matched
System, packing become finished product vaccine.
Another aspect of the invention provides a kind of method preparing rabies combined vaccine, the described method comprises the following steps：
(a) rabies VLP stostes are made through recombinantly expressing, purifying；
(b) rabies VLP stostes prepared by step (a) are subjected to chemical key connection with carrier protein and purified, is made mad
Dog disease VLP- carrier protein immunogenic conjugates.
(c) rabies VLP- carrier protein immunogenic conjugates prepared by step (b) are diluted, prepared, dispensed
As finished product vaccine.
Another aspect of the invention provides a kind of method preparing rabies combined vaccine, the described method comprises the following steps：
(a) hydrophobin fixed to seed culture of viruses inoculating cell, culture, harvest virus liquid, inactivation, that rabies are made in purifying is complete
Virion stoste；
(b) hydrophobin outer membrane is made through cracking, purifying in rabies whole virus particles stoste prepared by step (a)
Section stoste；
(c) hydrophobin outer membrane segment stoste and the carrier protein for preparing step (b) carry out chemistry key connection and pure
Change, hydrophobin outer membrane segment-carrier protein immunogenic conjugates are made.
(d) be diluted hydrophobin outer membrane segment-carrier protein immunogenic conjugates prepared by step (c),
It prepares, packing becomes finished product vaccine.
Another aspect of the invention provides a kind of method preparing rabies combined vaccine, the described method comprises the following steps：
(a) purified that rabies virus glucoprotein stoste is made；
(b) rabies virus glucoprotein stoste prepared by step (a) is subjected to chemical key connection with carrier protein and purified,
Rabies virus glucoprotein-carrier protein immunogenic conjugates are made.
(c) rabies virus glucoprotein-carrier protein immunogenic conjugates prepared by step (b) are diluted, matched
System, packing become finished product vaccine.
In term of the present invention, rabies immune originality conjugate (Rabies Immunogenic Conjugate RIC)
It is the conjugate with immunogenicity specifically referred to for hydrophobin.Rabies immune originality conjugate includes rabies disease
Malicious antigen and carrier protein are keyed between Rabies Virus Antigen and carrier protein through chemistry, carrier protein and rabies disease
Malicious antigen is keyed through chemistry, and chemical bond connects Rabies Virus Antigen to form Rabies Virus Antigen-load with carrier protein
Body protein immunogenic conjugates.In other words, rabies immune originality conjugate system's Rabies Virus Antigen is passed through with carrier protein
Rabies Virus Antigen-carrier protein conjugate that chemistry key connection is formed.
In term of the present invention, rabies combined vaccine (Rabies Conjugate Vaccine RCV) is specifically to prevent
Or the vaccine for the treatment of rabies virus infection.The antigen of rabies combined vaccine is rabies immune originality conjugate, rabies
Immunogenic conjugates include Rabies Virus Antigen and carrier protein, through chemistry between Rabies Virus Antigen and carrier protein
Key connection, carrier protein and Rabies Virus Antigen are keyed through chemistry, and chemical bond is by Rabies Virus Antigen and carrier protein
Connection forms Rabies Virus Antigen-carrier protein immunogenic conjugates.In other words, rabies combined vaccine system rabies disease
Malicious antigen is formed with carrier protein through the chemical Rabies Virus Antigen-carrier protein conjugate for being keyed formation is formulated again.
In term of the present invention, Rabies Virus Antigen (RabAg) refers to that can generate specifically to be immunized for hydrophobin
The substance of reaction, Rabies Virus Antigen contain one or more function that chemical key connection can be carried out with carrier protein
Property group.The functional groups that Rabies Virus Antigen carries out chemical key connection with carrier protein include hydroxyl (- OH) and/or carboxylic
Base (- COOH) and/or aldehyde radical (- CHO) and/or amino (- NH2).Rabies Virus Antigen can be natural, such as from warp
The rabies whole virus particles of culture, can also be the substance of recombinant expression, can also be semi-synthetic or synthesis substance.It is mad
Dog disease viral antigen includes rabies whole virus particles or includes the rabies VLP or entirely sick including rabies through recombinant expression
Outer membrane segment that malicious particle is prepared after cracking includes the glycoprotein isolated and purified from hydrophobin or including from mad dog
The nucleoprotein of sick virus isolation includes the phosphoprotein isolated and purified from hydrophobin or including from hydrophobin
The stromatin that isolates and purifies includes the polymerase isolated and purified from hydrophobin or includes the mad dog through recombinant expression
Sick viral glycoprotein includes rabies virus nucleoprotein through recombinant expression or includes the hydrophobin through recombinant expression
Phosphoprotein includes hydrophobin stromatin through recombinant expression or includes the hydrophobin poly through recombinant expression
Enzyme.
In term of the present invention, carrier protein (Carrier Protein CP) is connected with chemical bond with haptens or antigen
Connect and have the protein matter of immunogenicity, haptens or antigen, which are connected to, enhances exempting from for haptens or antigen on carrier protein
Epidemic focus and the reaction of T cell dependent immune response.Carrier protein contains one or more can be with haptens or antigen
Carry out the functional groups of chemical key connection.Carrier protein carries out the functional groups of chemical key connection with Rabies Virus Antigen
Including hydroxyl (- OH) and/or carboxyl (- COOH) and/or aldehyde radical (- CHO) and/or amino (- NH2).Carrier protein can be day
Right, such as the albumen from bacterium or virus, it can also be the substance of recombinant expression, can also be semi-synthetic or synthesis
Substance.Carrier protein includes tetanus toxoid (TT) or including diphtheria toxoid (DT) or including the nontoxic variation of diphtheria toxin
Body (CRM197) or including B group meningitis coccis outer membrane protein (OMP) or including Pneumococal surface protein A (PspA) or
Including PsaA (PsaA) or including pneumolysin (Ply) or including haemophilus influenzae D eggs
In vain (PD) or including pertussis toxin (PT) or including pertussis filamentous hemagglutinin (FHA) or including pertussis adhesin
(PRN) or including cholera toxin (CT) or including muramyl dipeptide (MDP) or including E.coli LT or including large intestine bar
Bacterium ST or including purified protein derivative of tuberculin (PPD) or including Pseudomonas aeruginosa exotoxin A (PEA) or including ovum
Albumin or including keyhole limpet hemocyanin (KLH) or including bovine serum albumin(BSA) (BSA) or including hepatitis B virus surface
Antigen (HBsAg) or including hepatitis B virus core antigen (HBcAg) or including Tetanus Toxin Fragment C (Tetanus
Toxin Fragment C,TTC)。
In term of the present invention, chemistry key connection refers in Rabies Virus Antigen-carrier protein immunogenic conjugates
Rabies Virus Antigen and carrier protein be with chemistry key connection (Linking) or crosslinking (Crosslinking) or bridging
(Bridging) form exists.In other words, chemical bond Rabies Virus Antigen is connected or is crosslinked with carrier protein or bridging at
Rabies Virus Antigen-carrier protein immunogenic conjugates.Therefore, in term of the present invention, chemistry key connection is equal or phase
It is crosslinked with chemical bond, chemistry key connection is also equal or identical chemical bond bridging.
In term of the present invention, linking arm (Linker Arm) is Rabies Virus Antigen-carrier protein immunogenicity knot
Close in object with the substance of chemistry key connection Rabies Virus Antigen and carrier protein, be Rabies Virus Antigen and carrier protein it
Between the new atom that creates or recruit.Therefore, in term of the present invention, linking arm is equal or same intervals arm (Spacer
Arm), linking arm is also equivalent or identical bridging molecule (Bridging Molecule).
It is former that the present invention provides the rabies immune that a kind of Rabies Virus Antigen and carrier protein are formed through chemistry key connection
Property conjugate, the present invention provide again it is a kind of by rabies immune originality conjugate it is formulated again made of rabies combined vaccine.
