CN108175855A - A kind of rabies immune originality conjugate - Google Patents

Abstract

The present invention relates to a kind of rabies immune originality conjugates（Rabies Immunogenic Conjugate,RIC）, including Rabies Virus Antigen（RabAg）And carrier protein（Carrier Protein,CP）It is characterized in that, the Rabies Virus Antigen includes at least and Rabies Virus Antigen of the carrier protein through chemistry key connection, the carrier protein is included at least to be connected to form Rabies Virus Antigen carrier protein immunogenic conjugates with carrier protein with carrier protein of the Rabies Virus Antigen through chemistry key connection, the chemical bond by Rabies Virus Antigen.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, has preferable effect.

Description

A kind of rabies immune originality conjugate

The application is application No. is 201710381237.2, and the applying date is on May 25th, 2017, entitled " a kind of The divisional application of rabies immune originality conjugate and vaccine and preparation method thereof ".

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 the human rabies case hair more than 95% 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 be 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 hydrophobin is exposed to 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). The incubation period of report 416 cases of Anhui Province's rabies such as Cao Minghua is most only 2 days short, and (such as Cao Minghua pacify within up to 5606 days Emblem preventive medicine magazine .2013,19 (1):1‐4).The incubation period of report 2065 cases of Guizhou Province's rabies such as Yu Chun is most short only It is 2 days, the 13 years (such as Yu Chun medical faunaes prevention .2016 (2) of longest: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 the effect of PEP (Wilde H.Vaccine.2007,25 (44):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, it is impossible to 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.The crude vaccine or present purified vaccine of early stage, 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 in vivo by MHC class Ⅱmolecules, 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).Rabies Working Committee of Advisory Board analysis 1976- is immunized in the U.S. The vaccine clinical research that 12 are delivered between 2008, the whole virus particles vaccine of existing rabies inactivation all generate in human body The 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).

It is that rabies cannot be prevented dead only by the PEP of vaccine in the case of no immunoglobulin joint injection (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, then as a result it is shown in no use in conjunction with rabies vaccine for immunization again and is resisted Under serum profiles, domestic and international commercially available Antirabic Vaccine's protective rate is only 0~30% (the auspicious grade China experiment of vast stretch of wooded country and clinic 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 The immune system of itself is induced to generate protective effect and removes virus.The whole virus particles vaccine of inactivation is not due to can induce inflammation Reaction is so as 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 that the rabies whole virus particles vaccine of existing inactivation is carried out 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 causes 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 are 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 excites the activity of lymphocyte to a greater extent to influence cancer cell, and propose Rabies vacciness can be as a kind of method of new treating cancer and the effect (Faiderbe of 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, and then generate stronger immune response effect with humoral immunity realization.Hence, it is believed that this The rabies immune originality conjugate of invention can be also used for preventing treat or auxiliary treatment include 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 Yi Shang patent Take precautions against the technological deficiency and deficiency more than being respectively provided with.

In conclusion the rabies immune originality conjugate emphasis of the present invention solves existing rabies whole virus particles epidemic disease Seedling does not generate cellular immunity or cellular immunity is faint and generate the defects of 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.It is relative to its difference of the prior art and advantageous effect：

(1) rabies immune originality conjugate of the present invention and the mad dog prepared according to this rabies immune originality conjugate Antigenic structure contained by virus combined vaccine is different from the prior art and document.In existing literature report and above-mentioned patent text institute In disclosed scheme, Rabies Virus Antigen and the albumen using tetanus toxoid as representative are to exist in hybrid form, i.e., mad Without chemistry key connection between dog disease viral antigen and tetanus toxoid, Rabies Virus Antigen and tetanus toxoid do not have yet There is the connection on recurring structure, Rabies Virus Antigen and tetanus toxoid 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 contained by virus combined vaccine is different from the prior art and document.Existing literature report and it is foregoing In scheme disclosed in patent text, the albumen using tetanus toxoid as representative is more the function of playing the part of adjuvant, to reach 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 so as 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, the albumen using tetanus toxoid as 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 are semi-finished product stostes 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 rabies combined vaccine is configured to using rabies immune originality conjugate as stoste, the method includes following Step：

