CN1744818A - Immune t-cell stimulation. - Google Patents

Abstract

The present invention is a novel approach for stimulating or enhancing immune T-cells in an animal. In particular, the invention is directed to an egg, and more specifically, a fraction thereof of a size less than 3000 daltons, obtained from an avian that has been hyperimmunized with one or more immunogens. The ingestion by an animal of an effective amount of the hyperimmune egg or fraction thereof causes a significant increase in the overall T-cell population in that animal.

Description

Immune t-cell stimulation

Related application

The application requires the priority of the U.S. Provisional Application No.60/381570 that submitted on May 16th, 2002.

Invention field

The present invention relates to stimulate or strengthen the method for the immune t-cell activity in the receptor by the composition of natural generation.More particularly, the present invention relates to, thereby can stimulate or strengthen the activity of immune t-cell in the animal from the ovum of hyperimmune birds or the administration of its fraction.

Background of invention

The stimulation of T-cell and enhancing have important function to the generation of immune response, the survival of mature T-cell and the growth of thymocyte.It is to eliminate by an important component part of the specificity acquired immune system of the cell of infected by microbes.It has also constituted the basis of host self recognizability simultaneously.The T-cell has passed through positive and negative the selection to himself recognizability, and wherein the MHCp complex is not activated fully, thereby has stoped the immune response at host tissue.

The stimulation of T-cell can increase cytotoxic T lymphocyte (CTL) colony, and wherein CTL has the ability of cracking target cell, is the lymphocytic a kind of important effector molecules activity of T.People T-cytositimulation is that a kind of acquisition is to having the effective ways of Cytotoxic T-cell monoid from the intrinsic tumour cell of body.This can increase the amount of the helper T lymphocyte that relates to human B lymphocyte activation and immunoglobulin generation equally.

The antigen-specific immune response of original T-cell relates to a series of very complicated collaborative incidents.This activation comprises differentiation, propagation and the effector function of T-cell.The various parameters of common this atomization of domination are determined, for example, and the characteristic of antigen presentation approach and dosage, microenvironment, antigen presenting cell (APC) and antigen.The initial critical event of immune t-cell activation is to be interacted with the antigenic peptide that combines with the MHC molecule on the APC by the specific t-cell receptor on the T cell (TCR) to cause.These incidents are vital for the generation of cell factor such as IL-2 and the expression of cytokine receptor.This will determine the degree and the duration of T cell proliferation again.The adjusting of IL-2 being produced by activation T-cell is one of key character of T cell activation adjusting.

Each kind of Aves, for example chicken (gallus domesticus), turkey and duck can produce in blood and ovum and anti-ly can cause the immunogenic of bird disease and anti-other immunogenic antibody.For example, LeBacqVerheyden etc. (Immunology 27:683 (974)) and Leslie, G.A. etc. (J.Med.130:1337 (the 1969)) immunoglobulin to chicken have carried out quantitative analysis.Polson etc. (Immunological Communications9:495-514 (1980)) carry out the natural mixture that immunity makes its anti-numerous protein and protein to hen, and detect IgY antibody in its yolk.Fertel etc. (Biochemicaland Biophysical Research Communications 102:1028:1033 (1981)) immune hen makes its energy antiprostaglandin, and detects antibody in its yolk.Jensenius etc. (Journal of Immunological Methods 46:63-68 (1981)) provide a kind of method that yolk IgG is used for Application in Immunodiagnosis of separating.Polson etc. (Immunological Communications 9:475-493 (1980)) have described from by isolated antibody the hen yolk of various plant virus immunity.

Developed hyperimmune ovum at present, but and be proved its excess and produce antibody and some biotic factors.Some examples for this class progress are as follows:

U.S. Patent No. 4,357 discloses the technical scheme of separation antibody from the yolk that derives from the hyperimmune hen in 272.Described antibody response is to cause by injecting the immunogene that derives from plant virus, human IgG, tetanus antitoxin, snake antivenin and Serameba repeatedly.

U.S. Patent No. 4,550 discloses the technical scheme that the immunogene that has at least 30,000 molecular weight or a particle weight from employing is carried out separation antibody in the yolk that the antibody of hyperimmune hen produces in 019.The immunogene that is used for the hyperimmune chicken is selected from: plant virus, human immunoglobulin(HIg), tetanus toxin and snake venom.

U.S. Patent No. 4,748 discloses the mammiferous method of passive immunity in 018, and it comprises that parenteral derives from the antibody purified of the ovum of the birds of using the corresponding antigens immunity, and described mammal has obtained the immunity to above-mentioned ovum.

