CN1438245A - Infectious causative-agent related egg-yolk antibody preparation and use thereof - Google Patents

Abstract

The invention refers to a method of making yolk antibody relative to contagious pathogens, including a method of making yolk multiclonal antibody IgY aimed at pathogens immunogen, anti-peculiarity natibody and specific expressing antigen of pathogens, and in addition the use of the yolk antibody. It uses laying fowls, especially hens as immune reactor, uses one or multiple pathogens immunogens for single or united immune injection, so that there is any one or their combination of IgY, anti-peculiarity antibody and the specific expressing antigen in the eggs. It is applied to immune diagnosis, therapy and prevention of contagions, especially to very harmful ones to the human beings: AIDS, hepatitis B, third hepatitis, tuberculosis and so on.

Description

The preparation and the application of the relevant yolk antibody of infectious agent

Technical field

The invention belongs to biological technical field, particularly relate to the preparation method of the relevant yolk antibody of infectious agent, also relate to the purposes of this yolk antibody in addition.

Background technology

The preparation of specific antibody has very important significance to diagnosis, treatment and the prevention of communicable disease, thereby is subjected to the extensive concern on domestic and international biomedical boundary, has carried out a large amount of work and has obtained bigger progress.In the prior art diagnosis and biotherapies of adopting monoclonal antibody to be applied to communicable disease more, wherein the MONOCLONAL ANTIBODIES SPECIFIC FOR method generally comprises and obtains communicable disease pathogenic agent related antigen, immune mouse and three steps of fusion hybridoma, that is a at first utilizes biochemical and immunochemical method is obtained the pathogenic agent related antigen that carries communicable disease pathogenic agent relevant information, b then with this pathogenic agent related antigen repeatedly duplicate injection go in the mouse body, after treating that the specific immune response certain hour takes place mouse, take out its spleen and isolate the primed lymphocyte that has pathogenic agent related antigen information, again this primed lymphocyte and murine myeloma cell are fused into hybridoma, filter out the positive hybridoma cell that is directed to the pathogenic agent related antigen again and carry out amplification in vitro or be inoculated in the mouse peritoneal, can from culture supernatant or mouse ascites, obtain required monoclonal antibody.

Said monoclonal antibody (abbreviation monoclonal antibody) or many monoclonal antibodies (being called for short how anti-) as the diagnostic reagent or the detection agent of communicable disease, are used widely in medical science and field of biology in recent years.The principle of these class methods is based on monoclonal antibody or many anti-high specific recognition capabilities to corresponding communicable disease pathogenic agent related antigen design, in conjunction with the existence that shows communicable disease pathogenic agent related antigen with corresponding marker (enzyme mark, gold mark, selenium mark, fluorescence, luminous and radio-labeling etc.) whether and measure how much.

Monoclonal antibody also obtains impressive progress as the research of therapeutical agent in recent years.Monoclonal antibody medicine (monoclonal antibody agents) can be applicable to treat tumour, viral infection, cardiovascular diseases and other illness, in particular for the treatment of communicable disease, has demonstrated good prospect.

Antibody drug generally comprises two classes, and the one, antipathogen monoclonal antibody or how anti-; The 2nd, antipathogen monoclonal antibody or how anti-conjugate, or claim immune conjugate (1mmunoconjugate).The immune conjugate molecule so be called targeted drug, is called biological missile or " magic bullet " (magic bullets) by monoclonal antibody or many anti-and " bullet " medicine two portions formations again, and corresponding with it methods of treatment is called targeted therapy.Monoclonal antibody or many anti-at target be generally the related antigen on pathogenic agent surface or specific acceptor.Mainly contain three classes as the material of " bullet ", i.e. radionuclide, medicine and toxin, its with monoclonal antibody or how anti-the connection constitute radioimmunity conjugate, chemo-immunity conjugate and immunotoxin respectively.The especially antitumor monoclonal antibody medicine of enantiopathy substance has carried out big quantity research since the eighties, particularly since nineteen ninety-seven, Rituxan, Herceptin are in the granted in succession clinical cancer therapy that is used for of the U.S., the research and development of monoclonal antibody medicine have had new growth momentum, become new focus (Zhen Yongsu, the present Research and the prospect of monoclonal antibody drug treatment tumour, Chinese Academy of Medical Sciences's journal of biotech drug, 2000,22:9-12).

Although the clinical study result of monoclonal antibody medicine shows good prospect for it is applied to treat tumour, but still have many problems need further research and solve (Nguyen DT, Amess JA, Doughty H, et al.IDEC-C2B8 anti-CD20 (rituximab) immunotherapy in patients with low-grade non-Hodgkin ' s lymphoma and lymphoproli ferative disorders:evaluation of response on 48 patients.Eur J Haematol, 1999,62:76-82).The problem that monoclonal antibody medicine exists relates generally to immunology and pharmacology two aspects.The key to the issue of immunology aspect is the anti-mouse Ig of people antibody (HAMA) reaction, prepares because be used for the most mouse monoclonal antibodies that use of the monoclonal antibody medicine of clinical study for many years, often causes the HAMA reaction.In addition, the heterogeneity of pathogenic agent aspect antigenicity, the antigenic modulation of pathogenic agent etc. also can influence the curative effect of monoclonal antibody medicine.The problem of pharmacology aspect mainly is the dose deficiency that arrives pathogenic agent.Monoclonal antibody medicine transports in vivo and is subjected to multiple factor affecting in the process.Because it is foreign protein, can be absorbed by reticuloendothelial system, have a great deal ofly will accumulate in liver, board and marrow; Moreover be exactly that body produces antibody and reduces its effectiveness this foreign protein.Monoclonal antibody medicine is a macromolecular substance, by the capillary endothelium layer and penetrate pathogenic agent and all be restricted.(Zhen Yongsu, the present Research and the prospect of monoclonal antibody drug treatment tumour, Chinese Academy of Medical Sciences's journal, 2000,22:9-12).

The communicable disease immunotherapy be utilize that pathogen antigen carries out that active immunity excites, enhancing body is to the active specific immunotherapy reaction of pathogenic agent.The pathogen vaccines of being studied at present mainly is recombinant vaccine, peptide vaccine, nucleic acid vaccine, antiidiotypic antibody vaccine.Wherein a pathogen gene engineered vaccine is by gene recombination technology, goal gene is imported recipient cell and the vaccine for preparing.The b peptide vaccine is based on that antigen is degraded to small peptide in the immune response in endochylema, forms the final activated cell toxic T lymphocyte of peptide-MHC-TCR complex body (CTL) reaction at last.The c nucleic acid vaccine is can cause that by coding the antigen gene fragment and the vector construction of protective immunological reaction form, and comprises dna vaccination and RNA vaccine, studies the more dna vaccination that is at present.Nucleic acid vaccine can stimulate body to produce humoral immunization and cellular immunization simultaneously, induces many subunit vaccines can not inductive CTL effect.D antiidiotypic antibody vaccine (Ab2 vaccine) is to have analogue antigen and immunoregulatory dual function according to antiidiotypic antibody, can overcome simultaneously immunity of organism suppresses, break immunological tolerance so can replace pathogen antigen to bring out specificity response to active immunization (Bhattacharya CM, Mukerjee S, biddle W, et alMurine monoclonal anti-idiotype antibldy as a potential netword antigen for human carcinoembryonicantigen.J Immumol, 1990,145:2758).

In above-mentioned pathogen vaccines immunotherapy method, because the antigenicity of pathogen antigen is strong and weak different, and the pathogenic agent host often is in immunosuppressive condition, is difficult to excite effective antipathogen reaction.Although the research of antiidiotypic antibody has obtained many progress, things is two aspects always.The Ab2 vaccine is used and is had many difficulties, though because Ab2 β can simulate micromolecule polypeptide antigen, be difficult to accurately reflect the antigenic determinant of macro-molecular protein, especially Ab2 β monoclonal antibody.Moreover because pathogen antigen is extremely complicated, modulation etc. often takes place, the pathogen specific idiotypic vaccine difficulty of preparation wide spectrum is very big.The Ab2 β that the more important thing is present use is mainly from mouse hybridoma, be foreign protein, can excite human immune system to produce people's anti-mouse Ig antibody (HAMA), neutralization reaction and toxigenicity immunocomplex take place, and lessen the curative effect, can cause serum sickness reaction when serious.