(a) it is 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 Into 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 to add in people 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 add in 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 conjugates(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 with carrier protein through chemistry, and the carrier protein is at least Including 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, and it is cell-mediated to can induce Th1 to Rabies Virus Antigen-carrier protein immunogenic conjugates Immune anamnestic reaction, more B cells is promoted to generate specific antibodies；Secondly, Rabies Virus Antigen-carrier protein is immunized Originality conjugate increases relative to Rabies Virus Antigen molecular weight, advantageously anti-with the immune response in quick inductor It should；In addition, Rabies Virus Antigen is connected directly chemical bond with being prepared by way of utilizing amalgamation and expression 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 Protein structure respective with carrier protein and effective action site maintain the immunogenicity of Rabies Virus Antigen；Gram 2) Taken larger fusion protein be difficult to express, purify and purification process in the defects of being easy to 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, cell immune response and humoral immune reaction is made to reach 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 The single self-existent and/or Rabies Virus Antigen-carrier protein conjugate of 2 or more, 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 is not by the chemical bond between carrier protein and other lists A self-existent Rabies Virus Antigen establishes the connection in structure, n >=1, and the Rabies Virus Antigen of described 2 or more leads to The chemical bond crossed between carrier protein establishes the connection in structure each other.The single self-existent and/or mad dog of 2 or more 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, more B cells is promoted to generate specific antibody, make 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 and other single self-existent Rabies Virus Antigens establish connection in structure.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 generates Rabies Virus Antigen-carrier protein immunogenic conjugates stronger be immunized should It answers, and can induce the cell-mediated immune anamnestic reactions of Th1, more B cells is promoted 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 resists Containing the rabies virus antigen of 2 or more 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 " for Rabies Virus Antigen, " CP " represent carrier protein, "-" as connection 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.The rabies of 2 or more 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 Rabies Virus Antigen-carrier protein immunogenic conjugates stronger immune response, and The cell-mediated immune anamnestic reactions of Th1 are can induce, more B cells is promoted to generate specific antibody, 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.

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 The functional groups of chemistry key connection 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 generation 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 to not include linking arm (Linker Arm), spacerarm (Spacer Arm) and bridging molecules at least (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, the chemical bond for connecting Rabies Virus Antigen and carrier protein includes newly between 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 two thiobis (succinyl Asia 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), ethylene glycol-bis- (succinimide ester succinic acid Ester) (EGS), ethylene glycol-bis- (sulfosuccinimide ester succinate) (sulfo-EGS), double amber imide glutarate (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), 4- Malaysias 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-be -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 Sour four fluoro- long-chain-biotin (MTS-ATF-biotin) of 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 with carrier protein by chemical bond after the Rabies Virus Antigen is activated It connects.

Any of the above-described is preferably, and is connected again with Rabies Virus Antigen by chemical bond 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 for preventing, treat or auxiliary treatment Cancer.

The present invention also providesA kind of method for preparing rabies immune originality conjugate, the method includes following steps Suddenly：

(a) it is purified that Rabies Virus Antigen stoste is made；

(b) Rabies Virus Antigen stoste prepared by step (a) and carrier protein are subjected to chemical key connection, 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, more B cells is promoted 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 with preparing Rabies Virus Antigen-carrier protein immunogenic conjugates phase using the mode of amalgamation and expression Than there is following and technique effect and overcome following defect：1) since Rabies Virus Antigen and carrier protein lead to 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 The defects of being easy to 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), hydrophobin is is fixed by the Rabies Virus Antigen stoste 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 obtains 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 The functional groups of chemistry key connection 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 generation 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 to not include linking arm (Linker Arm), spacerarm (Spacer Arm) and bridging molecules at least (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 two thiobis (succinyl Asia 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), ethylene glycol-bis- (succinimide ester succinic acid Ester) (EGS), ethylene glycol-bis- (sulfosuccinimide ester succinate) (sulfo-EGS), double amber imide glutarate (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), 4- Malaysias 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-be -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 Sour four fluoro- long-chain-biotin (MTS-ATF-biotin) of 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 with carrier protein by chemical bond 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 used to prevent, treat or assist Treating cancer.