In a preferred embodiment of the present invention, the hydrophobin fixed virus for preparing Rabies Virus Antigen is selected from PAS plants, PV
Strain, PM plants, CVS plants, Nishigara plants, Flury plants, Vnukovo-32 plants, CTN-1V plants, aGV plants or other hydrophobins
Any strain in fixed virus.Hydrophobin fixed virus is PV plants more preferable, PM plants, Flury plants, CTN-1V plants, aGV plants.It is mad
Most preferably PM plants, Flury plants, CTN-1V plants of dog disease viropexis strain.Unite States Standard biology collecting center (ATCC), China's food
Product drug assay research institute sells hydrophobin fixed virus.
It is former that the present invention provides the rabies immune that a kind of Rabies Virus Antigen and carrier protein are formed through chemistry key connection
Property conjugate, the present invention provide again it is a kind of by rabies immune originality conjugate it is formulated again made of rabies combined vaccine.
In a preferred embodiment of the present invention, the Rabies Virus Antigen is selected from rabies whole virus particles, through recombinant expression
Outer membrane segment that rabies VLP, rabies whole virus particles are prepared after cracking, the sugared egg isolated and purified from hydrophobin
In vain, the nucleoprotein that is isolated and purified from hydrophobin, the phosphoprotein isolated and purified from hydrophobin are detached from hydrophobin
The stromatin of purifying, the polymerase isolated and purified from hydrophobin, the rabies virus glucoprotein through recombinant expression, through weight
The rabies virus nucleoprotein of group expression, the hydrophobin phosphoprotein through recombinant expression, the hydrophobin through recombinant expression
Stromatin, the hydrophobin polymerase through recombinant expression.The more preferable rabies whole virus particles of Rabies Virus Antigen, warp
Outer membrane segment that the rabies VLP of recombinant expression, rabies whole virus particles are prepared after cracking, detached from hydrophobin it is pure
The glycoprotein of change, the rabies virus glucoprotein through recombinant expression.Rabies Virus Antigen most preferably rabies whole virus particles.
If meeting needs, other Rabies Virus Antigens can also be used, Rabies Virus Antigen provided herein is used for the purpose of example
The property shown illustrates the present invention.
It is former that the present invention provides the rabies immune that a kind of Rabies Virus Antigen and carrier protein are formed through chemistry key connection
Property conjugate, the present invention provide again it is a kind of by rabies immune originality conjugate it is formulated again made of rabies combined vaccine.
In a preferred embodiment of the present invention, the carrier protein is selected from tetanus toxoid (TT), diphtheria toxoid (DT), diphtheria
Toxin non-toxic variant (CRM197), B group meningitis coccis outer membrane protein (OMP), Pneumococal surface protein A (PspA), lung
Scorching coccus surface adhesion element A (PsaA), pneumolysin (Ply), haemophilus influenzae D albumen (PD), pertussis toxin
(PT), pertussis filamentous hemagglutinin (FHA), pertussis adhesin (PRN), cholera toxin (CT), muramyl dipeptide (MDP), big
Enterobacteria LT, Escherichia coli ST, purified protein derivative of tuberculin (PPD), Pseudomonas aeruginosa exotoxin A (PEA), keyhole blood
Azurin (KLH), bovine serum albumin(BSA) (BSA), hepatitis b virus s antigen (HBsAg), hepatitis B virus
Former (HBcAg), Tetanus Toxin Fragment C (TTC).Carrier protein more preferable TT, DT, CRM197, PD and OMP.Carrier protein is most
It is preferred that TT and CRM197.If meeting needs, other protein carriers can also be used, protein carrier provided herein is used for the purpose of
The present invention is illustrated.
In the present invention, the preparation of Rabies Virus Antigen and method of quality control and standard are those skilled in the art
It is well known.In a preferred embodiment of the present invention, technical staff can be according to document (the JOURNAL OF MICROBIOLOGYs such as Li Zhiqiang
.2010.30(1):96-99；The CHINA virus .2004.19 such as happy prestige (4):373-375), the people that the World Health Organization promulgates
It is manufactured with rabies inactivated vaccine and vertification regulation (WHO Technical Report Series No 941,2007) and China
The technological documents such as people's republic's pharmacopeia three (version in 2015) prepare hydrophobin whole virus particles stoste and carry out quality
Calibrating.
In the present invention, the preparation of Rabies Virus Antigen and method of quality control and standard are those skilled in the art
It is well known.In a preferred embodiment of the present invention, technical staff can be according to document (Fontana D, et
al.Vaccine.2014.32:2799-2804；Fontana D,et al.Vaccine.2015.33:4238-4246；Qi Ying
Structure, preparation and the Changchun the Efficacy evaluation Jilin University .2015 of beautiful jade hydrophobin sample particles), the World Health Organization
The human rabies inactivated vaccine of promulgation manufactures and vertification regulation (WHO Technical Report Series No 941,
2007) rabies VLP stostes are prepared with technological documents such as Pharmacopoeias of People's Republic of China three (version in 2015) and carries out quality
Calibrating.
In the present invention, the preparation of Rabies Virus Antigen and method of quality control and standard are those skilled in the art
It is well known.In a preferred embodiment of the present invention, technical staff can be according to another inventive technique scheme of the present inventor
(CN100413536C), the manufacture of human rabies inactivated vaccine and vertification regulation (WHO that the World Health Organization promulgates
Technical Report Series No 941,2007) and《Pharmacopoeia of People's Republic of China》Skills such as (three versions in 2015)
Art file prepares hydrophobin outer membrane segment stoste and carries out quality arbitration.
In the present invention, the preparation of Rabies Virus Antigen and method of quality control and standard are those skilled in the art
It is well known.In a preferred embodiment of the present invention, technical staff can be according to document (Amann R, et al.Journal of
Virology.2013.87(3):1618-1630；The such as Wang Xiaolei Nanjing Medical University journal (natural science edition) .2015.35
(6):772-776；The biotechnology communications .2015.26 such as Kan Haiping (4):493-496；The straits the such as Zhang Jianming preventive medicine is miscellaneous
Will .2009,15 (2):1-4), the manufacture of human rabies inactivated vaccine and vertification regulation (WHO that the World Health Organization promulgates
Technical Report Series No 941,2007) and the technologies such as Pharmacopoeia of People's Republic of China three (version in 2015)
File prepares rabies virus glucoprotein stoste and carries out quality arbitration.
In the present invention, the principle of Rabies Virus Antigen and carrier protein through chemistry key connection and method be can refer into life
Covalent bond coupling reaction technology (Hermanson G. (2008) Bioconjugate between object macromolecular and large biological molecule
Technigues,2nd Edition.Academic Press,London and New York.).In currently preferred reality
It applies in scheme, the chemical bond connection method of Rabies Virus Antigen and carrier protein is selected from carbodlimide method (EDC), mixed acid anhydride
Method (chloromethyl isobutyl ester process), N- hydroxyl succinimides ester process, glutaraldehyde method, diazotising method, succinic anhydride method, carbonyl dimidazoles
Method, maleimide method, disulfide bond method, periodate oxidation method, carboxymethyl hydroxylamine assay etc..The more preferable carbon of chemical bond connection method two
Imines method, glutaraldehyde method, diazotising method, disulfide bond method, periodate oxidation method.Chemical bond connection method most preferably carbodiimide
Method, periodate oxidation method.If meeting needs, other chemical bond connection methods, chemistry key connection provided herein can also be used
Method is of the invention just for the sake of being illustrated.