In view of the existing problems of above technical scheme, for fear of or reduce the HAMA reaction, now normally make mouse source property monoclonal antibody humanization or develop human antibody completely.The monoclonal antibody humanization mainly is to prepare chimeric antibody (chimericantibody) or reshaping antibody (reshaping antibody) by genetic engineering technique.Chimeric antibody is that the Fc section is replaced into the humanized, and other parts still are mouse source property.Reshaping antibody is meant that other parts are the humanized except that complementary determining region (CDR) is mouse source property.Make the miniaturization of conjugate molecule,, develop efficient monoclonal antibody medicine to improve the seepage force of medicine to pathogenic agent.Local injection is to improve the concentration of monoclonal antibody medicine at the pathogenic agent position.Multiple antibody mixes to be used to remedy the heterogeneous problem of monoclonal antibody.Wherein human antibody has higher technical difficulty to mouse source property monoclonal antibody humanization with developing completely, is difficult to popularization and application.

Bird especially chicken has very big-difference with Mammals in phylogeny, immunoglobulin (Ig) of chicken and mammalian immune sphaeroprotein also have a great difference on immunological characteristic.The immunity system of chicken can be discerned more immune response site and more antigenic determinant, so the chicken immune system can produce mammiferous albumen or biomolecules and has higher specific antibody of tiring with avidity.The chicken immune system is directed to the IgY that extrinsic protein produces and not only has higher immunologic opsonin, the more important thing is that this specific IgY mainly is stored in the egg yolk, therefore can utilize the hen that lays eggs to come mass production to have the antibody drug of important clinical using value as immune reactor.

Well-known egg is as the mankind's common food and cosmetic material, and no matter taking orally with external application does not all have any bio-toxicity and the bad sheet of doing to human body.Therefore, utilize bird to come the mass production antibody drug, and be applied to the diagnosis and the immunotherapy of communicable disease, will have extremely wide application prospect as immune reactor.The immune drug and the antiidiotypic antibody vaccine (Ab2 vaccine) that are feedstock production with the relevant yolk antibody IgY of infectious agent can solve above-mentioned monoclonal antibody conjugate or existing HAMA reaction of above-mentioned pathogen vaccines and heterogeneous problem well.

Goal of the invention

The objective of the invention is to overcome above-mentioned shortcoming with not enough, the preparation method of the relevant yolk antibody of a kind of infectious agent is provided, the preparation method who comprises the yolk polyclonal antibody IgY, antiidiotypic antibody and the pathogen specific antigen expressed that are directed to pathogenic agent, especially at yolk polyclonal antibody IgY, antiidiotypic antibody and the pathogen specific antigen expressed of multiple pathogenic agent in the preparation method of Multiple Combination thing, the purposes that also relates to this yolk antibody in addition promptly adopts this yolk antibody to prepare related drugs, food or healthcare products.Technical scheme

Technical scheme of the present invention is achieved in that the preparation method of the relevant yolk antibody of a kind of infectious agent utilizes bird as immune reactor, by the pathogen specific immunogen (is comprised pathogenic agent, purifying or recombinant antigen or vaccine knurl seedling, pathogen specific DNA or mRNA and the recombinant chou of forming with respective carrier thereof, be directed to antigenic mono-clonal of pathogen specific or polyclonal antibody) or its composition is independent or joint injection is given the especially chicken of bird of laying eggs, make its generation be directed to any or its composition in the immunogenic yolk antibody IgY of pathogen specific or antiidiotypic antibody (comprising Ab2 α and Ab2 β) or the pathogen specific antigen expressed and be transported in large quantities and store in the yolk, the Non-Invasive then yolk needed pathogen antigen of purifying that utilizes (comprises pathogen specific yolk antibody IgY, any or its composition among pathogen specific antigen expressed and antiidiotypic antibody Ab2 α and the Ab2 β).Utilize the IgY antibody in this yolk antibody to be directed to antigenic immunodiagnosis medicament of pathogen specific and immunotherapy medicament for main raw material can be prepared into, comprise that the targeted drug of pathogenic agent external diagnosis reagent, internal guide diagnostic reagent and specificity yolk antibody mediated immunity therapeutical agent and specificity yolk antibody coupling medicine or toxin is a biological missile; Utilize the pathogen specific antigen expressed in this yolk antibody can be prepared into immunodiagnosis medicament and the immunotherapy medicament that is directed to pathogenic agent, comprise communicable disease external diagnosis reagent and pathogen specific vaccine knurl seedling; The immunodiagnosis medicament and the immunotherapy medicament that utilize antiidiotypic antibody Ab2 α in this yolk antibody and Ab2 β to be directed to communicable disease body pathogenic agent for main raw material can be prepared into, the targeted drug that comprises communicable disease external diagnosis reagent, internal guide diagnostic reagent (mainly adopting Ab2 α) and antiidiotypic antibody Ab2 α coupling drug or toxin is antiidiotype biological missile and antiidiotypic antibody vaccine (mainly adopting Ab2 β); The diagnosis, immunotherapy and the targeted therapy that are used for communicable disease.This yolk antibody also can be used as additive and makes an addition in food, beverage and the healthcare products in addition, is used for the prevention and the early stage prophylactic treatment of communicable disease.

Owing to adopt the inventive method to prepare the relevant yolk antibody of infectious agent, utilize the chicken immune system can produce and have higher tiring and the specific antibody of avidity heterology albumen or biomolecules, can induce the antigenic antibody of specificity antipathogen of high titre, and it is low to have cost, the output height, high specificity, the advantage of the wide scale operation that also can intensify of determinant, can realize at the yolk polyclonal antibody IgY of multiple pathogenic agent, the disposable preparation of the Multiple Combination thing in antiidiotypic antibody and the pathogen specific antigen expressed, the more important thing is that this yolk antibody can solve above-mentioned mouse source property monoclonal antibody conjugate or existing HAMA reaction of pathogen vaccines and heterogeneous problem well, and human body is had no side effect, have security preferably.In addition, the relevant yolk antibody of the prepared infectious agent of the present invention or its composition can be used as raw material, the especially raw material of infectious agent related biochip of infectious agent dependent diagnostic reagent; Moreover be exactly that the relevant yolk antibody of the prepared infectious agent of the present invention or its composition can brought into play its immunomodulatory and therapeutic action aspect passive immunization and the active immunity two jointly.The present invention is applicable to immunodiagnosis, immunotherapy and the prevention of communicable disease, be particularly useful for the mankind are had the passive immunization and the active immunity treatment of diseases such as sexually transmitted disease, viral enteritis, virus pneumonia, viral encephalitis, mycoplasma, chlamydia pneumonia, typhus fever, bleb, rubella influenza and parasitic infection such as very big hazardness transmissible disease such as acquired immune deficiency syndrome (AIDS), hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, tuberculosis, helicobacter pylori property gastritis, syphilis, gonorrhoea, will have extremely wide application prospect.

For further openly the present invention, just the present invention is further described in detail down.

The preparation method of the relevant yolk antibody of a kind of infectious agent, it is characterized in that: with lay eggs bird especially chicken as immune reactor, the recombinant chou of forming with the pathogen antigen of one or more purifying or deactivation or the pathogen specific antigen by the genetic engineering technique reorganization or vaccine knurl seedling or pathogen specific DNA or mRNA and with respective carrier or be directed to antigenic mono-clonal of pathogen specific or polyclonal antibody or its liposome complex as immunogen, carry out separately respectively or multiple the associating carried out the especially repeatedly repetition immunization of chicken of bird, and then append with Freund and to strengthen reaction, treat that bird after 5-20 days especially promptly has in the laying eggs of chicken the polyclonal antibody IgY that is directed to above-mentioned pathogen immunogenic or any or its composition in antiidiotypic antibody or the pathogen specific antigen expressed to exist and stores.Its concrete steps comprise:

A) pathogen immunogenic or its composition carry out repeatedly that the immunization bird is preferably chicken.Pathogen immunogenic described herein is antigenic mono-clonal of recombinant chou, pathogen specific or the polyclonal antibody that adopts the pathogen antigen of purifying or the pathogenic agent by deactivation, the pathogen specific antigen by the genetic engineering technique reorganization or vaccine knurl seedling, the pathogen specific naked DNA by extracting purifying or mRNA and form with respective carrier, also comprise the liposome particulate of above-mentioned pathogen immunogenic or even the immunization of nanoparticle in addition, by means of liposome to tissue and the affinity of cell with the reinforced immunological effect; Described bird comprises chicken, duck, goose, quail, and first-selection is a chicken; The injection site comprises muscle, subcutaneous, intracutaneous, blood vessel and genital cloaca capsule; Injecting method comprises above-mentioned pathogen immunogenic injected separately to unite with multiple pathogen immunogenic and repeatedly repeats immunization, and wherein said multiple pathogen immunogenic is united and carried out immunization and comprise with any or its composition in pathogen antigen, gene recombination pathogen specific antigen or vaccine knurl seedling, pathogen specific naked DNA or the mRNA and the recombinant chou thereof in different sorts and source; Also comprise any or its composition in the pathogen specific mono-clonal in different sorts and source or the polyclonal antibody; Described immunogen injection volume is 1-2ml (the incomplete freund adjuvant of immune commercial weight 10-500 μ g/ml+ equivalent, wherein the consumption of antibody A b1 is the incomplete freund adjuvants of 100～2000 μ g/ml+ equivalent); Described pathogenic agent comprises any or its combination in the parasites of human such as virus, chlamydozoan, mycoplasma, Rickettsiae, bacterium, spirochete, fungi and protozoon, worm, nematode; Described communicable disease pathogenic agent comprises any or its combination in the pathogenic agent of diseases such as sexually transmitted diseases such as acquired immune deficiency syndrome (AIDS), hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, tuberculosis, helicobacter pylori property gastritis, syphilis, gonorrhoea, viral enteritis, virus pneumonia, viral encephalitis, mycoplasma, chlamydia pneumonia, typhus fever, bleb, rubella influenza and parasitic infection.