The present invention also provides a kind of rabies combined vaccine (Rabies Conjugate Vaccine RCV), including mad Dog disease immunogenic conjugates, the rabies immune originality conjugate includes Rabies Virus Antigen and carrier protein, described Rabies Virus Antigen includes at least the Rabies Virus Antigen being keyed with carrier protein through chemistry, and the carrier protein is at least Including 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, and it is cell-mediated to can induce Th1 to Rabies Virus Antigen-carrier protein immunogenic conjugates Immune anamnestic reaction, more B cells is promoted to generate specific antibodies；Secondly, Rabies Virus Antigen-carrier protein is immunized Originality conjugate increases relative to Rabies Virus Antigen molecular weight, advantageously anti-with the immune response in quick inductor It should；In addition, Rabies Virus Antigen is connected directly chemical bond with being prepared by way of utilizing amalgamation and expression 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 Protein structure respective with carrier protein and effective action site maintain the immunogenicity of Rabies Virus Antigen；Gram 2) Taken larger fusion protein be difficult to express, purify and purification process in the defects of being easy to 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, cell immune response and humoral immune reaction is made to reach 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- It is CSF, CpG ODN, trehalose dimycolate (TDM), phonetic containing poly- inosinic acid (Poly I) and/or poly- born of the same parents At least one of substance of 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 The single self-existent and/or Rabies Virus Antigen-carrier protein conjugate of 2 or more, 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 " The chemical bond of carrier protein, "-" to connect Rabies Virus Antigen and carrier protein is represented for Rabies Virus Antigen, " CP ", The single self-existent Rabies Virus Antigen is not by the chemical bond between carrier protein and other single independences Existing Rabies Virus Antigen establishes the connection in structure, n >=1, the Rabies Virus Antigen of described 2 or more by with load Chemical bond between body protein establishes the connection in structure each other.The single self-existent and/or hydrophobin of 2 or more 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, more B cells is promoted to generate specific antibody, make 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 and other single self-existent Rabies Virus Antigens establish connection in structure.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 generates Rabies Virus Antigen-carrier protein immunogenic conjugates stronger be immunized should It answers, and can induce the cell-mediated immune anamnestic reactions of Th1, more B cells is promoted 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 resists Containing the rabies virus antigen of 2 or more 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 " for Rabies Virus Antigen, " CP " represent carrier protein, "-" as connection 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.The rabies of 2 or more 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 Rabies Virus Antigen-carrier protein immunogenic conjugates stronger immune response, and The cell-mediated immune anamnestic reactions of Th1 are can induce, more B cells is promoted to generate specific antibody, 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.

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 generation 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 to not include linking arm (Linker Arm), spacerarm (Spacer Arm) and bridging molecules at least (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 The chemical bond of object, connection Rabies Virus Antigen and carrier protein is between Rabies Virus Antigen and carrier protein comprising newly-increased Atom or molecule.

Any of the above-described is preferably, and the linking arm or spacerarm or bridging molecules include two thiobis (succinyl Asia 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), ethylene glycol-bis- (succinimide ester succinic acid Ester) (EGS), ethylene glycol-bis- (sulfosuccinimide ester succinate) (sulfo-EGS), double amber imide glutarate (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), 4- Malaysias 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-be -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 Sour four fluoro- long-chain-biotin (MTS-ATF-biotin) of 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 with carrier protein by chemical bond after the Rabies Virus Antigen is activated It connects.

Any of the above-described is preferably, and is connected again with Rabies Virus Antigen by chemical bond 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 for preventing, treat or auxiliary for treating cancer.

The present invention also provides a kind of methods for preparing rabies combined vaccine, the described method comprises the following steps：