U.S. Patent No. 5,772,999 disclose by the hyperimmune ovum of acceptor administration and/or breast or its grade being assigned to prevent, suppress or reduces the method that (NSAID-causes) stomach that chronic gastroenteropathy disease in the acceptor or NSAID cause damages.

U.S. Patent No. 6,420,337 disclose the new cell factor activation factor (CAF) of separating from the hyperimmune ovum, and it can raise some proinflammatory cytokines, comprises TNF α, IL-6 and IL-1 β, and can reduce TGF β.

But do not have one piece to disclose or inspired in these lists of references, have the ability that stimulates or strengthen the immune t-cell activity in the described animal when described ovum or its fraction are administered to animal.Do not have in these lists of references to disclose or inspired a kind of like this method that rational expection is provided yet, wherein adopt non-specific vaccine hyperimmune birds can induce birds to give birth to ovum with this ability when being administered to receptor.

The invention summary

The purpose of this invention is to provide by ovum product and stimulate the method that immune t-cell produces in the animal described animals administer effective dose.

Further purpose of the present invention provides by the ovum product to described animals administer effective dose increases the method that immune t-cell produces in the animal.

The accompanying drawing summary

Fig. 1 is a curve map, and it has described the stimulation index with the Jurkat cell of the various time phases of cultivating less than 3000 daltonian fractions of hyperimmune ovum.

Fig. 2 is a curve map, and it has described the stimulation index with the Daudi cell of the various time phases of cultivating less than 3000 daltonian fractions of hyperimmune ovum.

                         Detailed Description Of The Invention

The present invention adopts new approach to stimulate or strengthen the activity of the immune t-cell in the animal. The present invention includes the ovum or its fraction that derive from through the hyperimmune birds of one or more immunogenes. Animal can cause the significant stimulation of T-cell monoid in the described animal to the absorption of the hyperimmune ovum of effective dose or its fraction.

Ovum product among the present invention is the product with special attraction, because it is fully natural, and itself can stimulate or strengthen T cytoactive in the animal through administration, and need not to worry the negative interaction that the immunostimulation product of many present employings is followed usually. Clearly, ovum there is allergia or has and to take in hyperimmune egg product in some form of medication to the animal of the tolerance of ovum.

The inventor believes that above-mentioned beneficial characteristics (being T cytositimulation and enhancing) is because some immunological factors that cause or strengthen through the hyperimmune process cause.These immunological factors are not considered to antibody because as described in the embodiment, the size of hyperimmune ovum less than 3000 daltonian grades of proportion by subtraction whole eggs this in stimulate and enhance immunity T-cytoactive aspect more effective.Clearly, the fraction less than 3000 dalton's sizes does not contain antibody.Therefore, the inventor thinks that these factors have replaced some immune-regulating factor type, has effectively induced the cell arm of immune response, has caused the stimulation of immune t-cell.

Definition

Term " ovum " or " whole egg " are all represented any complete ovum, no matter are edible ovum or hyperimmune ovum or other ovum.

Term " ovum product " or " its fraction " represent to derive from the spawn or the fraction of ovum respectively.

Term " edible ovum " or " edible ovum product " or " its fraction " are represented complete ovum respectively, or the spawn in its source or fraction, and these all derive from the animal that lays eggs that remains on non-hyperimmune state.

Term " hyperimmune ovum " or " hyperimmune ovum product " or " its fraction " are represented complete ovum respectively, or the spawn in its source or fraction, and these all derive from the animal that lays eggs that remains on hyperimmune state.

Term " immunogene " be meant can the inductor humoral antibody and/or cell-mediated immune responses and can with the product of its generation, as antibody, the material that reacts.

Term " immune-regulating factor " is meant and can influences immune material except antibody.

Term " immunogene in combination source " is meant by making up synthetic mode produces molecular diversity between immunogene new method.

Term " biotechnology immunogene " is meant by the gene clone technology of the insertion that allows to produce the coding nucleotide with immunogenic epi-position and the immunogene that method obtained of genetic manipulation.

Term " gene vaccine " is meant usually by nucleic acid vaccine recombinant technique production and that can cause immune response.

Term " administration " is meant any method that individuality is given material, and it comprises in oral, the nose, intraocular, parenteral (intravenous, intramuscular or subcutaneous), rectum or local administration.

The definition of term " animal " the expression animal kingdom.

Term " target animal " is meant the animal with the product function of laying eggs or lay eggs.

Term " receptor " is meant its administration by the ovum of target animal generation or the animal of ovum product.