B) the above-mentioned pathogen immunogenic full adjuvant that toos many or too much for use carries out supplementary immunization reaction.

C) treat that bird after 5-20 days especially promptly has the yolk antibody that is directed to above-mentioned pathogen immunogenic to exist in the laying eggs of chicken and stores.Wherein said yolk antibody comprises any or its composition among the yolk polyclonal antibody IgY that is directed to above-mentioned pathogen antigen, pathogenic agent, reorganization pathogen antigen or vaccine knurl seedling or its liposome or nanoparticle; Be directed to any or its composition in the pathogen specific antigen expressed of pathogen specific naked DNA or mRNA and recombinant chou thereof or its liposome or nanoparticle; Be directed to any or its composition among the antiidiotypic antibody Ab2 α of pathogen specific mono-clonal or polyclonal antibody or above-mentioned yolk polyclonal antibody IgY or its liposome or nanoparticle and the Ab2 β.

The present invention is with the above-mentioned pathogen immunogenic injecting immune especially chicken of bird of laying eggs, and with the yellow source of laid eggs as the antipathogen antibody that is directed to above-mentioned pathogen immunogenic, these class methods have lot of advantages such as bird especially chicken be easy to raise, expense is not high, collection is laid eggs conveniently, need not to twitch thing blood, not damaged meets modern protection of animal rule; Also have the little advantage of the required antigen amount of effective immune response of generation in addition, the heterologous protein of high conservative is to having stronger immunogenicity usually apart from bird far away especially chicken on the phylogenetics.

The preparation method of the relevant yolk antibody of a kind of infectious agent, its feature also is: the extracting and purifying method of this yolk antibody is to adopt water dilution method, multigelation method or ammonium sulfate, sodium sulfate, PEG, T 500, sodiun alginate or sad precipitation or lixiviate, extraction, concentrate, the method of membrane filtration is extracted purifying and is directed to any or its composition in the polyclonal anticancer antibody IgY of above-mentioned pathogen immunogenic or antiidiotypic antibody or the pathogen specific antigen expressed from above-mentioned bird yolk especially egg yolk, then the yolk antibody of this purifying is added that the respective secondary material package is drawn together trehalose and any formulation of being prepared on the pharmaceutics comprises tablet, injection, oral liquid and sprays.Its concrete steps comprise:

A) get birds, beasts and eggs especially egg shell and get Huang, remove the protein ingredient that adheres on the membrane of yolk, then yolk is placed sterile purified water, repetitive scrubbing three times is further removed egg on the membrane of yolk from component, punctures membrane of yolk after draining, and collects yolk.

B) above-mentioned collection yolk is stirred after, add the 0.1mol/L PH7.2 phosphoric acid salt-trehalose diluent of 5 times of volumes of yolk homogenate, make the dilution egg yolk liquid.The weight percent of trehalose is 5-30% in this dilution egg yolk liquid.

C) above-mentioned dilution egg yolk liquid was placed ice chest (20 ℃) freezing 1-5 hour, take out then in room temperature and melt again and low-speed centrifugal (500-1000r/min) back drop goes out to collect supernatant liquor, and then in throw out, add the 0.1mol/L PH7.2 phosphoric acid salt-trehalose diluent of 2-5 times of volume, abundant mixing dissolution precipitation thing, comply with last method freeze thawing collection supernatant liquor once more, so multigelation is 3-5 time, and the supernatant liquor that each time of merging collection obtains is standby.

D) in above-mentioned collection supernatant liquor, add any or its mixture in ammonium sulfate, sodium sulfate, PEG, T 500, sodiun alginate or the sad precipitation agent in proportion, its throw out of centrifugal collection promptly contains any or its composition in the polyclonal antibody IgY that is directed to above-mentioned pathogen immunogenic or antiidiotypic antibody or the pathogen specific antigen expressed in this throw out.The pathogenic agent of the batch preparations yolk antibody of being correlated with, every egg can extract the pathogen specific yolk antibody of 57～171mg, and average every egg can extract 89mg, and its purity is 95%.

E) above-mentioned throw out is redissolved back dialysis or ultrafiltration and concentration or gel chromatography with distilled water, further separation and purification is directed to any or its composition in the polyclonal antibody IgY of above-mentioned pathogen immunogenic or antiidiotypic antibody or the pathogen specific antigen expressed.

F) yolk antibody to above-mentioned purifying adds corresponding auxiliary material in proportion, and its any formulation that is prepared on the pharmaceutics is comprised tablet, injection, oral liquid and sprays.Auxiliary material described herein comprises trehalose.

The preparation method of the relevant yolk antibody of a kind of infectious agent, its feature also is: the modification treatment process of this yolk antibody IgY or antiidiotypic antibody Ab2 α is to adopt the method for coupling or covalent attachment radionuclide, medicine and toxin and other cytotoxic substances to prepare immune guiding medicine or biological missile; Be to adopt the method for coupling or covalent attachment radionuclide, luminophore, Radioactive colloidal gold or electroselenium or enzyme to prepare that immunodiagnosis medicament and external immune diagnostic reagent comprise immunohistochemical methods reagent and biochip in the body; Be to adopt the method for in above-mentioned yolk antibody especially antiidiotypic antibody Ab2 β or pathogen specific antigen expressed, adding Freund or other components to prepare pathogen specific vaccine (comprising antiidiotypic antibody vaccine and polypeptide vaccine).

The major parts one of guided missile is the navigationsystem that can follow the trail of the objective, the 2nd, and the bomb of destruction specific objective.Use this principle and prepare a kind of " weapon " that can follow the trail of pathogenic agent and can kill and wound to specificity pathogenic agent, be biological missile, its basic element of character is can discern the antibody of pathogen antigen target specifically and connect the drug toxicity that kills and wounds pathogenic agent at the end of antibody.So theoretically, as long as can develop the antibody that to discern pathogenic agent, and make it be with drug toxicity just can be crowned with success.But it is not in fact so simple, it at first is the antibody problem, early stage people are with the complete antibody behind the higher relatively pathogen antigen immune mouse of purity and specificity (promptly by the Fab fragment of identification epitope cluster and the immunoglobulin (Ig) that the Fc fragment is formed), make its " tail end " (Fc fragment) carry radioactive nuclide iodine (125I); The foundation of hybridoma technology in 1975 and the appearance of monoclonal antibody make combining of monoclonal antibody and radionuclide, have promoted the progress of targeted therapy.Yet, through experiment after a while, monoclonal antibody and many anti-results of treatment in vivo, do not have tangible difference, its major cause is: 1. monoclonal antibody and resist all are complete antibodies more, and in a single day they enter body, will be intersected by healthy tissues and adsorb, the concentrated area arrives target relatively; And be not easy to remove and excrete; 2. because the molecular weight of complete antibody is big, the physiologic barrier (as reticular tissue etc.) that is difficult for by body enters the pathogenic agent centre; 3. because complete antibody all derives from mouse, be a kind of heterology albumen, be neutralized so can produce anti-antibody in vivo to human body.Owing to have above problem, therefore make complete antibody be used for targeted therapy and be restricted.