(a) it is 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 Into 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, more B cells is promoted 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 with preparing Rabies Virus Antigen-carrier protein immunogenic conjugates phase using the mode of amalgamation and expression Than there is following and technique effect and overcome following defect：1) since Rabies Virus Antigen and carrier protein lead to 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 The defects of being easy to 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 isolates and purifies 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 At least one of the hydrophobin stromatin of group expression, hydrophobin polymerase through recombinant 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 obtains 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- It is CSF, CpG ODN, trehalose dimycolate (TDM), phonetic containing poly- inosinic acid (Poly I) and/or poly- born of the same parents At least one of substance of 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 The functional groups of chemistry key connection 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 generation 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 to not include linking arm (Linker Arm), spacerarm (Spacer Arm) and bridging molecules at least (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 two thiobis (succinyl Asia 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), ethylene glycol-bis- (succinimide ester succinic acid Ester) (EGS), ethylene glycol-bis- (sulfosuccinimide ester succinate) (sulfo-EGS), double amber imide glutarate (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), 4- Malaysias 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-be -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 Sour four fluoro- long-chain-biotin (MTS-ATF-biotin) of 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 with carrier protein by chemical bond 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 for preventing, 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 connects formed Rabies Virus Antigen-carrier protein immunogenic conjugates.The present invention is again including mad dog Carrier protein is connected formed Rabies Virus Antigen-carrier protein immunogenicity by chemical bond and combined by sick viral antigen Object.The invention also includes chemical bonds to connect carrier protein with Rabies Virus Antigen formed 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 " is represented and carried Body protein, "-" are to connect the chemical bond of Rabies Virus Antigen and carrier protein.Therefore, the present invention includes single be 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 the chemical bond between carrier protein 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 the Rabies Virus Antigen-carrier protein of 2 or more, the Rabies Virus Antigen of described 2 or more is jointly and carrier protein It is keyed through chemistry, carrier protein is keyed the Rabies Virus Antigen of 2 or more 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 the mad dog of 2 or more Sick viral antigen-carrier protein form is present in rabies immune originality conjugate, the Rabies Virus Antigen-carrier egg Containing the Rabies Virus Antigen of 2 or more 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 the Rabies Virus Antigen of 2 or more through chemical bond Connect to form rabies immune originality conjugate with carrier protein, the Rabies Virus Antigen of described 2 or more 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 respectively and/or jointly with carrier protein through chemistry, 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 with carrier protein through chemistry 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 it is self-existent and/ Or the Rabies Virus Antigen of 2 or more, 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 single to be 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 the connection of rabies whole virus particles is formed into rabies whole virus particles-carrier protein immunogenic conjugates.It is described Rabies whole virus particles are hydrophobins through cell culture, inactivation, the Rabies Virus Antigen purified.

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, rabies VLP connections are formed rabies VLP- by the present invention including carrier protein by chemical bond 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 The connection of hydrophobin outer membrane segment is 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 passes through including carrier protein Rabies virus glucoprotein connection is 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 states rabies combined vaccine and includes rabies immune originality conjugate, 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 is resisted hydrophobin by chemical bond including carrier protein Original connects formed Rabies Virus Antigen-carrier protein immunogenic conjugates formulated rabies combination epidemic disease formed again Seedling.The present invention again including Rabies Virus Antigen by chemical bond by carrier protein connect formed Rabies Virus Antigen- The carrier protein immunogenic conjugates formulated rabies combined vaccine formed again.The invention also includes chemical bonds by carrier egg It is white connected with Rabies Virus Antigen formed Rabies Virus Antigen-carrier protein immunogenic conjugates it is formulated again and Into rabies combined vaccine.The rabies combined vaccine of the present invention prevents and treats 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 connection of rabies whole virus particles is formed mad dog by the present invention including carrier protein by chemical bond Sick whole virus particles-carrier protein immunogenic conjugates formulated rabies totivirus combined vaccine formed again.It is described mad Dog disease whole virus particles are hydrophobins through cell culture, inactivation, the Rabies Virus Antigen purified.

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, and the rabies VLP is with carrying 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 Rabies VLP connections form to rabies VLP- carrier proteins immunogenic conjugates are formulated again to be formed by chemical bond 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 Hydrophobin outer membrane segment is connected to form hydrophobin outer membrane segment-carrier with carrier protein by connection, the chemical bond Protein immunogenic conjugate.In other words, the present invention is connected hydrophobin outer membrane segment by chemical bond including carrier protein Connecing formed hydrophobin outer membrane segment-carrier protein immunogenic conjugates, the formulated rabies cracking formed combines again 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 is formed rabies virus glucoprotein connection by chemical bond including carrier protein Rabies virus glucoprotein-carrier protein immunogenic conjugates formulated rabies glycoproteins combined vaccine formed 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 for preparing rabies immune originality conjugate, and the method includes following Step：

(a) it is 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 Into Rabies Virus Antigen-carrier protein immunogenic conjugates.

Another aspect of the invention provides a kind of method for 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 for 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 for 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 for preparing rabies immune originality conjugate, and the method includes following Step：

(a) it is 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 for preparing rabies combined vaccine, the described method comprises the following steps：

(a) it is 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 Into 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 for 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 for 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 for 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 for preparing rabies combined vaccine, the described method comprises the following steps：

(a) it is 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 Rabies Virus Antigen is connected to form Rabies Virus Antigen-load with carrier protein by chemical bond 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 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 or the substance of recombinant expression, can also be semi-synthetic or synthesis substance.It is mad Dog disease viral antigen is entirely sick including rabies whole virus particles or including the rabies VLP through recombinant expression or including rabies Outer membrane segment that malicious particle is prepared after cracking or the glycoprotein including being isolated and purified from hydrophobin or including from mad dog The nucleoprotein of sick virus isolation or the phosphoprotein including being isolated and purified from hydrophobin or including from hydrophobin The stromatin or the polymerase including being isolated and purified from hydrophobin that isolate and purify or including the mad dog through recombinant expression Sick viral glycoprotein or including the rabies virus nucleoprotein through recombinant expression or including the hydrophobin through recombinant expression Phosphoprotein or including the hydrophobin stromatin through recombinant expression or including the hydrophobin poly through recombinant expression Enzyme.