" T-cell " or " T-lymphocyte " is meant in thymus gland ripe, and a quasi-lymphocyte that has important function in immune response.It is divided into many subclass: killer T-cell is responsible for killing the cell that is infected by the virus; Helper T-cell induces other cell (B-lymphocyte) to produce antibody.

The preparation of hyperimmune ovum product

Should keep firmly in mind: hyperimmune is handled and both can be carried out in birds, handles the active ingredient that obtains thereby its ovum can contain hyperimmune; Also can in ox, carry out, handle the active ingredient that obtains thereby its breast can contain hyperimmune.Simultaneously can collect its hyperimmune breast though conceived the hyperimmune that identical technical scheme also is applicable to ox, following explanation only limits to the hyperimmune of birds.

Described hyperimmune ovum product can be produced by any animal that lays eggs.Preferred described animal is selected from Aves, perhaps in other words, is a kind of birds.In Aves, the poultry of preferably raising and train, but other species in this guiding principle, for example turkey, duck and goose also are the suitable sources of hyperimmune ovum product.

Described hyperimmune ovum product provides with spray-dired ovum powder, and it derives from the hen that lays eggs (referring to embodiment 1) of having adopted lineup's enteropathogen to carry out vaccine inoculation.Can think that any immunogene or immunogene set can be used for hyperimmune process of the present invention.Atomized drying can minimize the damage of antibody in the ovum and immune-regulating factor through the process of the ovum liquid of pasteurization, thereby thereby obtains a kind ofly to have high nutritive value, and can produce the product that passive protection demonstrate the ability that can reduce inflammation to possible enteric infection.Antibody as a monoid, has special resistance to the degraded of conventional enzyme, and after oral administration takes in digestion, but important fraction complete sum has the active enteron aisle approach that passes through.Many research reports, the antibody capable of oral absorption digestion produces protection at specific enteron aisle cause of disease.

In addition, when making this animal that lays eggs by following approach, the periodic booster immunization of antigen for example, when being in specific immune state, this animal will give birth to the ovum with beneficial characteristics that costimulatory receptor animal T-cell increases, when described ovum is taken in by receptor.

Under the situation of the knowledge aspect the necessary condition of known development and maintenance hyperimmune state, those skilled in the art know can adjust the immune commercial weight of administration according to the kind of the animal that lays eggs of being adopted, thereby makes animal remain in hyperimmune state.

Preferably produce hyperimmune state by any immunogene or combinations of immunogens.Preferably by repeatedly being exposed to multiple immunogene, repeatedly being exposed to single immunogene or single exposure and realizing hyperimmune in the immunogene storehouse.

Except carrying out originally the immunity, also can realize immunity originally by the synthetic immunity that obtains of combinatorial chemistry process by naturally occurring immunity.Thereby its elementary tactics is the combination generation of set number of chemical building stone has multifarious elements collection.Recently, develop many methods and be used for oligomer (Fodor, S. etc., Science 251:767 (1991); Houghton, R. etc., Nature 354:82 (1991)) and little organic molecule library (Bunin, B.﹠amp; Ellman, J., J.Am.Chem.Soc.114:10997 (1992)) solid phase and liquid phase combination synthetic.Synthetic can be used as of multiple fast peptide and oligomer made up the immunogenic source of deriving.In addition, thus optionally strategy will allow organic building stone to be added into raising immunogenicity on the backbone molecule in the mode of combination.

The alternate model that animal is produced in hyperimmune oviparity can be used for substituting the immunogenicity vaccine, comprises the application of gene vaccine.Especially, any DNA construct (generally including a promoter region and an immunogene coded sequence) all can cause immune response.Gene vaccine comprises immunogene code carrier, exposed dna fragmentation, plasmid DNA, DNA-RNA antigen, DNA-protein combination body, DNA-liposome combination, DNA expression library and the virus and the DNA of bacteria that are used to produce immune response that imports.The method that DNA imports comprises particle bombardment, directly injection, viral vectors, liposome and jet injection, reaches other method.When adopting these introduction methods, only need less amount and usually cause more lasting immunogene to produce.When adopting this genetic manipulation, import the method for optimizing of DNA for DNA is injected its chest muscle by the mode of intramuscular injection to birds.

Preferred hyperimmune operation

The following is one is used to make the animal that lays eggs to be in the list of steps of immune rising state preferred operations example:

1. select one or more immunogenes.

2. in the animal that lays eggs, cause immune response by initial immunity.