Problem according to above existence, the researchist had managed to study the antibody fragment of preparing the application small molecular weight in recent years, and manage personnel selection-people's hybridoma and prepare people-human monoclonal antibodies, prepare genetic engineering antibody with engineered method again at present, this is a kind ofly not contain the segmental Fab micromolecule polypeptide of Fc, and has set up the bifunctional antibody technology.The latter has the both arms (promptly two Fab fragments being combined) of specific recognition, therefore dual recognition capability is arranged, and promptly both can combine with pathogenic agent, can be connected with the medicine bullet again.Because genetic engineering antibody does not contain the Fc fragment, therefore reduced non-special absorption, and the mouse derived component can have been minimized; Micromolecular genetic engineered product, helping killing and wounding material, to enter pathogenic agent inner and be difficult for being identified as foreign matter and producing anti-antibody by the host; This bifunctional antibody makes bullet directly hit pathogenic agent, if with bifunctional antibody and LAK, the til cell combined utilization then can obtain bigger effect, thus the development of control disease effectively.But these class methods all have higher technical difficulty, are difficult to promote.

The present invention utilizes the modification of relevant yolk antibody IgY of infectious agent or antiidiotypic antibody Ab2 α to handle promptly to adopt the method for its coupling or covalent attachment radionuclide, medicine and toxin and other cytotoxic substances to prepare immune guiding medicine or biological missile in order to overcome above problem.Its concrete steps comprise:

A) enzyme decomposition or the coupling activity group of relevant yolk antibody IgY of infectious agent or antiidiotypic antibody Ab2 α are modified processing in advance, wherein the enzyme decomposition is to utilize stomach en-or ficin that relevant yolk antibody IgY of infectious agent or antiidiotypic antibody Ab2 α are decomposed, be reduced to the Fab small pieces with DTT or beta-mercaptoethanol again, so that it has better tissue penetration; Or with different activating reagents with the amino on the relevant yolk antibody IgY of infectious agent or antiidiotypic antibody Ab2 α and the Fab small pieces thereof or carboxyl or sulfydryl or aldehyde radical activation, as it being become active ester with 1-ethyl-3-(3-dimethylamino) carbodiimide hydrochloride (EDC) and N-hydroxy thiosuccinimide (NHS), and then with various drug molecule coupling.

B) the pre-modification of coupling drug is handled and is comprised radionuclide, medicine and lps molecule are activated with different activating reagents, as it being become active ester with 1-ethyl-3-(3-dimethylamino) carbodiimide hydrochloride (EDC) and N-hydroxy thiosuccinimide (NHS), so adopt similar known shared method, can be respectively the coupling drug activation that contains groups such as hydroxyl, amino, aldehyde radical, diazanyl, sulfydryl, so that it can relevant yolk antibody IgY with infectious agent or antiidiotypic antibody Ab2 α and Fab small pieces generation covalent attachment thereof.

C) relevant yolk antibody IgY of infectious agent or antiidiotypic antibody Ab2 α and Fab small pieces thereof comprise with the coupling drug of activation treatment becomes communicable disease specific immunity targeted drug or biological missile with radionuclide, medicine and lps molecule covalent attachment.

The present invention is that the modification processing of relevant yolk antibody IgY of employing infectious agent or antiidiotypic antibody Ab2 α promptly adopts the method for coupling or covalent attachment radionuclide, luminophore, Radioactive colloidal gold or electroselenium or enzyme to prepare the interior immunodiagnosis medicament of body and external immune diagnostic reagent comprises immunohistochemical methods reagent and biochip.Its concrete steps comprise:

A) the relevant yolk antibody IgY of infectious agent or antiidiotypic antibody Ab2 α are adopted known shared method carry out radionuclide, luminophore, Radioactive colloidal gold or electroselenium or enzyme labelling and prepare its corresponding marker.

B) cooperate other components to prepare immunodiagnosis medicament and external immune diagnostic reagent (comprising immunohistochemical methods reagent and biochip) in the body radionuclide, luminophore, Radioactive colloidal gold or the electroselenium of relevant yolk antibody IgY of infectious agent or antiidiotypic antibody Ab2 α or in the enzyme labelling thing any.

The present invention adopts the yolk antibody IgY in the relevant yolk antibody of infectious agent to prepare the pathogen specific immunotherapeutic agent.Its concrete steps comprise: add Freund and other components to above-mentioned mesolecithal antibody IgY, and then carry out immunization, the immunity body produces the antiidiotypic antibody Ab2 β that is directed to the relevant yolk antibody IgY of infectious agent, thereby induces body to produce antiviral specific immune response.

The present invention adopts antiidiotypic antibody Ab2 β or pathogen specific antigen expressed in the relevant yolk antibody of infectious agent to prepare the specific pathogen vaccine.Its concrete steps comprise: add Freund in any or its composition in above-mentioned antiidiotypic antibody Ab2 β, pathogen specific antigen expressed and other components prepare the specific pathogen vaccine.Antiidiotypic antibody Ab2 β vaccine described herein adopts the infectious agent associated antibodies IgY that especially injects in the yolk antibody to bird especially chicken injection pathogen specific mono-clonal or polyclonal antibody to prepare.

Can stimulate body to produce corresponding anti-Id antibody (Ab2) at idiotype (Id) on antibody molecule (Ab1) variable region of exotic antigen.Wherein Ab2 β has sequence of amino acid similar to exotic antigen or sterie configuration, and it can simulate the antigenic effect of initiating in vivo, so be considered to image ＂ in the ＂ of outside antigen in body immune system (internal image).Use this characteristic of Ab2 β, can on purpose prepare Ab2 β, be used to induce body to produce antiviral specific immune response as antigenic surrogate it, promptly so-called antiidiotype vaccine (anti-idiotype vaccine).It is to replace antigen as immunogen, so such vaccine is specially adapted to can't cultivate or cultivate very difficult at present and the very low pathogenic agent of output with anti-Id type antibody (Ab2); Directly with the adventurous pathogenic agent of pathogenic agent seedling; And immunogenicity is low and the pathogenic agent polyose antigen that can not produce with recombinant DNA technology.

The preparation method of the relevant yolk antibody of a kind of infectious agent, its feature also is: above-mentioned yolk antibody IgY or antiidiotypic antibody Ab2 α are prepared into the method that the above-mentioned cytotoxic substance of liposome particulate or even nanoparticle and then coupling becomes the immune guiding medicine, to increase its affinity and perviousness to focus; Above-mentioned antiidiotypic antibody Ab2 β or pathogen specific antigen expressed are prepared into the method for the specific pathogen vaccine of liposome particulate or even nanoparticle.

Liposome technology be called " biological missile " the 4th generation the target administration technology, liposome belongs to the nano-grade medicine carrier, this technology is utilized the monopolizing characteristic of liposome, toxic side effect is big, poor stability, the fast pharmaceutical pack of degraded are rolled in the liposome in blood, bigger according to vascular endothelial cell gap, body foci position, liposome medicament can see through this gap and arrive lesions position, piles up in focus portion to discharge, thereby reaches targeted delivery of drugs.The main auxiliary material of liposome is a phosphatide, and that phosphatide is eliminated in blood is very slow, so liposome medicament is long in circulation of blood system retention time, the effect that lesions position is fully treated.Utilize this technology large quantities of known high toxicity active medicines can be applied to clinical treatment safely and effectively, anticarcinogen, Tri-Biocin, antimycotic medicine, parasiticide class medicine, protein or polypeptide drug are wherein arranged, greatly improve the clinical treatment level, alleviated patient's slight illness.Monoclonal antibody can be connected on the liposome simultaneously,, the drug-loaded liposome orientation be sent into by means of the specific reaction of antigen and antibody.The present invention utilizes the relevant yolk antibody of above-mentioned infectious agent to comprise any or its prepared liposome particulate or even nanoparticle of composition in yolk antibody IgY, antiidiotypic antibody Ab2 α, antiidiotypic antibody Ab2 β and the pathogen specific antigen expressed, can effectively arrive the target area, bring into play its active and passive immunotherapy effect jointly and different pathogens is brought into play the immunotherapy effect jointly.

The preparation method of the relevant yolk antibody of a kind of infectious agent, its feature also is: two or more component with identical or close effect in the above-mentioned yolk antibody is mixed into composition according to a certain ratio with other auxiliary materials, makes corresponding yolk antibody component be directed to pathogenic agent performance synergy.

The purposes of the relevant yolk antibody of a kind of infectious agent is characterized in that: any formulation of any in the above-mentioned yolk antibody or arbitrary combination separately or unite diagnosis, treatment and the prevention that is used for transmissible disease.