In the present invention, the chemistry key connection of Rabies Virus Antigen and carrier protein is no linking arm (Linker
Arm) or spacerarm (Spacer Arm) or bridging molecules (Bridging Molecule) are directly connected to, also known as zero-length
Connect (Zero-Length Linking) or zero-length crosslinking (Zero-Length Crosslinking) or zero-length bridging
(Zero-Length Bridging) connects the chemical bond between Rabies Virus Antigen and carrier protein and not comprising newly-increased
Atom or molecule.In a preferred embodiment of the present invention, the chemistry key connection of Rabies Virus Antigen and carrier protein is that do not have
There are being directly connected to for linking arm or spacerarm, bridging agent to be selected from 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides
(EDC), 1- cyclohexyl -3- (2-N- morpholinyl ethyls) carbodiimide (CMC), dicyclohexylcarbodiimide (DCC), N, N'- bis-
Diisopropylcarbodiimide (DIC), 2- ethyl -5- phenyl isoxazole -3'- sulfonate (Woodward ' s reagent K), N, N'-
Carbonyl dimidazoles (CDI) and schiff bases generate and the reagent such as sodium cyanoborohydride (NaBH3CN) or boron of reductive amination process
Sodium hydride (NaBH4).Bridging agent more preferable EDC, CMC, DCC, NaBH3CN, NaBH4.Bridging agent most preferably EDC, NaBH3CN.
If meeting needs, other bridging agents can also be used, bridging agent provided herein is of the invention just for the sake of being illustrated.
In the present invention, the chemistry key connection of Rabies Virus Antigen and carrier protein be by linking arm or spacerarm or
The connection of bridging molecules, the linking arm or spacerarm or bridging molecules newly created is by Rabies Virus Antigen and carrier protein through changing
It learns key connection and forms Rabies Virus Antigen-carrier protein immunogenic conjugates, that is, connect Rabies Virus Antigen and carrier
Chemical bond between albumen includes new adatom or molecule.In a preferred embodiment of the present invention, Rabies Virus Antigen and
The chemistry key connection of carrier protein is the connection by linking arm or spacerarm or bridging molecules, linking arm or spacerarm or bridging
Molecule is selected from two thiobis (succinyl phosphorons amino propyl acid ester) (DSP), two thiobis (sulfosuccinimide base propionic ester)
(DTSSP), disuccinimidyl suberate (DSS), bis- (thiosuccimide base) suberate sodium salt (BS3), tartaric acid
Two succinimides (DST), tartaric acid disulfo succinimide ester (sulfo-DST), bis- (2- (succinimidyloxycarbonyls
Oxygen) ethyl) sulfone (BSOCOES), bis- (2- (sulfosuccinimide oxygen carbonyl oxygen) ethyl) sulfones (sulfo-BSOCOES), second two
Bis- (sulfosuccinimide ester the succinate) (sulfo- of alcohol-bis- (succinimide ester succinate) (EGS), ethylene glycol-
EGS), double amber imide glutarate (DSG), N, oneself two sub- acyls of bis- succinimidyl carbonates of N'- (DSC), dimethyl
Amine ester (DMA), heptanedioyl imines dimethyl phthalate (DMP), dimethyl-octa dinitrate (DMS), bis- the third imidic acids of thiobis of 3,3'-
Dimethyl ester (DTBP), 1,4- bis- (3'- (2'- disulfide groups pyridine) propionic acid acylamino-) butane (DPDPB), dimaleimide base oneself
Alkane (BMH), difluorodinitrobenzene (DFDNB), difluorodinitrobenzene base sulfone (DFDNPS), (β-(the 4- nitrine salicyloyls of curing two
Amino) ethyl) (BASED), formaldehyde, glutaraldehyde, 1,4- butanediols glycidol ether, adipic acid dihydrazide (ADH), carbohydrazide,
Diamino dimethyl diphenyl, benzidine, nitrogen-amber star argon ammonia -3 (2- pyridines two are thio)-acid esters (SPDP), long-chain-nitrogen -
Amber star argon ammonia -3 (2- pyridines two are thio)-acid esters (LC-SPDP), -3 (two sulphur of 2- pyridines of sulfo group long-chain-nitrogen-amber star argon ammonia
Generation)-acid esters (sulfo-LC-SPDP), succinimido oxo carbonyl-methyl-(2- pyridylthios) benzene (SMPT), sulfo group
Long-chain-succinimido oxo carbonyl-methyl-(2- pyridylthios) benzene (sulfo-LC-SMPT), 4- (N- maleimides
Aminomethyl) cyclohexane-carboxylic acid N-hydroxy-succinamide ester (SMCC), 4- (N- maleimidomethyls) hexamethylene -1- carboxylics
Thio-N- succinimides the ester sodium salt (sulfo-SMCC) of sour 3-, maleimide yl benzoic acid succinimide ester (MBS),
M- maleimidobenzoyls succinimide ester (sulfo-MBS), N- succinimides (4- iodoacteyls) aminobenzoic
Sour (SIAB), sulfo-N-succinimidyl (4- iodoacteyls) aminobenzoic acid (sulfo-SIAB), 4- (4- maleimides
Aminocarbonyl phenyl) butyric acid succinimide ester (SMPB), sulfosuccinimide base -4- (P- maleimidophenyls) butyrate
(sulfo-SMPB), 4- maleimidobutyric acids-N- succinimide esters (GMBS), sulfo group maleimidobutyric acid-N-
Succinimide ester (sulfo-GMBS), succinimido -6- ((iodoacetyl) amino) capronate (SIAX), succinyl are sub-
Amido -6- (6- (((4- iodoacteyls) amino) hexanoyl) amino) caproic acid (SIAXX), succinimido -4- (((iodo second
Acyl group) amino) methyl) hexamethylene -1- carboxylic acids (SIAC), succinimido -6- ((((4 (iodoacteyl) amino) methyl)
Hexamethylene-be -1- carbonyls) amino) caproic acid (SIACX), 4- nitro phenyl ester iodoacetic acid (NPIA), 4- (4-N- maleimide benzene
Methylamine ester) butyric acid hydrazides (MPBH), 4-N- maleimidomehyl hexamethylene -1- carboxyl hydrazides (M2C2H), 3- (2- pyridyl groups two
It is thio) propionyl hydrazides (PDPH), n-hydroxysuccinimide -4- azidos salicylic acid (NHS-ASA), N- weight ratio succinyls
Imines -4- azidos salicylic acid (sulfo-NHS-ASA), sulfosuccinimide -4- nitrine salicyl aminocaproic acids (sulfo-
NHS-LC-ASA), sulfosuccinimide base -2- (P- azidos-salicyloyl amino) ethyl -1,3'- disulfide group propionic esters
(SASD), succinimido -4- triazobenzenes formic acid esters (HSAB), sulfosuccinimide base -4- triazobenzene formic acid esters
(sulfo-HSAB), N- succinimidos -6- (4'- azido -2'- nitro-phenylaminos) capronate (SANPAH), sulfo group
Succinimido -6- (4'- azido -2'- nitro-phenylaminos) capronate (sulfo-SANPAH), 5- azido -2- nitre
Yl benzoic acid-N- succinimide esters (ANB-NOS), sulfosuccinimide -2- (M- nitroazides base-benzamido) -
Ethyl -1,3'- dithio dipropyls acid esters (SAND), N- succinimidos-(4- azidos phenyl) -1,3'- dithiopropionic acids
Ester (SADP), N- sulfosuccinimides base-(4- azidos phenyl) -1,3'- dithiopropionic acids ester (sulfo-SADP), sulfo group
Succinimido -4- (P- azidophenyls) butyric acid (Sulfo-SAPB), sulfosuccinimide -2- (7- nitrine -4- methyl first
Butylcoumariii -3- acetamides) ethyl -1,3'- dithiopropionic acids ester (SAED), sulfosuccinimide -7- azido -4- methyl
Cumarin -3- acetic acid esters (Sulfo-SAMCA), p-nitrophenyl diazonium pyruvic acid (pNPDP), p-nitrophenyl -2- diazonium 3,
3,3- trifluoroacetic acids (PNP-DTP), 1- (p- nitrine salicyloyls amino) -4- (iodacetyl amido) butane (ASIB), N- (4- (p-
Azidosalicylamides base) butyl)-3'- (two sulphur of 2'- pyridyl groups) propionamide (APDP), UVINUL MS 40-iodoacetamide, hexichol
Ketone -4- maleimides, p- azido benzoyls hydrazine, 4- (P- nitrine salicyloyls amino)-butylamine (ASBA), P- azidophenyl second
Dialdehyde (APG), 4- nitrine -2- nitrobenzophenone biotin -4- nitrobenzenes base esters (ABNP), sulfosuccinimide base -2- (6-
(biotin amido group) -2- (p- azido benzoyls amino) acylamino-s) ethyl -1,3- disulfide group propionic esters (sulfo-
SBED), four fluoro- long-chain-biotin (MTS-ATF-biotin) of methane thiosulfonic acid azido, methane thiosulfonic acid azido four
Fluorine biotin (MTS-ATF-LC-biotin), three (hydroxymethyl) hydrogen phosphide (THP), three (hydroxymethyl) phosphorus base propionic acid
(THPP).Linking arm or spacerarm or bridging molecules more preferable DTSSP, BS3、sulfo-DST、sulfo-BSOCOES、sulfo-
EGS, DSG, DSC, DMA, DMP, BMH, glutaraldehyde, ADH, carbohydrazide, sulfo-LC-SPDP, sulfo-LC-SMPT, sulfo-
SMCC、sulfo-MBS、Sulfo-SIAB、sulfo-SMPB、Sulfo-GMBS、MPBH、M2C2H、PDPH、Sulfo-NHS-LC-
ASA、Sulfo-HSAB、Sulfo-SANPAH、Sulfo-SADP、ASIB、ABH、ASBA、sulfo-SBED.Linking arm or interval
Arm or bridging molecules most preferably DTSSP, DMP, ADH, carbohydrazide, sulfo-LC-SPDP, sulfo-SMCC, sulfo-MBS,
MPBH、PDPH、sulfo-SBED.If meeting needs, other linking arms or spacerarm or bridging molecules can also be used, give herein
The linking arm or spacerarm or bridging molecules gone out is of the invention just for the sake of being illustrated.