3. the immunogenic booster immunization vaccine of the suitable dosage of administration is used to induce and keep hyperimmune state.

Step 1:Can adopt any immunogene or combinations of immunogens as vaccine.Wherein immunogene can be bacterium, virus, protozoa, fungi, cell, other any material that maybe can cause that the immune system of the animal that lays eggs replys.The key point of this step is that immunogene must bring out immunity and hyperimmune state in the animal that lays eggs.Though only there is single antigen to play vaccine in the method for the invention, preferred vaccine is to be selected from the polyvalent bacterial of following antigen family and the mixture of viral antigen: enteric bacilli and bacteroid, pneumococcus (Pneumococci), pseudomonad (Pseudomonas), salmonella (Salmonella), streptococcus (Streptococci), bacillus (Bacilli), staphylococcus (Staphylococci), Neisseria (Neisseria), clostridium (Clostridia), mycobacterium (Mycobacteria), actinomycetes (Actinomycetes), Chlamydia (Chlamydiae) and mycoplasma (Mycoplasma).Though other viral antigen family also produces effect, viral antigen preferably is selected from following antigen family: adenovirus, picornavirus and herpes virus.In a particular preferred embodiment, adopted the polyvaccine that is called PL-100.The bacterium that is included in the PL-100 vaccine is listed in the table 1 of embodiment 1.

Step 2:Described vaccine both can be for inactivated vaccine also can be an attenuated vaccine, and can come administration by any method that can cause immune response.Preferred by mode administration immunity realization immunity originally through intramuscular injection.The muscle that preferably is used to inject in birds is chest muscle.Adoptable other medications comprise intravenous injection, intraperitoneal injection, intradermal, rectal suppository, spray or oral administration.When dna technique is used for the hyperimmune operation,, be generally the 1-100 microgram with needs amount still less.

The many methods that can know by the those of skill in the art in immune field determine whether to have caused immune response in the animal that lays eggs.Its example comprises enzyme linked immunosorbent assay (ELISA) (ELISA), and the detection that the antibody of excitant antigen exists is used for estimating the experiment of host's immunocyte to the responsibility of antigen.The required immunogenic minimum dose of induce immune response depends on the vaccine inoculation operation of being adopted, and it comprises the type of adjuvant and the immunogen preparation that is adopted and as the type of host's the animal that lays eggs.

Step 3:Can be preferably induce and keep hyperimmune state by at interval repeating to strengthen the administration proper dosage at a fixed time at target animal.The time interval preferably during 6-12 month in the interval in 2-8 week.Preferred dose is the immunogenicity vaccine of 0.05-5 milligram.But booster immunization must not can be led the immunogenicity tolerance.Well known this generic operation.

Can also adopt other the hyperimmune maintenance operating procedure or the combination of operating procedure, for example, initial immunity is carried out in intramuscular injection, and booster immunization is carried out in intravenous injection.Other step comprise administration microcyst simultaneously with the liquid immunogene, or intramuscular injection carries out initial immunity, and comes the administration booster by oral administration or through the mode of microcyst approach parenteral.The known many first and hyperimmune compound modes of those skilled in the art.

The processing of hyperimmune ovum and administration

, preferably collect the ovum of these animals and process generation hyperimmune ovum product after the sufficient hyperimmune of animal via in case lay eggs.Subsequently, hyperimmune ovum product can be administered to receptor.

Ovum of the present invention and/or ovum product are administered to receptor by any way that can stimulate or strengthen immune t-cell activity in the receptor.Preferably carry out administration by directly feed ovum or its any active ingredient.Ovum and yolk are natural food compositions, and except to those allergy sufferers, itself nontoxic and safety.

A kind of preferable methods of processing ovum relates to becomes the ovum powder with the ovum drying.Though the method for known various dry ovum, but atomized drying is a preferable methods.The method of atomized drying ovum is methods known in the art.

In a preferred embodiment, the hyperimmune ovum is with food product that contains many nutrient components such as vitamin and mineral matter or food supplement administration.Though so say, the ovum powder of doing also can mix in the beverage, and wherein beverage is following form: for example, and the beverage of albumen powder, powder composition, albumen replenishers and any other nutrition, the product that motion is relevant.

In another embodiment, described ovum powder can be used in baking mixture, powder rod, candy, the biscuit etc.Other examples of ovum processing comprise: make the egg thin pancake, boil soft or boil hard egg, roasting egg, if or be ready that ovum liquid can be eaten something rare or be processed into to described ovum.