Human body has certain immunizing power to transmissible disease, but different people, immunizing power have have by force a little less than.Immunotherapy is to manage to transfer various active defense factors in the human body, improves the immunizing power of body, with remaining pathogenic agent after the elimination pharmacological agent, with recurrence and the deterioration that wards off disease.The immunotherapy of transmissible disease is summarized can be divided into specific active immunotherapy and non-specific immunity treatment two big classes.Immunotherapy is a kind of measure of setting upright, and few side effects is welcome so be subject to patient.The passive immunotherapy of transmissible disease is an immunologic effector substance of showing body infusion external source, brings into play therapeutic action by these exogenous effector substances in body.Mainly contain following two big classes at present:

A) the antipathogen targeted therapy promptly utilizes the monoclonal antibody of high degree of specificity to be carrier, takes Cytotoxic murderer to affected area, can kill and wound pathogenic agent specifically.At present according to the different in kind of used murderer, the targeted therapy of transmissible disease is divided into: 1. radioimmunotherapy, the high energy radionuclide is connected with monoclonal antibody, radionuclide can be brought to the focus pathogen kill; 2. antibody targeted chemotherapy, the immune conjugate that antipathogen medicine and monoclonal antibody are formed by chemically crosslinked can kill and wound pathogenic agent with the drug targeting focus, and commonly used have methotrexate (MTX), a Zorubicin etc.; 3. immunotoxin therapy (immunotoxintherapy) links to each other toxin with monoclonal antibody, and the immunotoxin of preparation has specific strong killing activity to pathogenic agent.Toxin commonly used has two classes: a class is a plant poison, comprises comb numb seed toxin (RT), abrin (abrin), momordin (MD), Oxymatyine (OXY) etc.Another kind of is cytotoxin, comprises diphtheria toxin (DT), Pseudomonas aeruginosa extracellular toxin (PE).But used monoclonal antibody mostly is the mouse resource monoclonal antibody at present, is applied to understand the antibody that produces anti-mouse resource monoclonal antibody behind the human body, and it can not be used has repeatedly influenced its curative effect.Use engineered method, make mouse source antibody humanization can reduce this problem, but technical difficulty is higher, extensively promotes limited.

B) adoptive immunotherapy (adoptive immunotherapy) is to get the immune donor lymphocyte of pathogenic agent is transferred to the patient, or the immunocyte of getting patient self is after external activation, propagation, transfer again in patient's body, make it in patient's body, bring into play the antipathogen effect.The effector cell of adoptive immunotherapy has heterogeneity, all works in killing and wounding pathogenic agent as CTL, NK cell, scavenger cell, lymphokine activated killer cell (LAK) and wetting property lymphocyte (TIL) etc.

The purposes of the relevant yolk antibody of a kind of infectious agent, its feature also is: any in polyclonal anticancer antibody IgY in the above-mentioned yolk antibody or antiidiotypic antibody Ab2 α and fluorescence thereof, luminous, enzyme, the metal mark thing is used to prepare the external use immune diagnostic reagent; The radioisotope labeling thing of polyclonal anticancer antibody IgY in the above-mentioned yolk antibody or antiidiotypic antibody Ab2 α and liposome even nanoparticle is used to prepare immune guiding diagnostic reagent in the body.Above-mentioned described immune diagnostic reagent comprises immunohistochemical methods reagent and biochip.

The purposes of the relevant yolk antibody of a kind of infectious agent, its feature also is: polyclonal antibody IgY in the above-mentioned yolk antibody or antiidiotypic antibody Ab2 α and liposome thereof even nanoparticle are used to prepare the therapeutic immunization preparation; The conjugate of polyclonal antibody IgY in the above-mentioned yolk antibody or antiidiotypic antibody Ab2 α and liposome thereof even nanoparticle and radionuclide, medicine and toxin is used to prepare immune guiding medicine or biological missile.

The purposes of the relevant yolk antibody of a kind of infectious agent, its feature also is: the pathogen specific antigen expressed in the above-mentioned yolk antibody is used to prepare polypeptide vaccine; Antiidiotypic antibody Ab2 β in the above-mentioned yolk antibody is used to prepare the antiidiotypic antibody vaccine.

The specificity active immunity treatment is by activating specific immunity system, reach the purpose of eliminating pathogen, and this treatment does not influence normal surrounding tissue.Theoretically,, can activate specific lymphocyte (B cell and (or) T cell by the specific active immunotherapy of vaccine mediation) reaction, produce antipathogen effect at once, and pathogenic agent is in the future produced anamnestic response.At first, utilize pathogenic agent to prepare vaccine; Afterwards, pathogen antigen became the material of preparation vaccine.Along with the progress of molecular biology method, but utilize the genetic modification pathogen antigen of antigen expressed to come enhancing immunity originality or utilize modifying factor to be used for vaccine production with technology such as secrete cytokines.Simultaneously, also can utilize purification pathogenic agent related antigen, dna encoding proteantigen and (or) protein derived peptide prepares vaccine.The final purpose of vaccine inoculation is certain part of activating immune system, as antibody or lymphocyte, kills pathogenic agent.At present, the specificity active immunity treatment has a lot of methods, as the mixture of adjuvants such as complete pathogenic agent and bacille Calmette-Guerin vaccine; Change pathogenic agent with genetic method.In addition, single antigen prepd vaccine such as also available peptide, mucoprotein and carbohydrate.Many recombiant vaccines can be expressed various antigens such as hepatitis B surface antigen.Utilize recombiant vaccine, other genes (for example gene of coding cooperation stimulating factor molecule) can also the transduction of pathogenic agent genes involved.Also can adopt antiidiotypic antibody at specific antibody, because the antigenicity difference of pathogen antigen, and the pathogenic agent host often is in immunosuppressive condition, is difficult to excite effective antipathogen reaction.Since the eighties, many scholars can overcome immunosuppression by the experimentation on animals proof with image type Ab2 (claiming the antiidiotype vaccine again) in the antigen, induce body to produce specific immune response.The immunological network theory thinks that idiotype (Id) antigenic determinant that exists on antibody (Ab1) molecule can stimulate body to produce a series of production of antibodies such as A-Id antibody (Ab2) and Ab3, thereby constitutes the immunological network of a complexity.According to antiidiotypic antibody and the characteristics of antibody idiotope reaction and the function of antiidiotypic antibody, A-Id can be divided into Ab2 α, Ab2 β, Ab2 γ, Ab2 δ.Wherein Ab2 β can discern that Ab1 goes up and antigen complementary determinant, can suppress combining of antigen and Id fully.It has similar antigenic structure, can simulate ＂ antigen by ＂, produces the biological effect identical with antigen, is considered to outside antigen " interior image " (mternal image) in body immune system.Ab2 β has following characteristics; A) individual antibody (Ab1) reaction that produces of energy specificity and different genera; B) energy specificity and inhomogeneity antibody (Ab1) reaction; C) Ab2 β can combine Ab1 competitively with antigen, has competitive inhibition between the two; D) can induce the individual specific immune response that produces of different genera.

Antiidiotypic antibody with interior imaging characteristic can be simulated the pathogenic agent related antigen and be induced body to produce active immunity to reply, and its answering is extremely complicated, but relates generally to humoral immunization and cellular immunization two aspects.Ab2 β activates helper T lymphocyte subgroup (Th1 and Th2) as thymus-dependent antigen (TD), secretes a series of cytokines, produces INF-γ, IL-10, TNF and IL-2 as the Th1 cell; The Th2 cell produces IL-4, IL-5, IL-6, IL-10 etc.The cytokine influence that the Th2 cell produces and differentiation of promotion bone-marrow-derived lymphocyte and propagation produce specific antibody Ab3, and an Ab3 part can be simulated mutually with Ab1, promptly has and antigen-binding, is referred to as antipathogen antibody (Ab1 ').The generation of total Ab3 be proved disappear with small pathogenic agent focus and/or patient sb.'s illness took a favorable turn is proportionate.The Th1 cell that is activated discharges cytokine, can be as INF-γ by influencing the NK cell and the T cell function is brought into play the antipathogen effect, IL-2 can promote T cell growth, the amplification of clone's property and enhancing body's immunological function, also can induce or promote various kinds of cell toxic cell activity, as NK, CTL, LAK and TIL etc., performance immunosurveillance and antipathogen effect.IL-2 also can work in coordination with simultaneously stimulates B cell proliferation and secretory antibody, acts on performance ADCC and CDC antipathogen effect (Gong Feili etc., Medical Immunology, second edition, Wuhan: Science Press, 2000) mutually with complement and effector cell.Immunity system is a system that is in self running balance in the human body.In the intravital immunity system of patients with infectious diseases, antiidiotypic antibody is crucial factor of system, and quality and quantity with artificial method change antiidiotypic antibody will inevitably make system change, and reach new balance again on higher level.The system balancing that makes the pathogenic agent immunity is according to the direction development and change that help the patient of prediction in theory, in some sense this antiidiotypic antibody key in application place just.