In a currently preferred embodiment, cyanidization agent (1- cyano -4-dimethylaminopyridine tetrafluoro boron is first used
Acid esters (CDAP) or cyanogen bromide (CNBr)) activation process is carried out to carrier protein, then with adipic acid dihydrazide (ADH) be homologous double
Function connects arm is added in the carrier protein activated, forms carrier protein-ADH derivatives, adds Rabies Virus Antigen,
Condensation carrier protein-ADH derivatives by carbodiimide-mediated pass through chemical bonds, shape with Rabies Virus Antigen
It is further purified to obtain for preparing rabies combination at Rabies Virus Antigen-carrier protein immunogenic conjugates
The antigen component of vaccine.In another preferred embodiment of the present invention, cyanidization agent (1- cyano -4- dimethylaminos are first used
Pyridinium tetrafluoroborate (CDAP) or cyanogen bromide (CNBr)) activation process is carried out to Rabies Virus Antigen, then with adipoyl hydrazine
(ADH) it is that homologous difunctional linking arm is added in the Rabies Virus Antigen activated, forms Rabies Virus Antigen-ADH and spread out
Biology adds carrier protein, the condensation Rabies Virus Antigen-ADH derivatives by carbodiimide-mediated and carrier
Albumen forms Rabies Virus Antigen-carrier protein immunogenic conjugates by chemical bonds, further purified to obtain
The antigen component of rabies combined vaccine must be used to prepare.In another preferred embodiment of the present invention, cyanidization agent is first used
(1- cyano -4-dimethylaminopyridine tetrafluoro boric acid ester (CDAP) or cyanogen bromide (CNBr)) is respectively to carrier protein and rabies
Viral antigen carries out activation process, then is separately added into and has been activated with the homologous difunctional linking arm of adipic acid dihydrazide (ADH)
In carrier protein and Rabies Virus Antigen, it is respectively formed carrier protein-ADH derivatives and Rabies Virus Antigen-ADH derives
Object, then carrier protein-ADH derivatives and Rabies Virus Antigen-ADH derivatives are mixed, by the contracting of carbodiimide-mediated
Cooperation carrier protein-ADH derivatives, by chemical bonds, form rabies disease with Rabies Virus Antigen-ADH derivatives
Malicious antigen-carrier protein immunogenic conjugate, further purified antigen of the acquisition for preparing rabies combined vaccine
Component.The chemistry key connection of Rabies Virus Antigen and carrier protein can be there are many method, and connection method described herein is only
It is only for illustrating the present invention.So various equivalent methods, which may be used, realizes connection of the present invention, therefore any use
It is likely in the method and technique or process of Rabies Virus Antigen and carrier protein chemistry key connection for implementing this hair
It is bright.
It is provided by the invention it is a kind of by rabies immune originality conjugate it is formulated again made of rabies combined vaccine
Include effective dose and with Rabies Virus Antigen of the carrier protein through chemical bonds.It is promulgated according to the World Health Organization
Human rabies inactivated vaccine manufacture and vertification regulation (WHO Technical Report Series No 941,2007),
European Pharmacopoeia Antirabic Vaccine manufactures vertification regulation (European Pharmacopoeia.Rabies vaccine for
Human use in prepared cell cultures) and《Pharmacopoeia of People's Republic of China》(three versions in 2015) people uses
Rabies vacciness manufactures the requirement of vertification regulation, and the Antirabic Vaccine of a dosage includes >=hydrophobin of 2.5IU
Antigen.WHO, European Pharmacopoeia and Chinese Pharmacopoeia have promulgated the NIH methods and standard of Antirabic Vaccine's efficacy determinations.At this
In invention preferred embodiment, the potency of rabies immune originality conjugate and rabies combined vaccine is through NIH effect
Test method measures, and potency unit is denoted as >=2.5IU/ agent, and every dose is 0.5ml or 1ml.
It is provided by the invention it is a kind of by rabies immune originality conjugate it is formulated again made of rabies combined vaccine,
In the preferred embodiment of the invention, rabies combined vaccine includes human albumin as protective agent or stabilizer.In the present invention
In preferred another embodiment, with carrier protein of the Rabies Virus Antigen through chemical bonds completely instead of existing mad dog
It needs that function of the human albumin as protective agent or stabilizer is added in disease vaccine.The main work(of human albumin in the prior art
Can one of be by preventing hydrophobin particle aggregation, and then avoiding the reduction in Rabies Virus Antigen site in vaccine,
To realize the function of protective agent or stabilizer as Rabies Virus Antigen.Rabies Virus Antigen in the present invention due to
It is the antigen through chemical bonds carrier protein, the Rabies Virus Antigen through chemical bonds carrier protein is because of carrier
The space obstacle of albumen, prevents the aggregation of Rabies Virus Antigen, and then avoids Rabies Virus Antigen site in vaccine
Reduction, therefore the present invention rabies combined vaccine include human albumin.In currently preferred another embodiment,
Rabies combined vaccine do not include and the functionally similar protective agent of human albumin or stabilizer yet.