At last, yolk known in this field and/or egg white have partly contained the composition of the above-mentioned observed or beneficial effect mentioned.The very clear and definite understanding of those of ordinary skill in the art can provide more virtuous fraction or remove unfavorable composition by further separation, and also allow other administering mode, for example in parenteral, subcutaneous, intravenous, intramuscular, endoperitoneal, nose, oral or local mode administration ovum product.This further separation will provide the ability that adopts described ovum or its grade to assign to prepare capsule product and pharmaceutical composition.

Thereby to effective stimulation of a certain amount of hyperimmune ovum of receptor administration product energy immunity or enhance immunity T-cell is crucial.As indicated above, size is proved the most effective less than 3000 daltonian hyperimmune egg-development stage branches (referring to the preparation of embodiment 2).Consider this point, the inventor finds size and the body weight according to receptor, and every receptor administration divides from the hyperimmune egg-development stage less than 3K of any dosage of 0.1 μ g/mL to 1000mg/mL can effectively stimulate or strengthen the T-cell.But, the about 0.2 μ g/mL of preferred every animals administer is to the hyperimmune egg-development stage branch less than 3K of about 200 μ g/mL, further preferred 0.5 μ g/mL to 20 μ g/mL.If the time and intensity that treatment continues will be according to the health of receptor, whether show disease or illness and show then the state of development of this disease or illness etc. decide.Consider these, thereby hyperimmune ovum product will carry out administration with the amount of energy effective stimulus or enhancing T-cytoactive enhance immunity system's Fighting Disease or illness.Should be clear and definite, though preferably less than the hyperimmune egg-development stage branch of 3K, bigger fraction and whole egg are effective too.Can determine the day constant of administration according to the particular condition of receptor, wherein day constant for from less than 1 in the scope of several complete hyperimmune ovum (or contain and less than a hyperimmune ovum product) to several complete hyperimmune ovum equivalences.According to the separable and concentrated more effective fraction of method well known in the art.

By illustrating that with reference to following being used to embodiments of the invention can further embody beneficial characteristics of the present invention.

Embodiment

Embodiment 1

The preparation of PL-100 vaccine

The polyvaccine that contains bacterium that is called PL-100 as shown in table 1 (deriving from American type culture collection (American Type Culture Collection)) is adopted the medium reprovision of 15ml and 37 ℃ of following overnight incubation.In case the good growth of acquisition is got only about half of bacterial suspension and is used for being seeded to 1 liter broth bouillon and cultivation under 37 ℃ subsequently.Remaining suspension is transferred to the ethylene glycol test tube of sterilization and reaches 6 months most-20 ℃ of following preservations.

When as seen well growing in the culture, by centrifugal results bacterium.The bacterial precipitation thing is resuspended in the sterile physiological salting liquid and with bacteria samples centrifugal 3 times to wash the cell part off.After washing for the third time, the sediment of acquisition is resuspended in a spot of distilled water.

By the suspension in the glass flask being placed 80 ℃ water be incubated overnight heat inactivation not contain the bacterial suspension of medium.Adopt a spot of heat-killed bacterium to detect the vigor of broth bouillon.Adopt heat-killed bacterium to inoculate meat soup, cultivated 5 days down at 37 ℃, and detect its growth every day, because described bacterium must be inactivated as vaccine.

Heat-killed bacterium is through being refrigerated to bone dry.Subsequently, bacterium and the sterile saline solution with drying is mixed to 2.2 * 10 ⁸Individual bacterial cell/ml salt solution (1.0 optical density (OD) under 660nm).

Table 1

Antigen in the PL-100 vaccine

Title	Medium	Title	Medium
				Imitation staphylococcus (Staphylococcus simulans)	BHI	Pseudomonas aeruginosa (Pseudomonas aeruginosa)	BHI
Staphylococcus epidermidis (Staphylococcus epidermidis)	BHI	Friedlander (Klebsiella pneumoniae)	BHI
				Streptococcus pyrogenes (Streptococcus pyogenes), the A1 type	APT	Salmonella typhimurium (Salmonella typhimurium)	BHI
Streptococcus pyrogenes, the A3 type	APT	Hemophilus influenzae (Haemophilus influenzae)	BHI
				Streptococcus pyrogenes, the A5 type	APT	Streptococcus mitis (Streptococcus mitis)	APT
Streptococcus pyrogenes, the A8 type	APT	Common sex change bacterium (Proteus vulgaris)	BHI
				Streptococcus pyrogenes, the A12 type	APT	Shigella dysenteriae (Shigella dysenterlae)	BHI
Streptococcus pyrogenes, the A14 type	APT	Diplococcus pneumopniae (Diplococcus pneumoniae)	APT
				Streptococcus pyrogenes, the A18 type	APT	Sore blister rod bacillus (Propionibacter acnes)	Broth bouillon
Streptococcus pyrogenes, the A22 type	APT	Streptococcus sanguis (Streptococcus sanguis)	APT
				Colon bacillus (Escherichia coli) (ATCC#26)	BHI	Streptococcus salivarius (Streptococcuss alivarus)	APT
Colon bacillus (ATCC#884)	BHI	Streptococcus mutans (Streptococcus mutans)	BHI
				Intestines salmonella (Salmonnella enteritidis)	BHI	Streptococcusagalactiae (Streptococcus agalactiae)	APT