In term of the present invention, carrier protein (Carrier Protein CP) is to be connected with haptens or antigen with chemical bond 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 or the substance of recombinant expression, it 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 into 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 the substance with chemistry key connection Rabies Virus Antigen and carrier protein in object, 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 isolates and purifies 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 Beautiful jadeThe structure of hydrophobin sample particle, preparation and Efficacy evaluation Changchun Jilin University .2015), the World Health Organization Promulgation human rabies inactivated vaccine manufacture 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 such as Zhang Jianming straits 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 two of chemical bond connection method 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 does not include 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 is being directly connected to for linking arm or spacerarm, bridging agent is selected from1- (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- ethyls -5- phenyl isoxazole -3'- sulfonate (Woodward ' s reagent K), N, N'- Carbonyl dimidazoles (CDI) and the reagent of schiff bases generation and reductive amination process are such asSodium cyanoborohydride(NaBH3CN) orBoron 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 Alcohol-bis- (succinimide ester succinate) (EGS), ethylene glycol-bis- (sulfosuccinimide ester succinate) (sulfo- EGS), oneself two sub- acyls of double amber imide glutarate (DSG), bis- succinimidyl carbonate of N, 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-(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), sulfo group long-chain-Nitrogen--3 (two sulphur of 2- pyridines of 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- maleimidobenzoyl succinimide esters(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 is 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 ester (SASD)、Succinimido -4- triazobenzene formic acid esters(HSAB), sulfo groupSuccinimido -4- triazobenzene formic acid esters (sulfo‐HSAB)、N- succinimidos -6- (4'- azido -2'- nitro-phenylaminos) capronate(SANPAH), sulfo groupSuccinimido -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- azidophenyls 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, first with cyanidization agent (1- cyano -4-dimethylaminopyridine tetrafluoro boron Acid esters (CDAP) or cyanogen bromide (CNBr)) activation process is carried out to carrier protein, then with adipic acid dihydrazide (ADH) be homologous double In the carrier proteins that the addition of function connects arm has activated, carrier protein-ADH derivatives are formed, add Rabies Virus Antigen, Condensation carrier protein-ADH derivatives by carbodiimide-mediated pass through chemical bonds, shape with Rabies Virus Antigen Into Rabies Virus Antigen-carrier protein immunogenic conjugates, further purified acquisition combines for preparing rabies The antigen component of vaccine.In another preferred embodiment of the present invention, first with cyanidization agent (1- cyano -4- dimethylaminos Pyridinium tetrafluoroborate (CDAP) or cyanogen bromide (CNBr)) activation process is carried out, then with adipoyl hydrazine to Rabies Virus Antigen (ADH) it in the Rabies Virus Antigen activated for homologous difunctional linking arm addition, forms Rabies Virus Antigen-ADH and spreads out Biology adds carrier protein, condensation Rabies Virus Antigen-ADH derivatives and carrier by carbodiimide-mediated Albumen forms Rabies Virus Antigen-carrier protein immunogenic conjugates by chemical bonds, further purified to obtain It must be used to prepare the antigen component of rabies combined vaccine.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 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 acquisition is for preparing the antigen of 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 to implement this hair in the method and technique or process of Rabies Virus Antigen and carrier protein chemistry key connection 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 to add in function of the human albumin as protective agent or stabilizer in disease vaccine.The main work(of human albumin in the prior art Can one of be by preventing hydrophobin particle aggregation, and then avoid the reduction in Rabies Virus Antigen site in vaccine, So as to fulfill the protective agent or the function of 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 not include human albumin.In currently preferred another embodiment, Rabies combined vaccine does not also include protective agent functionally similar with human albumin or stabilizer.