The present invention is exactly the specific active immunotherapy that adopts prepared yolk pathogen specific antigen expressed of aforesaid method and antiidiotypic antibody Ab2 β to be used for transmissible disease as the pathogen specific vaccine, can activate specific lymphocyte (B cell and (or) T cell) reaction, produce corresponding antipathogen effect, and pathogenic agent in the future produced anamnestic response, activate body immune system even readjust the system balancing of immunity of organism, transfer humoral immunization and immunization of cell, produce specific pathogen antibody or killer lymphocyte, kill pathogenic agent jointly, reach the purpose of immunity treatment transmissible disease.

The purposes of the relevant yolk antibody of a kind of infectious agent, its feature also is: any of the polyclonal antibody IgY in the above-mentioned yolk antibody, pathogen specific antigen expressed, antiidiotypic antibody Ab2 α, Ab2 β or arbitrary combination are used to prepare food, beverage and the healthcare products that keep off infection.Promptly adopt any or arbitrary combination among the polyclonal antibody IgY among the present invention, pathogen specific antigen expressed, antiidiotypic antibody Ab2 α, the Ab2 β are made an addition in food, beverage and the healthcare products according to a certain ratio, be used for the prevention and the early stage prophylactic treatment of transmissible disease.Technique effect

The present invention utilizes the IgY antibody in the relevant yolk antibody of infectious agent to be directed to antigenic immunodiagnosis medicament of pathogen specific and immunotherapy medicament for main raw material is prepared at above problem, comprises that the targeted drug of external diagnosis reagent, internal guide diagnostic reagent and specificity yolk antibody mediated immunity therapeutical agent and specificity yolk antibody coupling medicine or toxin is a biological missile; Utilize the pathogen specific antigen expressed in this yolk antibody can be prepared into immunodiagnosis medicament and the immunotherapy medicament that is directed to pathogenic agent, comprise external diagnosis reagent and specificity vaccine knurl seedling and nucleic acid vaccine; The immunodiagnosis medicament and the immunotherapy medicament that utilize antiidiotypic antibody Ab2 α in this yolk antibody and Ab2 β to be directed to pathogenic agent for main raw material can be prepared into, the targeted drug that comprises external diagnosis reagent, internal guide diagnostic reagent (mainly adopting Ab2 α) and antiidiotypic antibody Ab2 α coupling drug or toxin is antiidiotype biological missile and antiidiotypic antibody vaccine (mainly adopting Ab2 β); The diagnosis, immunotherapy and the targeted therapy that are used for transmissible disease.This yolk antibody IgY and antiidiotypic antibody also can be used as additive and make an addition in food, beverage and the healthcare products in addition, the prevention and the early stage prophylactic treatment that are used for transmissible disease are in particular for having the passive immunization and the active immunity treatment of sexually transmitted disease, viral enteritis and influenzas etc. such as very big hazardness transmissible disease such as acquired immune deficiency syndrome (AIDS), hepatitis B, hepatitis C, tuberculosis, helicobacter pylori property gastritis, syphilis, gonorrhoea to the mankind.

Owing to adopt the inventive method to prepare the relevant yolk antibody of infectious agent, utilize the chicken immune system can produce and have higher tiring and the specific antibody of avidity heterology albumen or biomolecules, can induce the antiviral antibody of specificity of high titre, and it is low to have cost, the output height, high specificity, the advantage of the wide scale operation that also can intensify of determinant, can realize at the yolk polyclonal antibody IgY of multiple pathogenic agent, the disposable preparation of the Multiple Combination thing in antiidiotypic antibody and the pathogen specific antigen expressed, the more important thing is that this yolk antibody can solve above-mentioned mouse source property monoclonal antibody conjugate or existing HAMA reaction of tumor vaccine and heterogeneous problem well, and human body is had no side effect, have security preferably.In addition, the relevant yolk antibody of the prepared infectious agent of the present invention or its composition can be used as raw material, the especially raw material of infectious agent related biochip of infectious agent dependent diagnostic reagent; Moreover be exactly that prepared yolk antibody of the present invention and composition thereof can brought into play its immunotherapy effect jointly aspect passive immunization and the active immunity two.The present invention is applicable to immunodiagnosis, treatment and the prevention of transmissible disease, is particularly useful for the mankind are had the passive immunization and the active immunity treatment of sexually transmitted disease, viral enteritis and influenzas etc. such as very big hazardness transmissible disease such as acquired immune deficiency syndrome (AIDS), hepatitis B, hepatitis C, tuberculosis, helicobacter pylori property gastritis, syphilis, gonorrhoea.

Embodiment

Further specify the present invention below in conjunction with embodiment.

Embodiment 1: comprise pathogen specific antigen with having immunocompetent pathogen specific immunogen, vaccine knurl seedling, any or its mixture immunity in DNA or mRNA and antibody or its liposome complex especially chicken of bird of laying eggs, and then append reinforcement with Freund and react, treat that bird after 5-20 days especially promptly has in the laying eggs of chicken the polyclonal antibody IgY that is directed to above-mentioned pathogen immunogenic or any or its composition in antiidiotypic antibody or the pathogen specific antigen expressed to exist and stores, its concrete steps comprise: get 6 monthly age SPF pure lines Lay boat bird inlay and raise in standard I I level cleaning level Animal House, every chicken is in the above-mentioned pathogen immunogenic 1-2ml of chest belly multi-point injection (the incomplete freund adjuvant of immune commercial weight 10-500 μ g/ml+ equivalent, wherein the consumption of antibody A b1 is the incomplete freund adjuvant of 100～2000ug+ equivalent), 2～4 week back booster immunizations injections 3-5 time treat promptly to contain in immune hen produced after 5-20 days the immune egg pathogenic agent yolk antibody of being correlated with.

Embodiment 2: after the preparation of the relevant yolk antibody of infectious agent goes yolk behind the egg white to stir above-mentioned immune egg, the 0.1mol/L PH7.2 phosphoric acid salt-trehalose diluent that adds 5 times of volumes of yolk homogenate, placed ice chest (20 ℃) then freezing 1-5 hour, take out in room temperature and melt again and low-speed centrifugal (500-1000r/min) back drop goes out to collect supernatant liquor, the 0.1mol/L PH7.2 phosphoric acid salt-trehalose diluent that in throw out, adds 2-5 times of volume again, abundant mixing dissolution precipitation thing, comply with last method freeze thawing collection supernatant liquor once more, so multigelation is 3-5 time, merge and collect the supernatant liquor adding sodium sulfate that each time obtains, make the final concentration of sodium sulfate reach 10-30%, its throw out of centrifugal collection, and redissolve this throw out back with distilled water to salt dialysis back ultrafiltration and concentration, gel-filtration, the relevant yolk antibody of pathogenic agent that lyophilize promptly obtains purifying comprises any or its composition in yolk polyclonal antibody IgY or antiidiotypic antibody or the pathogen specific antigen expressed.Experimental result shows: the pathogenic agent of the batch preparations yolk antibody of being correlated with, and every egg can extract the pathogen specific yolk antibody of 57～171mg, and average every egg can extract 89mg, and its purity is 95%.

Embodiment 3: the detection of the detection hepatitis B reorganization surface antigen specificity yolk antibody IgY of the relevant yolk antibody of infectious agent adopts the ELISA indirect method to detect, with hepatitis B reorganization surface antigen liquid coated elisa plate hole, adopt the mouse-anti chicken IgY monoclonal antibody of horseradish peroxidase-labeled anti-as two; The ELISA double antibody sandwich method is adopted in the detection of specific expressed hepatitis B surface antigen(HBsAg), with hepatitis B surface antibody coated elisa plate hole, adopts the hepatitis B surface antibody of the horseradish peroxidase-labeled antibody that serves as a mark; The ELISA indirect method is adopted in the detection of hepatitis B reorganization surface antigen specificity yolk antiidiotypic antibody, with hepatitis B surface antigen specificity yolk antibody IgY coated elisa plate hole, adopts the mouse-anti chicken IgY monoclonal antibody of horseradish peroxidase-labeled anti-as two; Above-mentioned ELISA detects and carries out according to routine operation.Experimental result shows: the tiring 1 of hepatitis B reorganization surface antigen specificity yolk antibody: 1280-1: between 10240, wherein hepatitis B reorganization surface antigen specificity yolk antibody IgY on average tiring about 1: 5120; On average tiring about 1: 2560 of specific expressed hepatitis B surface antigen; On average tiring about 1: 5120 of hepatitis B reorganization surface antigen specificity yolk antiidiotypic antibody.