The immunologic process of hyperimmune ovum product

The pathogene of preparation deactivation as indicated above.As inoculation for the first time, described bacterium mixes with complete Freund ' s adjuvant, and the bacterial components of 5.6mg is injected in the pigeon breast portion muscle.For remaining vaccine, bacterium is prepared product and incomplete Freund ' s adjuvant mix to be incorporated in and be injected in the chicken body with the interval in two weeks 6 middle of the month.

Collect the ovum that the hyperimmune hen gives birth to, and it is spray dried to Powdered form.In spray-drying process, the porch temperature is no more than 320 degrees Fahrenheits, and the temperature of discharge is consistent with the powder that whole humidity with 3.0 to 4.0 percentages produces, and pumping pressure remains on about 2500 to 4000P.S.I.Adopted the low temperature in the 100-160F scope, thereby and in dry run the moisture content of monitoring sample make the end-product obtain any desirable denseness.

Embodiment 2

The method for optimizing of the partial purification fraction of preparation ovum

Following embodiment has put down in writing the method (being suitable for large scale purification) that a kind of acquisition has lower molecular weight and is the partially purified egg fraction of non-aggregated forms.The whole egg of edible ovum of hyperimmune and contrast separates egg white also spray-dried separately with yolk after fragmentation.By obtaining the hyperimmune ovum as embodiment 1 described method.Thereby independent processing egg white powder obtains its moisture fraction and is used for ultrafiltration.

Thereby carry out the minimum infection that whole purification steps makes bacterium or pyrogen.Adopt sterile water to come obtain solution and all glassware all through formerization of reducing phlegm and internal heat.In addition, solution is carried out aseptic filtration.

Derive from the goods of yolk

Solvent extraction

Thereby will carry out the liquid flux extraction with propane or butane as the daiyan of preparation as described in the embodiment 1 and from moisture yolk fraction, separate lipid components.Briefly, 500 gram daiyan powder are placed post, and to wherein adding 4 liters of petrogas solvents.Take out the lipid of cleer and peaceful extraction on the solvent.Thereby six lipids extract and have carried out other solvent extraction step six times altogether.

Play filter

Adopt the dry degrease yolk of 4 liters sterile distilled water dilution 400 grams, and adopt Virtis (handishear) to make its homogenize.Both can under the speed of 24RPM, carry out centrifugally for yolk mixture, and can allow its maintenance freezing again until undissolved yolk solids precipitation.The moisture fraction that Amicon RA1000 ultrafiltration system that 3000 dalton tackle spiral stretch film comes ultrafiltration to obtain is equipped with in employing.Keep rate of pumping, inlet pressure is 20psi, and outlet pressure is 15psi.Adopt penetrant and the freeze drying or freezing storage, biologicall test or the further purifying of being used for of disposable sterilized Nalgene filter aseptic filtration＜3,000 Dalton molecular weights of 0.45 μ m.

Comprise having low-molecular-weight and be the partially purified egg-development stage branch of non-aggregated forms less than 3000 daltonian molecule types in the yolk.Since the initial substance of 400 grams, the output of the daltonian partially purified fraction of＜3K is about 12 grams or accounts for 3% of total amount.

Derive from the goods of egg white

Adopt 4 liters deionized water to dilute the 400 gram egg whites that from edible ovum, separate to obtain as embodiment 1 described hyperimmune ovum and contrast.Make mixture mixing homogenize and carry out ultrafiltration through the filter filtration of 40 μ m and through 3KDa MW C0 ultrafiltration system.From the egg white powder of 400g, recyclable 8.6g or 2.15% less than the daltonian partially purified egg-development stage branch of 3K.