It is provided by the invention it is a kind of by rabies immune originality conjugate it is formulated again made of rabies combined vaccine,
In the preferred embodiment of the invention, rabies combined vaccine also may include adjuvant commonly used in the art.Preferred adjuvant has aluminium
Adjuvant, calcium phosphate adjuvant, cholera toxin (CT), choleratoxin B subunit (CTB), pertussis toxin (PT), pertussis toxin B
Subunit (PTB), pertussis filamentous hemagglutinin (FHA), pertussis adhesin (PRN), Soap chitins QS-21, alpha-tocopherol, spiny dogfish
Alkene, lipoid, liposome (liposomes), Monophosphate Lipid A (MPL-A), MF59, viruslike particle proteoliposome
(Virosomes), polyglycolide (PLA) microballoon, polylactic acid-glycollic acid (PLGA) microballoon, lipid-cholesterol (DC-Chol), two
Methyl double octadecyl quaternary ammonium bromides (DDA), immunostimulating complex (ISCOM), Montanide ISA50, Montanide
ISA51, Montanide ISA206, Montanide ISA720, Montanide ISA series of adjuvants, AS01, AS02, AS03,
AS04, AS series of adjuvants, muramyl dipeptide (MDP), bacteria lipopolysaccharide (OM-174), e. coli heat-labile toxin (LT), IL-
1, IL-2, IL-6, IL-12, IL-15, IL-18, IFN-γ, GM-CSF, CpG ODN, trehalose dimycolate
(TDM), contain the pick up adjuvant of poly- inosinic acid (Poly I) and/or poly- cytidylic acid (Poly C).It is more excellent
The adjuvant of choosing be QS-21, MPL-A, MF59, Virosomes, Montanide ISA50, Montanide ISA51,
Montanide ISA206, Montanide ISA720, AS01, AS02, AS03, AS04, CpG, pick up adjuvant.Most preferred assistant
Agent is MF59, Virosomes, Montanide ISA51, AS03, AS04, CpG, pick up adjuvant.
It is provided by the invention it is a kind of by rabies immune originality conjugate it is formulated again made of rabies combined vaccine,
In the preferred embodiment of the invention, rabies combined vaccine includes preservative, and preferred preservative is thimerosal, 2- benzene oxygen second
Alcohol, benzyl alcohol.Preferred preservative is 2- Phenoxyethanols.In currently preferred another embodiment, rabies combine
Vaccine is the vaccine not comprising any preservative free.
It is provided by the invention it is a kind of by rabies immune originality conjugate it is formulated again made of rabies combined vaccine
In, the dosage form and compatibility of rabies combined vaccine are determined according to the technical standard well known to pharmaceutical field those skilled in the art.
In a preferred embodiment of the present invention, rabies combined vaccine dosage form be preferably liquid dosage form, freeze-dried formulation, pill, tablet,
Capsule formulation, more preferably liquid dosage form or freeze-dried formulation, most preferably freeze-dried formulation.
It is provided by the invention it is a kind of by rabies immune originality conjugate it is formulated again made of rabies combined vaccine
In, rabies combined vaccine route of inoculation includes intracutaneous injection, hypodermic injection, intramuscular injection, intraperitoneal injection, further includes intranasal
Chamber, oral cavity, anus, vagina, oromucosal route administration.
Explanation：
The linking arm or spacerarm or bridging molecules of new establishment of the present invention, refer in Rabies Virus Antigen and load
The linking arm or spacerarm or bridging molecules that body protein is formed in the reaction process through chemistry key connection；The new adatom divides
Son refers to Rabies Virus Antigen and carrier in the reaction process of Rabies Virus Antigen and carrier protein through chemistry key connection
The atom or molecule newly formed between albumen.
The biological or chemicals such as hydrophobin fixed virus of the present invention, adjuvant, chemical bond bridging agent, protide reagent
Preparation is existing by commercially available product in addition to specified otherwise preparation method and technology.In the present invention, described mad
Dog disease viropexis strain is purchased from Chinese drug and food calibrating research institute or Unite States Standard biology collecting center (ATCC) or U.S.
Disease prevention and control center of state (CDC).
Disclosure above has carried out general description to the present invention, can be but unrestricted by following examples, lifts
Example further understands perhaps spiritual in core of the invention.The embodiment be used merely to explain idea of the invention and it is several preferably
Embodiment, should not be construed as limiting the scope of the invention in any way.It should be understood that the present invention is not limited to have as follows
Body embodiment, and to following examples carry out a variety of variations, adjustment, modification, modification, change, improvement, improvement, reduction,
Simplifying, increasing, increasing etc. can be implemented by those skilled in the art, without departing from the scope or spirit of the invention.The present invention's
Other embodiments are predicted under the premise of without departing substantially from core of the invention by those skilled in the art.
Description of the drawings
Fig. 1 is that the rabies immune originality conjugate column chromatography of the preferred embodiment of the present invention 3 purifies collection of illustrative plates
Specific implementation mode
The present invention can be by being discussed further, by Rabies Virus Antigen and carrier protein in conjunction with following examples
It learns bond to close, rabies immune originality conjugate is made, then formulated at rabies combined vaccine, rabies combined vaccine is through dynamic
Object is tested and laboratory verification is proved with good safety and outstanding immunogenicity and remarkable Vaccine effectiveness.The reality
It applies example and has selected several preferred embodiments, only for more preferably illustrating the present invention, rather than invention content is limited.
Embodiment 1
Rabies whole virus particles antigen stock prepares and vaccine formulation：
It takes Vero cell works library cell to be recovered in 37 DEG C, amplification cultivation, and 3-5 generations, cell density is passed through continuous
Reach 1.0 × 105~1.0 × 106/ ml, the bioreactor for being seeded to the microcarrier containing 1~25g/L carry out tank stream culture.Micro- load
Body high density suspension culture 5~7 days, cell density reaches 1.0 × 106~1.0 × 107/ ml, uses maintaining liquid instead.By 0.001~
0.1MOI virus inoculation titres are not less than 7.5lg LD50CTN-1V plants of the hydrophobin fixed virus of/ml.Use maintaining liquid instead,
In 33~35 DEG C of tank stream cultures.Start within 3rd day continuous harvest virus liquid, can continuously harvest virus liquid 20 days or more.The disease of harvest
The clarified filtering of venom, 100~300KD films are concentrated by ultrafiltration 10~30 times.By 1:2000~1:8000 are added beta-propiolactone, set 4
DEG C 18~24 hours inactivation of viruses, then through 37 DEG C of water-baths 2 hours so that remaining beta-propiolactone complete hydrolysis, and through carrying out virus
Inactivation verification.Virus liquid after inactivation is purified with Sepharose 4FF gel filtration chromatographies, collects the V containing whole virus particles0Peak,
It is rabies whole virus particles antigen stock that 1~10% human serum albumin of antigen stabilizer, which is added, in virus liquid after purification.
With ELISA measure rabies whole virus particles antigen stock in rabies virus antigen content, be added disodium hydrogen phosphate,
Sodium dihydrogen phosphate, sodium chloride, water for injection, formulated, tune pH7.0~8.0, packing, as vaccine.It is issued by the World Health Organization
The Antirabic Vaccine of cloth manufactures and vertification regulation (WHO Technical Report Series, No.941,2007),《In
Magnificent people's republic's pharmacopeia》Antirabic Vaccine's manufacture that (version in 2015) is promulgated and vertification regulation prescriptive procedure and standard into
Row is examined and determine, and is human rabies whole virus particles vaccine (RV-Vero) after assay approval.
Embodiment 2
The preparation of carrier protein-tetanus toxoid (TT)
By the lockjaw freeze-drying lactobacillus breakdown of Cord blood, it is seeded to the agar semisolid culturemedium of beef infusion broth containing tryptone,
It is cultivated 24~48 hours in 33~37 DEG C.Transferred species is cultivated 24~48 hours to semisolid culturemedium in 33~37 DEG C.Transferred species is extremely again
Zengjing Granule is carried out in the seed bottle of the fluid nutrient medium of beef infusion broth containing tryptone, is cultivated 24~48 hours in 33~37 DEG C.By bacterium
In kind culture in glassware object transferred species to the fermentation tank of double peptone fluid nutrient mediums, cultivated 60~80 hours in 33~37 DEG C.Fermentation finishes,
The formaldehyde sterilization treatment of final concentration 0.1~0.5% is added, through supernatant is collected by centrifugation.Supernatant is clear through 0.45~1.2 μm of filter
Clear filtering, then be concentrated by ultrafiltration through 50~100KD films.It is separately added into NaHCO3To final concentration 0.2~0.8%, (NH4)2SO4To end
Concentration 15.0~20.0% stirs 30 minutes, sets and be stored at room temperature 12~48 hours.Supernatant is collected by centrifugation.In the supernatant of collection
(the NH of final concentration 6.0~12.0% is further added by liquid4)2SO4, stir 30 minutes, set and be stored at room temperature 12~24 hours.Centrifugation is received
Collection precipitates and is dissolved in 0.1%NaHCO3In solution, ammonium is removed with 50~100KD film ultrafiltration.NaCl is added to final concentration 0.85%,
Formaldehyde is added to final concentration 0.15~0.25%, sets 37 DEG C of detoxifications 15~31 days.Formaldehyde is removed with 50~100KD film ultrafiltration,
It is stoste through the purifying of Sephacryl S-300 gel filtration chromatographies, then through 0.22 μm of filter aseptic filtration.The original of detoxification after purification
Liquid is pressed《Pharmacopoeia of People's Republic of China》(version in 2015)《Tetanol》Gainer and method calibrating, assay approval
It is afterwards tetanus toxoid stoste, sets 2~8 DEG C and save backup.