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- 1st, IL-2, IL-6, IL-12, IL-15, IL-18, IFN-γ, GM-CSF, CpG ODN, trehalose dimycolate (TDM), the pick up adjuvant containing 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, it is 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 the core content or spirit of the present 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 the core of the present 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 embodiment

The present invention can be by being discussed further, by Rabies Virus Antigen and carrier protein with reference to following examples It learns bond to close, rabies immune originality conjugate is made, then formulated into 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 being limited.

Embodiment 1

Rabies whole virus particles antigen stock prepares and vaccine formulation：

Vero cell works library cell is taken to be recovered in 37 DEG C, amplification cultivation, and 3-5 generations, cell density are 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 inoculations titre is 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 add in beta-propiolactone, put 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 virus liquid after purification, which adds in 1~10% human serum albumin of antigen stabilizer,.

With ELISA measure rabies whole virus particles antigen stock in rabies virus antigen content, add in disodium hydrogen phosphate, Sodium dihydrogen phosphate, sodium chloride, water for injection, formulated, tune pH7.0~8.0, as packing, vaccine.It is issued by the World Health Organization Cloth Antirabic Vaccine manufacture 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, the agar semisolid culturemedium of beef infusion broth containing tryptone is seeded to, 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 in, 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, puts 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, put 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 the ultrafiltration of 50~100KD films.NaCl is added in final concentration 0.85%, Formaldehyde is added to final concentration 0.15~0.25%, puts 37 DEG C of detoxifications 15~31 days.Formaldehyde is removed with the ultrafiltration of 50~100KD films, 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, puts 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 0.1M PBS that pH is 7.5.Add in 100mg/ml 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 in, TEA additions are 1~3 with CDAP additions ratio:1 (v/v) adjusts pH to 9.0, Maintain reaction 2~5 minutes.Add in ADH (adipoyl hydrazine) NaHCO₃(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 the ultrafiltration of 50~100KD films, 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 contents ratio:1(w/w) Ratio, mixing rabies whole virus particles purifying stoste and tetanus toxoid-ADH derivative stostes.PH adjusted to 5.0~ 5.8, the sulfo-NHS (N- hydroxy thiosuccinimides) of final concentration of 10~25mM is added in, 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 Sepharose4FF 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, puts 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, add in 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, as packing 0.5-1.0ml/ agent, 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：

Rabies whole virus particles purifying stoste that is prepared by 1 method of the present embodiment and not adding human serum albumin is 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 chromatographies method, 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, adds in 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, as packing 0.5-1.0ml/ agent, 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 toQi YinglinMethod (Qi YinglinThe structure of hydrophobin sample particle, preparation and Efficacy evaluation Changchun 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 in 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, as packing 0.5-1.0ml/ agent, 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 (such as Zhang Jianming straits 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, adds in 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, as packing 0.5-1.0ml/ agent, 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 to 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, without depilation, tetanus signs such as no late period paralysis, and increased before every cavy weight ratio injection.

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, puts 37 DEG C and places 42 My god.The cavy 4 of every part of test specimen injection 250~350g weight, injecting pathway is subcutaneous for veutro, 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 paradoxical reaction is no different, 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, 7th, 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 result of the test 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, it is of the invention The rabies immune originality conjugate and vaccine of offer and the rabies immune originality knot prepared using the method for the invention 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, 0.5ml is immunized in every mouse peritoneal, 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 mouse in typical rabies brain symptom 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 (10)

1. a kind of rabies immune originality conjugate（Rabies Immunogenic Conjugate, RIC）, including rabies Viral antigen（RabAg）And carrier protein（Carrier Protein, CP）, which is characterized in that the Rabies Virus Antigen Including at least the Rabies Virus Antigen being keyed with carrier protein through chemistry, the carrier protein includes at least and rabies disease Rabies Virus Antigen is connected to form mad dog with carrier protein by carrier protein of the malicious antigen through chemistry key connection, the chemical bond Sick viral antigen-carrier protein immunogenic conjugates.

2. rabies immune originality conjugate according to claim 1, which is characterized in that the Rabies Virus Antigen packet It includes rabies whole virus particles, prepared after cracking through the hydrophobin sample particle of recombinant expression, rabies whole virus particles Hydrophobin outer membrane segment, isolate and purify from hydrophobin rabies virus glucoprotein, detach from hydrophobin The nucleoprotein of purifying, the phosphoprotein isolated and purified from hydrophobin, the stromatin isolated and purified from hydrophobin, from mad The polymerase of dog disease virus isolation, the rabies virus glucoprotein through recombinant expression, the hydrophobin through recombinant expression Nucleoprotein, the hydrophobin phosphoprotein through recombinant expression, the hydrophobin stromatin through recombinant expression and through recombinate table At least one of hydrophobin polymerase reached.