Embodiment 3

Immunostimulatory activity in the immunocyte system

All cell-line is all from ATCC, and cultivates in the medium of its recommendation.These cells are all cultivated in 96 orifice plates and are made it reach exponential phase.Suitably dilution 3k fraction, and adding was cultivated 24-96 hour in the hole separately.By adopting the blue staining cell of Alamar, thereby and read its fluorescence intensity in the excitation wavelength of 520nm by automatic microplate readers and determine cell viability.Stimulation index (SI) is represented the relative number of treated cell than the cell that does not stimulate control group.

Fig. 1 is a curve map, the stimulation index (SI) less than the Jurkat cell (T-chronic myeloid leukemia cell-line) in each stage (from 24 to 96 hours) of the fraction co-incubation of 3K of its expression and hyperimmune ovum.The SI value significantly increases in the concentration range of 5.5-50 μ M, and this hyperimmune of having represented the Jurkat cell stimulates.Cell activation 48,72 with 96 hour stage in this is consistent.

Fig. 2 is a curve map, the stimulation index (SI) of the Daudi cell (B lymphoblastoid cell line) in each stage (from 24 to 48 hours) of the fraction co-incubation of the 3K of its expression and hyperimmune ovum.The result shows that observing hyperimmune after cultivating 24 hours under the concentration of the 1.24-300 of 3k fraction μ M stimulates generation.

Therefore, the 3k fraction of hyperimmune ovum has shown strong immunostimulatory activity to the result that influences of Jurkat and Daudi cell-line.This points out us to carry out the 3H-thymidine in the T cytositimulation experiment of PHA mediation and mixes, as described in following embodiment 4.

Embodiment 4

Adopt the in-vitro multiplication experiment of the periphery lymphocyte of PHA processing

Employing derives from healthy contributor's periphery lymphocyte culture voluntarily and carry out this experiment.Adopt the PHA of variable concentrations to handle lymphocyte, and its growth kinetics is compared with the dynamics of the cell that adds normal saline solution.

The result shows, compared with the control (periphery lymphocyte adds normal saline solution) comprise 0.1%, 0.5%, 1.0% and 2.5% with at low concentration, can be observed the mitogenesis effect in the periphery lymphocyte of processing.Stimulation index corresponding to these concentration is respectively 1.6,3.02,2.83 and 1.8, and this shows that under experiment condition, total cellular score has increased by 160%, 302%, 283% and 180% respectively.

The blast transformation of T-cell

Prepare the PHA dilution in 1: 100 to 1: 1000 ratio, and dilution is added in the culture plate system of lymphocyte culture.Add phytohemagglutin phytolectin, Pockeweed antigen and A concanavalin and identify culture hole.Behind cultivation stage, mix experiment by the radioactivity thymidine and determine the lymphocytic propagation of T-.

The increase that PHA is strong the free short lymphocytic propagation of cell division.

The partially purified fraction (as embodiment 2 described fractions less than 3K) of complete hyperimmune ovum and hyperimmune ovum and the mitogenesis effect in the independent control medium have been compared in this research.Under the various concentration that comprise 0.24,0.98,3.9,15.6,62.5 and 250 μ g/ml, detect complete hyperimmune ovum.Under the concentration of 0.1,0.2,0.7,2.0,6.1,18.4,55.3 and 166.0 μ g/ml, detect the fraction less than 3K of the purifying of hyperimmune ovum.

Be respectively 0.9,0.8,0.9,0.9,0.8 with the stimulation index of the corresponding hyperimmune whole egg of above-mentioned concentration, this increases corresponding to 90%, 80%, 90%, 90%, 80% and 80% total cellular score respectively under experiment condition.Be respectively 1.1,1.2,1.5,1.7,1.4,1.3,1.0 and 1.2 (referring to table 2) with the stimulation index of the corresponding 3K purifying of above-mentioned concentration fraction, this increases corresponding to 110%, 120%, 150%, 170%, 140%, 130%, 100% and 120% total cellular score respectively under experiment condition.

These results show that in the concentration range of 0.24-250 μ g/ml complete hyperimmune ovum can not the external T-cell of significant stimulation.On the other hand, the purifying fraction less than 3K of hyperimmune ovum demonstrates significant stimulation T-cell under the concentration of 0.7 μ g/ml-20.0 μ g/ml.Table 1 has shown the actual number of the cell of every hole moderate stimulation.

This activity of the purifying fraction of hyperimmune ovum may be by specificity inducing the cell factor activation factor of the cell factor of the cell arm that relates to stimulating immune system to cause.