Embodiment 4: the relevant yolk antibody degerming after filtration of the stability test pathogenic agent of the relevant yolk antibody of infectious agent, in 10mg/ml packing small test tube,, regularly carry out its detection of tiring respectively at-20 ℃ of freezing, 4 ℃ and room temperature preservation.Experimental result shows: the relevant yolk antibody of pathogenic agent is in-20 ℃ of stored frozen, and its stability can keep more than 12 months at least; In 4 ℃ of preservations, its stability can keep more than 6 months at least; In room temperature preservation, its stability can keep more than 2 months at least.

Embodiment 5: dextran 1.0g is at first got in the coupling test of hepatitis B reorganization surface antigen specificity yolk antibody IgY and Oxymatyine, add sodium periodate 0.3g after adding 200mL distilled water, the lucifuge reaction is spent the night under the room temperature, make dextran fully be oxidized to many aldehyde radicals dextran, freeze-drying after distilled water is fully dialysed; Injection for curing is got in preceding 2 hours among the phosphoric acid buffer 2mL of many aldehyde radicals dextran 10mg adding 0.15mol/L (pH7.2), add oxidation matrine 10mg, reaction is after 24 hours under the room temperature, the hepatitis B reorganization surface antigen specificity yolk antibody IgY 10mg that adds purifying again, 4 ℃ of stirring reactions 24 hours; Add sodium borohydride 0.2mg then in above-mentioned reaction mixture, centrifugal after Sephadex G-150 chromatography column is crossed post at 4 ℃ of following reductase 12s hour, collecting first peak can be used for the treatment of.

Embodiment 6: result of treatment is observed by to 36 routine hepatitis B patients (chronic viral hepatitis B 26 examples wherein, reactivity hepatitis B 12 examples, hepatitis B virus carriers's 8 examples) give drug alone (Interferon, rabbit 3,000,000 units), hepatitis B reorganization surface antigen specificity yolk antibody IgY-Oxymatyine targeted therapy, hepatitis B reorganization surface antigen specificity yolk antibody IgY-Oxymatyine and hepatitis B reorganization surface antibody specificity yolk antiidiotypic antibody Ab2 β combination therapy respectively and carry out observation of curative effect and comparison.Experimental result shows: hepatitis B virus surface antigen HBsAg negative conversion rate combination therapy group (71.2%) is apparently higher than targeted therapy group (67.5%) and drug alone group (12.4%); Two groups (89.4%) obviously is better than drug alone group (42.5%) behind the hepatitis B virus HBeAg negative conversion rate; Two groups (59.2%) obviously is better than drug alone group (6.5%) behind the hepatitis B viruses (HBV) DNA negative conversion rate: back two groups of patients serum ALT, DBIL and TNF-alpha levels are starkly lower than the drug alone group.Show that hepatitis B reorganization surface antigen specificity yolk antibody IgY-Oxymatyine targeted therapy has advantages such as high specificity, toxic side effect are little; Hepatitis B reorganization surface antigen specificity yolk antibody IgY-Oxymatyine and hepatitis B reorganization surface antibody specificity yolk antiidiotypic antibody Ab2 β combination therapy have the effect that activates body cell and humoral immunization, thereby reach the purpose to hepatitis B virus resisting protection liver organization cell.

Claims (10)

1, the preparation method of the relevant yolk antibody of a kind of infectious agent, it is characterized in that: with lay eggs bird especially chicken as immune reactor, with pathogen specific antigen one or more purifying or that adopt the genetic engineering technique reorganization or vaccine knurl seedling or pathogen specific DNA or mRNA and correspondingly the recombinant chou formed of carrier be directed to the antigenic mono-clonal of pathogen specific or the liposome complex of polyclonal antibody or pathogenic agent or above-mentioned pathogen immunogenic as immunogen, carry out separately respectively or multiple the associating carried out the especially repeatedly repetition immunization of chicken of bird, and then append with Freund and to strengthen reaction, treat that bird after 5-20 days especially promptly has in the laying eggs of chicken the polyclonal antibody IgY that is directed to above-mentioned pathogen immunogenic or any or its composition in antiidiotypic antibody or the pathogen specific antigen expressed to exist and stores.Its concrete steps comprise:

A) pathogen immunogenic or its composition carry out repeatedly that the immunization bird is preferably chicken.Pathogen immunogenic described herein is by the pathogen antigen of purifying or the pathogenic agent by deactivation, pathogen specific antigen by the genetic engineering technique reorganization or vaccine knurl seedling, pathogen specific naked DNA by extracting purifying or mRNA and antigenic mono-clonal of recombinant chou, pathogen specific or the polyclonal antibody formed with respective carrier, also comprises the liposome particulate or even the nanoparticle of above-mentioned pathogen immunogenic in addition; Described bird comprises chicken, duck, goose, quail, and first-selection is a chicken; The injection site comprises muscle, subcutaneous, intracutaneous, blood vessel and genital cloaca capsule; Injecting method comprises above-mentioned pathogen immunogenic injected separately with former associating of panimmunity and repeatedly repeats immunization, and wherein said multiple pathogen immunogenic is united and carried out immunization and comprise with any or its composition in pathogen antigen, gene recombination pathogen specific antigen or vaccine knurl seedling, pathogen specific naked DNA or the mRNA and the recombinant chou thereof in different sorts and source; Also comprise any or its composition in the pathogen specific mono-clonal in different sorts and source or the polyclonal antibody; Described immunogen injection volume is 1-2ml (the incomplete freund adjuvant of immune commercial weight 10-500 μ g/ml+ equivalent, wherein the consumption of antibody A b1 is the incomplete freund adjuvants of 100～2000 μ g/ml+ equivalent).Described pathogenic agent comprises any or its combination in the parasites of human such as virus, chlamydozoan, mycoplasma, Rickettsiae, bacterium, spirochete, fungi and protozoon, worm, nematode; Described communicable disease pathogenic agent comprises any or its combination in the pathogenic agent of diseases such as sexually transmitted diseases such as acquired immune deficiency syndrome (AIDS), hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, tuberculosis, helicobacter pylori property gastritis, syphilis, gonorrhoea, viral enteritis, virus pneumonia, viral encephalitis, mycoplasma, chlamydia pneumonia, typhus fever, bleb, rubella influenza and parasitic infection.

B) the above-mentioned pathogen immunogenic full adjuvant that toos many or too much for use carries out supplementary immunization reaction.

C) treat that bird after 5-20 days especially promptly has the yolk antibody that is directed to above-mentioned pathogen immunogenic to exist in the laying eggs of chicken and stores.Wherein said yolk antibody comprises any or its composition among the yolk polyclonal antibody IgY that is directed to above-mentioned pathogen antigen, pathogenic agent, reorganization pathogen antigen or vaccine knurl seedling and liposome or nanoparticle; Be directed to any or its composition in the pathogen specific antigen expressed of pathogen specific naked DNA or mRNA and recombinant chou thereof and liposome thereof or nanoparticle; Be directed to any or its composition among the antiidiotypic antibody Ab2 α of pathogen specific mono-clonal or polyclonal antibody or above-mentioned yolk polyclonal antibody IgY and liposome or nanoparticle and the Ab2 β.

2, the preparation method of the relevant yolk antibody of a kind of infectious agent according to claim 1, its feature also is: the extracting and purifying method of the relevant yolk antibody of above-mentioned pathogenic agent is to adopt water dilution method, multigelation method or ammonium sulfate, sodium sulfate, PEG or sad precipitation or lixiviate, extraction, concentrate, membrane filtration method from above-mentioned bird especially egg yolk, extract purifying and be directed to any or its composition in the polyclonal antibody IgY of above-mentioned pathogen immunogenic or antiidiotypic antibody or the pathogen specific antigen expressed, then the relevant yolk antibody of pathogenic agent of this purifying is added that the respective secondary material package is drawn together trehalose and any formulation of being prepared on the pharmaceutics comprises tablet, injection, oral liquid and sprays.Its concrete steps comprise:

A) get birds, beasts and eggs especially egg shell and get Huang, remove the protein ingredient that adheres on the membrane of yolk, then yolk is placed sterile purified water, repetitive scrubbing three times is further removed the protein ingredient on the membrane of yolk, punctures membrane of yolk after draining, and collects yolk.

B) above-mentioned collection yolk is stirred after, add the 0.1mol/L PH7.2 phosphoric acid salt-trehalose diluent of 5 times of volumes of yolk homogenate, make the dilution egg yolk liquid.The weight percent of trehalose is 5-30% in this dilution egg yolk liquid.