Embodiment 3
Rabies whole virus particles-carrier protein immunogenic conjugates prepare and vaccine formulation：
Tetanus toxoid stoste is measured, 1~10mg/ml is diluted to the pH 0.1M PBS for being 7.5.100mg/ml is added
CDAP (1- cyano -4- dimethylaminos-pyridinium tetrafluoroborate) acetonitrile solutions react 30 to 0.5~1.5mg/mgTT of final concentration
After second, 0.2M TEA (triethylamine) are added, TEA additions are 1~3 with CDAP addition ratios:1 (v/v) adjusts pH to 9.0,
Maintain reaction 2~5 minutes.ADH (adipoyl hydrazine) NaHCO is added3(0.1M) solution is tieed up to 2.0~4.0mg/mgTT of final concentration
Hold reaction 120 minutes.Residual chemical agents are removed with 50~100KD film ultrafiltration, as tetanus toxoid-ADH derivatives are former
Liquid.Rabies whole virus particles purifying stoste that is being prepared according to 1 method of the present embodiment and not adding human serum albumin is measured, by mad
Dog disease whole virus particles stock protein content is 1~5 with tetanus toxoid-ADH derivative stock protein content ratios:1(w/w)
Ratio, mixing rabies whole virus particles purifying stoste and tetanus toxoid-ADH derivative stostes.PH is adjusted to 5.0~
5.8, the sulfo-NHS (N- hydroxy thiosuccinimides) of final concentration of 10~25mM is added, adding EDAC, (carbon two is sub-
Amine) to 25~100mM of final concentration, maintain reaction 240 minutes.Reaction terminates, and adjusts pH to 6.8~7.5, stands overnight.With
Sepharose 4FF gel filtration chromatographies purify, and collect the V containing whole virus particles-carrier protein immunogenic conjugates0Peak, warp
0.22 μm of filter aseptic filtration is rabies whole virus particles-carrier protein immunogenic conjugates stoste, sets 2~8 DEG C of guarantors
It deposits spare.
Fig. 1 shows rabies viruses whole virus particles-carrier protein immunogenic conjugates through Sepharose 4FF gels
Column chromatography purifies 280nm collection of illustrative plates, observes that the Forward of carrier protein signal is formed with rabies whole virus particles antigen after bonding
Conjugate, carrier protein combined with rabies whole virus particles to form immunogenic conjugates after it is unbonded almost without leaving
Floating preteins.
Rabies virus antigen in rabies whole virus particles-carrier protein immunogenic conjugates stoste is measured with ELISA
Content, be added polyoxyethylene sorbitan monoleate (50-300 μ g/ml), succinate buffer (300-800 μ g/ml), sodium chloride (7.5~
9.5mg/ml), water for injection, formulated, adjusting pH to 5.0~7.0, packing 0.5-1.0ml/ agent, as vaccine.It is defended by the world
Antirabic Vaccine's manufacture that raw tissue is promulgated and vertification regulation (WHO Technical Report Series, No.941,
2007)、《Pharmacopoeia of People's Republic of China》The Antirabic Vaccine's manufacture and vertification regulation regulation side that (version in 2015) is promulgated
Method and standard are examined and determine, and are human rabies whole virus particles combined vaccine (RCV-VP) after assay approval.
Embodiment 4
Hydrophobin outer membrane segment-carrier protein immunogenic conjugates prepare and vaccine formulation：
By 1 method of the present embodiment prepare and plus human serum albumin rabies whole virus particles purifying stoste by invention
Another invention the method (CN200610152928.7) of person through final concentration 0.2~1.0% Triton-100 cracking, 10~
60% sucrose density gradient centrifugation or the purifying of Sepharose 4FF gel filtration chromatography methods, 50~100KD films are concentrated by ultrafiltration, system
The standby hydrophobin outer membrane segment stoste containing furcella and stromatin.Hydrophobin outer membrane segment stoste is pressed into this implementation again
3 method of example prepares hydrophobin outer membrane segment-carrier with tetanus toxoid-ADH derivatives under EDAC condensations
Protein immunogenic conjugate stoste.
Further hydrophobin outer membrane segment-carrier protein immunogenic conjugates stoste is used by 3 method of embodiment
ELISA measures rabies virus antigen content in hydrophobin outer membrane segment-carrier protein immunogenic conjugates stoste, is added
Polyoxyethylene sorbitan monoleate (50-300 μ g/ml), succinate buffer (300-800 μ g/ml), sodium chloride (7.5~9.5mg/ml), injection
With water, formulated, adjusting pH to 5.0~7.0, packing 0.5-1.0ml/ agent, as vaccine.The people promulgated by the World Health Organization
Manufactured with rabies vacciness and vertification regulation (WHO Technical Report Series, No.941,2007),《The Chinese people
Republic's pharmacopeia》Antirabic Vaccine's manufacture of (version in 2015) promulgation and vertification regulation prescriptive procedure and standard are examined
It is fixed, after assay approval combined vaccine (RCV-split) is cracked for human rabies.
Embodiment 5
Hydrophobin sample particle (VLP)-carrier protein immunogenic conjugates prepare and vaccine formulation：
With reference to neat beautiful jade beautiful jade method (structure, preparation and the Efficacy evaluation Changchun of neat beautiful jade beautiful jade hydrophobin sample particles
Jilin University .2015) hydrophobin sample particle stoste is prepared, then hydrophobin sample particle stoste is pressed into 3 side of the present embodiment
Method prepares hydrophobin sample particle stoste-carrier protein with tetanus toxoid-ADH derivatives under EDAC condensations
Immunogenic conjugates stoste.
Hydrophobin sample particle stoste-carrier protein immunogenic conjugates stoste is further pressed into 3 method of embodiment
Rabies virus antigen content in hydrophobin sample particle-carrier protein immunogenic conjugates stoste is measured with ELISA, is added
Polyoxyethylene sorbitan monoleate (50-300 μ g/ml), succinate buffer (300-800 μ g/ml), sodium chloride (7.5~9.5mg/ml), injection
With water, formulated, adjusting pH to 5.0~7.0, packing 0.5-1.0ml/ agent, as vaccine.The people promulgated by the World Health Organization
Manufactured with rabies vacciness and vertification regulation (WHO Technical Report Series, No.941,2007),《The Chinese people
Republic's pharmacopeia》Antirabic Vaccine's manufacture of (version in 2015) promulgation and vertification regulation prescriptive procedure and standard are examined
It is fixed, it is people's hydrophobin sample particle combined vaccine (RCV-VLP) after assay approval.
Embodiment 6
Rabies virus glucoprotein-carrier protein immunogenic conjugates prepare and vaccine formulation：
With reference to the Zhang Jianming method (straits the such as Zhang Jianming preventive medicine magazine .2009,15 (2):1-4) Prepare restructuring is expressed
Rabies virus glucoprotein (GP) stoste, then by rabies virus glucoprotein stoste press 3 method of the present embodiment and tetanus
It is former that toxin-ADH derivatives prepare rabies virus glucoprotein-carrier protein immunogenic conjugates under EDAC condensations
Liquid.