3. rabies immune originality conjugate according to claim 2, which is characterized in that the rabies whole virus particles Hydrophobin through cell culture, inactivation, purifying Rabies Virus Antigen.

4. rabies immune originality conjugate according to claim 2, which is characterized in that the hydrophobin sample particle It is the Rabies Virus Antigen prepared through recombinant expression.

5. rabies immune originality conjugate according to claim 2, which is characterized in that the hydrophobin outer membrane Section is the Rabies Virus Antigen containing furcella and stromatin that rabies whole virus particles are prepared through cracking.

6. rabies immune originality conjugate according to claim 2, which is characterized in that the rabies virus glucoprotein It is the Rabies Virus Antigen being isolated and purified from hydrophobin or through recombinant expression containing rabies virus glucoprotein.

7. rabies immune originality conjugate according to claim 1, which is characterized in that the carrier protein includes broken Wind toxoid（TT）, diphtheria toxoid（DT）, diphtheria toxin non-toxic variant（CRM197）, B group meningitis cocci outer membrane proteins （OMP）, Pneumococal surface protein A（PspA）, PsaA（PsaA）, pneumolysin（Ply）、 Haemophilus influenzae D albumen（PD）, pertussis toxin（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）, ovalbumin, keyhole limpet hemocyanin（KLH）, bovine serum albumin(BSA)（BSA）, B-mode liver Scorching viral surface antigen（HBsAg）, hepatitis B virus core antigen（HBcAg）, Tetanus Toxin Fragment C（TTC）At least It is a kind of.

8. according to claim 1-7 any one of them rabies immune originality conjugates, which is characterized in that the rabies disease Malicious antigen is present in the form of Rabies Virus Antigen-carrier protein conjugate in the rabies immune originality conjugate, The Rabies Virus Antigen-carrier protein conjugate resists including single self-existent and/or 2 or more hydrophobin Original-carrier protein conjugate, containing single self-existent and/or 2 in the Rabies Virus Antigen-carrier protein conjugate A above rabies virus antigen, i.e., Rabies Virus Antigen is 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 shapes Formula is present in rabies immune originality conjugate, wherein " RabAg " for Rabies Virus Antigen, " CP " represent carrier protein, "-" is to connect the chemical bond of Rabies Virus Antigen and carrier protein, and the single self-existent Rabies Virus Antigen is simultaneously The company in structure is not established by the chemical bond between carrier protein and other single self-existent Rabies Virus Antigens It connects, n >=1, the Rabies Virus Antigen of described 2 or more is established in structure each other by the chemical bond between carrier protein Connection.

9. rabies immune originality conjugate according to claim 8, which is characterized in that the Rabies Virus Antigen with The form of Rabies Virus Antigen-carrier protein conjugate is present in the rabies immune originality conjugate, the mad dog Sick viral antigen-carrier protein conjugate is single self-existent Rabies Virus Antigen-carrier protein conjugate（RabAg- CP）, single Rabies Virus Antigen, i.e. hydrophobin are contained only in the Rabies Virus Antigen-carrier protein conjugate Antigen is present in the form of RabAg-CP in rabies immune originality conjugate, wherein " RabAg " for Rabies Virus Antigen, " CP " represents the chemical bond of carrier protein, "-" to connect Rabies Virus Antigen and carrier protein, single self-existent mad dog Sick viral antigen is built not by the chemical bond between carrier protein and other single self-existent Rabies Virus Antigens Connection in vertical structure.

10. rabies immune originality conjugate according to claim 8, which is characterized in that the Rabies Virus Antigen It is present in the form of Rabies Virus Antigen-carrier protein conjugate in the rabies immune originality conjugate, it is described mad Dog disease viral antigen-carrier protein conjugate is the Rabies Virus Antigen-carrier protein conjugate of 2 or more（(RabAg- CP)_n- RabAg, n >=1）, resist containing the rabies viruses of 2 or more in the Rabies Virus Antigen-carrier protein conjugate Original, 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 " is that Rabies Virus Antigen, " CP " represent carrier protein, "-" as connection Rabies Virus Antigen and carrier egg White chemical bond, n >=1, the Rabies Virus Antigen of described 2 or more are built each other by the chemical bond between carrier protein Connection in vertical structure.