C) above-mentioned dilution egg yolk liquid was placed ice chest (20 ℃) freezing 1-5 hour, take out then in room temperature and melt again and low-speed centrifugal (500-1000r/min) back drop goes out to collect supernatant liquor, and then in throw out, add the 0.1mol/L PH7.2 phosphoric acid salt-trehalose diluent of 2-5 times of volume, abundant mixing dissolution precipitation thing, comply with last method freeze thawing collection supernatant liquor once more, so multigelation is 3-5 time, and the supernatant liquor that each time of merging collection obtains is standby.

D) in above-mentioned collection supernatant liquor, add any and mixture thereof in ammonium sulfate, sodium sulfate, PEG, T 500, sodiun alginate or the sad precipitation agent in proportion, its throw out of centrifugal collection promptly contains any or its composition in the polyclonal antibody IgY that is directed to above-mentioned pathogen immunogenic or antiidiotypic antibody or the pathogen specific antigen expressed in this throw out.The pathogenic agent of the batch preparations yolk antibody of being correlated with, every egg can extract the pathogen specific yolk antibody of 57～171mg, and average every egg can extract 89mg, and its purity is 90-95%.

E) above-mentioned throw out is redissolved back dialysis or ultrafiltration and concentration or gel chromatography with distilled water, further separation and purification is directed to any or its composition in the polyclonal antibody IgY of above-mentioned pathogen immunogenic or antiidiotypic antibody or the pathogen specific antigen expressed.

F) the relevant yolk antibody of pathogenic agent to above-mentioned purifying adds corresponding auxiliary material in proportion, and its any formulation that is prepared on the pharmaceutics is comprised tablet, injection, oral liquid and sprays.Auxiliary material described herein comprises trehalose.

3, the preparation method of the relevant yolk antibody of a kind of infectious agent according to claim 1 and 2, its feature also is: the modification treatment process of relevant yolk antibody IgY of above-mentioned pathogenic agent or antiidiotypic antibody Ab2 α is that coupling or covalent attachment radionuclide, medicine and toxin prepare immune guiding medicine or biological missile; Be that coupling or covalent attachment radionuclide, luminophore, Radioactive colloidal gold or electroselenium or enzyme prepare the interior immunodiagnosis medicament of body and external immune diagnostic reagent comprises immunohistochemical methods reagent and biochip; Be in the relevant yolk antibody IgY of above-mentioned pathogenic agent, to add Freund to prepare the pathogen specific immunotherapeutic agent; Be in relevant yolk antibody of above-mentioned pathogenic agent especially antiidiotypic antibody Ab2 β or pathogen specific antigen expressed, to add Freund and other components prepare the pathogen specific vaccine.Its concrete steps comprise:

A) enzyme decomposition or the coupling activity group of relevant yolk antibody IgY of pathogenic agent or antiidiotypic antibody Ab2 α are modified processing in advance, wherein the enzyme decomposition is to utilize stomach en-or ficin that yolk antibody IgY or antiidiotypic antibody Ab2 α are decomposed, be reduced to the Fab small pieces with DTT or beta-mercaptoethanol again, so that it has better tissue penetration; Or with corresponding activating reagent with the amino on yolk antibody IgY or antiidiotypic antibody Ab2 α and the Fab small pieces thereof or carboxyl or sulfydryl or aldehyde radical activation, and then with various drug molecule coupling.

B) the pre-modification of coupling drug is handled and is comprised the corresponding activating reagent of radionuclide, medicine and lps molecule, respectively the coupling drug that contains groups such as hydroxyl, amino, aldehyde radical, diazanyl, sulfydryl is activated, and then make its relevant yolk antibody IgY or antiidiotypic antibody Ab2 α and Fab small pieces generation covalent attachment thereof with pathogenic agent.

C) relevant yolk antibody IgY of pathogenic agent or antiidiotypic antibody Ab2 α and Fab small pieces thereof comprise with the coupling drug of activation treatment radionuclide, medicine and lps molecule covalent attachment are prepared pathogen specific immune guiding medicine or biological missile.

D) the relevant yolk antibody IgY of pathogenic agent or antiidiotypic antibody Ab2 α are carried out radionuclide, luminophore, Radioactive colloidal gold or electroselenium or enzyme labelling and prepare its corresponding marker.

E) cooperate other components to prepare immunodiagnosis medicament and external immune diagnostic reagent (comprising immunohistochemical methods reagent and biochip) in the body radionuclide, luminophore, Radioactive colloidal gold or the electroselenium of relevant yolk antibody IgY of pathogenic agent or antiidiotypic antibody Ab2 α or in the enzyme labelling thing any.

F) in the relevant yolk antibody IgY of above-mentioned pathogenic agent, add Freund and other components, and then carry out immunization, the immunity body produces the antiidiotypic antibody Ab2 β that is directed to the relevant yolk antibody IgY of pathogenic agent, thereby induces body to produce antiviral specific immune response.

G) add Freund in any or its composition in above-mentioned antiidiotypic antibody Ab2 β, pathogen specific antigen expressed and other components prepare pathogen specific vaccine (comprising antiidiotypic antibody vaccine and polypeptide vaccine).

H) especially inject IgY antibody in the relevant yolk antibody of pathogenic agent to bird especially chicken injection pathogen specific mono-clonal or polyclonal antibody, prepare described antiidiotypic antibody Ab2 β vaccine.

4, according to the preparation method of the relevant yolk antibody of the described a kind of infectious agent of claim 1-3, its feature also is: above-mentioned yolk antibody IgY or antiidiotypic antibody Ab2 α are prepared into the above-mentioned cytotoxic substance of liposome particulate or even nanoparticle and then coupling and prepare the method for immune guiding medicine; Above-mentioned antiidiotypic antibody Ab2 β or pathogen specific antigen expressed are prepared into the method for the pathogen specific vaccine of liposome particulate or even nanoparticle.

5, according to the preparation method of the relevant yolk antibody of the described a kind of infectious agent of claim 1-4, its feature also is: two or more component with identical or close effect in the above-mentioned yolk antibody is mixed into composition according to a certain ratio with other auxiliary materials, makes the relevant yolk antibody component of corresponding pathogenic agent be directed to pathogenic agent performance synergy.

6, the purposes of the relevant yolk antibody of a kind of infectious agent is characterized in that: any formulation of any in the above-mentioned yolk antibody or arbitrary combination separately or unite diagnosis, treatment and the prevention that is used for transmissible disease.

7, the purposes of the relevant yolk antibody of a kind of infectious agent according to claim 6, its feature also is: any in polyclonal antibody IgY in the relevant yolk antibody of above-mentioned pathogenic agent or antiidiotypic antibody Ab2 α and fluorescence thereof, luminous, enzyme, the metal mark thing is used to prepare the external use immune diagnostic reagent; The polyclonal antibody IgY in the relevant yolk antibody of above-mentioned pathogenic agent or the radioisotope labeling thing of antiidiotypic antibody Ab2 α and liposome even nanoparticle are used to prepare immune guiding diagnostic reagent in the body.Above-mentioned described immune diagnostic reagent comprises immunohistochemical methods reagent and biochip.

8, the purposes of the relevant yolk antibody of a kind of infectious agent according to claim 6, its feature also is: polyclonal antibody IgY in the relevant yolk antibody of above-mentioned pathogenic agent or antiidiotypic antibody Ab2 α and liposome thereof even nanoparticle are used to prepare the therapeutic immunization preparation; The polyclonal antibody IgY in the relevant yolk antibody of above-mentioned pathogenic agent or the conjugate of antiidiotypic antibody Ab2 α and liposome even nanoparticle and radionuclide, medicine and toxin are used to prepare immune guiding medicine or biological missile.

9, the purposes of the relevant yolk antibody of a kind of infectious agent according to claim 6, its feature also is: the IgY antibody in the relevant yolk antibody of above-mentioned pathogenic agent is used to prepare the transmissible disease immunotherapeutic agent; Pathogen specific antigen expressed in the relevant yolk antibody of above-mentioned pathogenic agent is used to prepare the pathogenic agent polypeptide vaccine; Antiidiotypic antibody Ab2 β in the relevant yolk antibody of above-mentioned pathogenic agent is used to prepare pathogenic agent antiidiotypic antibody vaccine.

10, the purposes of the relevant yolk antibody of a kind of infectious agent according to claim 6, its feature also is: any or its composition of the polyclonal antibody IgY in the relevant yolk antibody of above-mentioned pathogenic agent, pathogen specific antigen expressed, antiidiotypic antibody Ab2 α, Ab2 β is used to prepare food, beverage and the healthcare products that keep off infection.