Further rabies virus glucoprotein-carrier protein immunogenic conjugates stoste is used by 3 method of embodiment
ELISA measures rabies virus antigen content in rabies virus glucoprotein-carrier protein immunogenic conjugates stoste, is added poly-
Sorb ester 80 (50-300 μ g/ml), succinate buffer (300-800 μ g/ml), sodium chloride (7.5~9.5mg/ml), injection
Water, formulated, adjusting pH to 5.0~7.0, packing 0.5-1.0ml/ agent, as vaccine.It is used by the people that the World Health Organization promulgates
Rabies vacciness manufacture and vertification regulation (WHO Technical Report Series, No.941,2007),《The Chinese people are total
With state's pharmacopeia》Antirabic Vaccine's manufacture of (version in 2015) promulgation and vertification regulation prescriptive procedure and standard are examined and determine,
It is people's rabies glycoproteins combined vaccine (RCV-G) after assay approval.
Embodiment 7
The inspection of rabies combined vaccine specific toxicities, Poison Reverse inspection
Separately sampled embodiment 3, embodiment 4, embodiment 5,4 kinds of hydrophobin-carrier proteins prepared by embodiment 6
Simultaneously equivalent is merged into 8 parts of test specimens for immunogenic conjugates stoste and 4 kinds of human rabies combined vaccines.Every part of test specimen
The cavy 4 of 250~350g weight is injected, injecting pathway is subcutaneous abdomen, and injection dosage is 2ml/, the 7th day after injection,
Carry out within 14th day and the 21st day observation cavy symptom.Observation period all test specimen injection sites take off without suppuration, without necrosis, nothing
Skin increased before being injected without depilation, the tetanus signs such as no late period paralysis, and every cavy weight ratio.
Separately sampled embodiment 3, embodiment 4, embodiment 5,4 kinds of hydrophobin-carrier proteins prepared by embodiment 6
Simultaneously equivalent is merged into 8 parts of test specimens for immunogenic conjugates stoste and 4 kinds of human rabies combined vaccines, sets 37 DEG C and places 42
It.The cavy 4 of every part of test specimen injection 250~350g weight, injecting pathway are that veutro is subcutaneous, injection dosage 5ml/
Only, observation cavy symptom is carried out within the 7th day, the 14th day and the 21st day after injection.Observation period all test specimen injection sites without
Suppurate, without necrosis, without decortication, without depilation, tetanus signs such as no late period paralysis, and before every cavy weight ratio injection
Increase.
Illustrate the 4 kinds of hydrophobins-carrier protein immunogenic conjugates stoste of the invention prepared and 4 kinds of people with mad dog
Tetanus toxoid carrier used in sick combined vaccine does not occur after a series of processing of special combination processing steps
Tetanus toxin specific toxicities react, and Poison Reverse phenomenon also do not occur.
Embodiment 8
Rabies combined vaccine security inspection
Separately sampled embodiment 3, embodiment 4, embodiment 5,4 kinds of hydrophobin-carrier proteins prepared by embodiment 6
Simultaneously equivalent is merged into 8 parts of test specimens for immunogenic conjugates stoste and 4 kinds of human rabies combined vaccines.Every part of test specimen
Selection 18~22g of weight mouse 5 and 250~350g of weight cavys 2 respectively, every mouse peritoneal injected sample 0.5ml, often
Guinea pig intraperitoneal injection 5.0ml is observed 7 days, records changes of weight.As a result show that the equal key of all mouse, cavy is deposited in the observation period,
And it is no different paradoxical reaction, there is increase before every mouse, the injection of cavy weight ratio when expiring.
Illustrate the 4 kinds of hydrophobins-carrier protein immunogenic conjugates stoste of the invention prepared and 4 kinds of people with mad dog
Without polluting exogenous toxicant in sick combined vaccine, also without there is unexpected uneasy total divisor.
Embodiment 9
Rabies combined vaccine immunogenicity-Antibody dynamics measure
Human rabies whole virus particles vaccine (RV- prepared by 3 traditional technologies of separately sampled embodiment 1 and embodiment
Vero) and human rabies totivirus combined vaccine (RCV-VP) and physiological sodium chloride solution that 6~8 week old BALB/c are immunized is small
Mouse, each sample are immunized 6 mouse, and only, immunization route is subcutaneous by immunizing dose 0.1ml/, immune programme 0,3,7,14 days, in 3,
7, it takes a blood sample through abdominal aorta within 14,28 days, centrifuges serum.With ELISA method measure the special IgG, IgG1 of serum anti-RabAg,
IgG2a antibody measures anti-rabies virus neutralizing antibody titers (Virus Neutralization with RFFIT methods
Antibody, VNA), it is as a result shown in Table 1, table 2, table 3, table 4 respectively, it is therein statistics indicate that being combined in human rabies totivirus
The IgG GMT of vaccine (RCV-VP) early stage are horizontal and Conversion rate, IgG1GMT are horizontal and Conversion rate, IgG2a GMT are horizontal and sun turns
Rate, VNA GMT are horizontal and Conversion rate is above Vero cell Antirabic Vaccines (RV- prepared by existing traditional technology
Vero).Rabies combined vaccine Antibody dynamics test result shows human rabies totivirus combined vaccine (RCV-VP) early stage
It generates antibody titer and Positive seroconversion rate is better than Vero cell Antirabic Vaccines prepared by traditional technology.Therefore, of the invention
The rabies immune originality conjugate and vaccine of offer, and the rabies immune originality knot using the method for the invention preparation
Closing object and vaccine has beneficial technique effect, can be in the immune response in inoculation early stage more quick inductor, favorably
Drug effect is played before early stage in incubation period or disease incidence after patient infects rabies viruses in realizing, saves the life of patient.
Compared with 1. rabies combined vaccine of table generates IgG GMT (Conversion rate) with Vero cell rabies
Compared with 2. rabies combined vaccine of table generates IgG1GMT (Conversion rate) with Vero cell rabies
Compared with 3. rabies combined vaccine of table generates IgG2a GMT (Conversion rate) with Vero cell rabies
Compared with 4. rabies combined vaccine of table generates VNA GMT (Conversion rate) with Vero cell rabies
Embodiment 10
Rabies combined vaccine Vaccine effectiveness measures
Separately sampled embodiment 1, embodiment 3, embodiment 4, embodiment 5,5 kinds of human rabies prepared by embodiment 6
Combined vaccine and human rabies press 1 with reference to vaccine:25、1:125、1:625 dilutions, with the human rabies knot of different dilutions
It closes vaccine and human rabies and 14~16g mouse 16 is immunized respectively with reference to vaccine, every mouse peritoneal is immunized 0.5ml, is immunized
Twice, it is spaced 7 days.After just exempting from 14 days, with containing 5-100LD50The rabies virus strain CVS progress intracerebral attacks of virus quantity, every
0.03ml.Observation 14 days, statistics dead and in typical rabies brain symptom mouse after the 5th day, calculates ED50Value, then calculate epidemic disease
Seedling potency.By the data in table 5 it is found that compared with RV-Vero prepared by existing conventional method, rabies combined vaccine has more
High potency.
5. rabies combined vaccine titration result of table
Claims (21)
1.A method of rabies immune originality conjugate is prepared, the described method comprises the following steps：
(a) purified that Rabies Virus Antigen stoste is made；
(b) the Rabies Virus Antigen stoste for preparing step (a) carries out chemical key connection with carrier protein, then purified system
At Rabies Virus Antigen-carrier protein immunogenic conjugates, carrier protein is tetanus toxoid (TT), chemistry key connection
It is to removePeptide connectsBeing connected chemically in addition, the chemical bond connection method of Rabies Virus Antigen and the carrier protein includes carbon two
Imines method, mixed anhydride method, N- hydroxyl succinimides ester process, glutaraldehyde method, diazotising method, succinic anhydride method, carbonyl dimidazoles
At least one of method, maleimide method, disulfide bond method, periodate oxidation method, carboxymethyl hydroxylamine assay.
2. according to the method described in claim 1, it is characterized in that, the Rabies Virus Antigen includes rabies totivirus
Grain, the hydrophobin outer membrane prepared after cracking through the hydrophobin sample particle of recombinant expression, rabies whole virus particles
Segment, the dog disease viral glycoprotein isolated and purified from hydrophobin, the nucleoprotein isolated and purified from hydrophobin, from mad dog
The phosphoprotein of sick virus isolation, is isolated and purified from hydrophobin the stromatin isolated and purified from hydrophobin
Polymerase, the rabies virus glucoprotein through recombinant expression, the rabies virus nucleoprotein through recombinant expression, through recombinant expression
Hydrophobin phosphoprotein, the hydrophobin stromatin through recombinant expression, the hydrophobin polymerase through recombinant expression
At least one of.
3. according to the method described in claim 2, it is characterized in that, in step (a), the Rabies Virus Antigen stoste is will
Hydrophobin fixes that seed culture of viruses inoculating cell, culture, harvest virus liquid, inactivation, rabies whole virus particles are former made of purifying
Liquid.
4. according to the method described in claim 2, it is characterized in that, in step (a), the Rabies Virus Antigen stoste is warp
Hydrophobin sample particle stoste made of recombinant expression, purifying.
5. according to the method described in claim 2, it is characterized in that, in step (a), the Rabies Virus Antigen stoste be by
According to the hydrophobin outer membrane segment stoste that following steps obtain, hydrophobin fixes seed culture of viruses inoculating cell, culture, harvest disease
Venom, inactivation, purifying and manufactured rabies whole virus particles stoste；The rabies whole virus particles prepared in above-mentioned steps are former
Hydrophobin outer membrane segment stoste is made through cracking, purifying in liquid.
6. according to the method described in claim 2, it is characterized in that, in step (a), the Rabies Virus Antigen stoste is warp
Rabies virus glucoprotein stoste made of purifying.
7. method according to any one of claims 1 to 6, which is characterized in that the Rabies Virus Antigen is according to choosing
Extremely from PAS plants, PV plants, PM plants, CVS plants, Nishigara plants, Flury plants, Vnukovo-32 plants, CTN-1V plants, aGV plants
Rabies Virus Antigen prepared by a kind of few hydrophobin fixed virus.
8. method according to any one of claims 1 to 6, which is characterized in that the Rabies Virus Antigen is according to choosing
At least from PAS plants, PV plants, PM plants, CVS plants, Nishigara plants, Flury plants, Vnukovo-32 plants, CTN-1V plants, aGV plants
Rabies Virus Antigen prepared by a kind of hydrophobin fixed virus DNA recombinant expression.
9. the method according to the description of claim 7 is characterized in that the Rabies Virus Antigen contains one or more
The functional groups of chemical key connection are carried out with carrier protein.
10. according to the method described in claim 9, it is characterized in that, the Rabies Virus Antigen contain one or two with
The upper functional groups that chemistry key connection is carried out with carrier protein include hydroxyl (- OH), carboxyl (- COOH), aldehyde radical (- CHO) and
Amino (- NH2) at least one.
11. the method according to the description of claim 7 is characterized in that the carrier protein contain one or more with it is mad
Dog disease viral antigen carries out the functional groups of chemical key connection.
12. according to the method for claim 11, which is characterized in that the carrier protein contain one or more with it is mad
The functional groups that dog disease viral antigen carries out chemical key connection include hydroxyl (- OH), carboxyl (- COOH), aldehyde radical (- CHO), ammonia
Base (- NH2) at least one.
13. the method according to the description of claim 7 is characterized in that the Rabies Virus Antigen and the carrier protein
Chemical bond bridging agent includes 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC), 1- cyclohexyl -3- (2-N-
Morpholinyl ethyl) carbodiimide (CMC), dicyclohexylcarbodiimide (DCC), N, N'- diisopropylcarbodiimide (DIC), 2-
Ethyl -5- phenyl isoxazole -3'- sulfonate (Woodward ' s reagent K), N, N'- carbonyl dimidazoles (CDI), schiff bases
Generate the reagent sodium cyanoborohydride (NaBH with reductive amination process3) or sodium borohydride (NaBH CN4At least one of).
14. the method according to the description of claim 7 is characterized in that the chemistry of the Rabies Virus Antigen and carrier protein
Key connection is to be directly connected to, described to be directly connected to not include linking arm (Linker Arm), spacerarm (Spacer Arm) and bridge
Join molecule (Bridging Molecule), Rabies Virus Antigen is that zero-length connects (Zero-Length with carrier protein
Linking) or zero-length is crosslinked (Zero-Length Crosslinking) or zero-length bridging (Zero-Length
Bridging), the chemical bond of connection Rabies Virus Antigen and carrier protein is between Rabies Virus Antigen and carrier protein
And not comprising newly-increased atom or molecule.
15. the method according to the description of claim 7 is characterized in that the chemistry of the Rabies Virus Antigen and carrier protein
Key connection is by the connection of linking arm, spacerarm or bridging molecules, and the linking arm, spacerarm or the bridging molecules that newly create will be mad
Dog disease viral antigen and carrier protein form Rabies Virus Antigen-carrier protein immunogenic conjugates through chemistry key connection,
The chemical bond for connecting Rabies Virus Antigen and carrier protein is former comprising increasing newly between Rabies Virus Antigen and carrier protein
Son or molecule.
16. the method according to claims 14 or 15, which is characterized in that the linking arm or spacerarm or bridging molecules are
Adipic acid dihydrazide (ADH).
17. according to claim 1 to 6,9 to 14 any one of them methods, which is characterized in that the Rabies Virus Antigen warp
It is keyed again by chemistry with carrier protein after activation.
18. according to claim 1 to 6,9 to 14 any one of them methods, which is characterized in that after the carrier protein is activated
It is keyed again by chemistry with the Rabies Virus Antigen.
19. according to claim 1 to 6,9 to 14 any one of them methods, which is characterized in that the Rabies Virus Antigen and
It is keyed again by chemistry after the carrier protein difference is activated.
20. according to any one of claim 1 to 6,9 to 14 the method, which is characterized in that the rabies immune being prepared is former
Property conjugate preparing the application prevented and treated on rabic drug.
21. according to any one of claim 1 to 6,9 to 14 the method, which is characterized in that the rabies immune being prepared is former
Property conjugate prepare prevent, treat or auxiliary for treating cancer drug on application.
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| CN201810179166.2A CN108187038B (en) | 2017-05-25 | 2017-05-25 | Rabies combined vaccine |
| CN201810179167.7A CN108295252B (en) | 2017-05-25 | 2017-05-25 | Method for preparing rabies combined vaccine |
| CN201810178918.3A CN108175855B (en) | 2017-05-25 | 2017-05-25 | Rabies immunogenic conjugate |
| CN201710381237.2A CN107137703B (en) | 2017-05-25 | 2017-05-25 | A kind of rabies immune originality conjugate and vaccine and preparation method thereof |
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| CN201810179167.7A Division CN108295252B (en) | 2017-05-25 | 2017-05-25 | Method for preparing rabies combined vaccine |
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| CN201710381237.2A Active CN107137703B (en) | 2017-05-25 | 2017-05-25 | A kind of rabies immune originality conjugate and vaccine and preparation method thereof |
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| CN112098640B (en) * | 2020-09-16 | 2021-12-14 | 浙江正熙生物技术股份有限公司 | Fluorescent protein and/or coupled protein monoclonal antibody marking method and kit thereof |
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