Table 1
											The 3H-thymidine mixes/hole [cpm]
Numbering	PHA [μg/ml]	Contrast	The fraction of＜3K [μ g/ml]
												5	0	166.00	55.33	18.44	6.15	2.05	0.68	0.23	0.08
1	101241	541	890	624	819	958	1050	806	754	644
											2	111452	714	765	578	637	759	890	689	794	517
3	98898	552	648	649	711	759	1010	944	639	688
											4	121212	633	705	600	967	697	806	698	573	636
5	97149	465	506	523	723	604	1009	809	494	667
											6	118218	406	437	436	475	813	1009	919	802	393
AVE	108028	552	659	568	722	765	962	811	676	591
											Neg	10365	111	167	78	166	118	94	107	127	114

Table 2
											Stimulation index
Numbering	PHA [μg/ml]	Contrast	PL-100[μg/ml]
												5	0	166.00	55.33	18.44	6.15	2.05	0.68	0.23	0.08
1	183.5	1.0	1.6	1.1	1.5	1.7	1.9	1.5	1.4	1.2
											2	202.0	1.3	1.4	1.0	1.2	1.4	1.6	1.2	1.4	0.9
3	179.2	1.0	1.2	1.2	1.3	1.4	1.8	1.7	1.2	1.2
											4	219.7	1.1	1.3	1.1	1.8	1.3	1.5	1.3	1.0	1.2
5	176.0	0.8	0.9	0.9	1.3	1.1	1.8	1.5	0.9	1.2
											6	214.2	0.7	0.8	0.8	0.9	1.5	1.8	1.7	1.5	0.7
AVE	195.8	1.0	1.2	1.0	1.3	1.4	1.7	1.5	1.2	1.1
											Neg	18.8	0.2	0.3	0.1	0.3	0.2	0.2	0.2	0.2	0.2

Repeat identical experiment.As follows, table 3 and 4 is an experimental result.Observe the similar increase of T-cytositimulation.

Table 3
											The 3H-thymidine mixes/hole [cpm]
Numbering	PHA [μg/ml]	Contrast	The fraction of＜3K [μ g/ml]
												5	0	166.00	55.33	18.44	6.15	2.05	0.68	0.23	0.08
1	102484	335	461	548	361	526	537	690	597	619
											2	114522	326	391	461	401	408	391	551	584	629
3	110201	454	501	393	635	494	533	433	524	500
											4	121811	397	494	521	344	405	477	538	514	807
5	97453	391	372	493	537	459	393	484	474	560
											6	112934	354	353	358	377	396	397	508	519	519
AVE	109901	376	429	462	443	448	455	534	535	606
											NEG	8746	48	65	74	117	54	70	87	46	111

Table 4
											Stimulation index
Numbering	PHA [μg/ml]	Contrast	The fraction of＜3K [μ g/ml]
												5	0	166.00	55.33	18.44	6.15	2.05	0.68	0.23	0.08
1	272.4	0.9	1.2	1.5	1.0	1.4	1.4	1.8	1.6	1.6
											2	304.4	0.9	1.0	1.2	1.1	1.1	1.0	1.5	1.6	1.7
3	293.0	1.2	1.3	1.0	1.7	1.3	1.4	1.2	1.4	1.3
											4	323.8	1.1	1.3	1.4	0.9	1.1	1.3	1.4	1.4	2.1
5	259.1	1.0	1.0	1.3	1.4	1.2	1.0	1.3	1.3	1.5
											6	300.2	0.9	0.9	1.0	1.0	1.1	1.1	1.4	1.4	1.4
AVE	292.2	1.0	1.1	1.2	1.2	1.2	1.2	1.4	1.4	1.6
											NEG	23.3	0.1	0.2	0.2	0.3	0.1	0.2	0.2	0.1	0.3

Describe and illustrate the present invention though combine particular herein, the qualification of the detail shown in the present invention is not subjected to.On the contrary, can in the purport of the equivalent of claim and scope, carry out all kinds of modifications and do not deviate from scope of the present invention.

Claims (20)

1. by the ovum product of described animals administer effective dose being stimulated the method for the immune t-cell in the animal.

2. the method for claim 1, wherein said ovum product comprises ovum or the egg-development stage branch that derives from birds, wherein said birds adopt at least a immunogene to carry out hyperimmune.

3. method as claimed in claim 2, wherein said immunogene is selected from:

The imitation staphylococcus; Staphylococcus epidermidis; Streptococcus pyrogenes; Colon bacillus; The intestines salmonella; Pseudomonas aeruginosa; Friedlander; Salmonella typhimurium; Hemophilus influenzae; Streptococcus mitis; Common sex change bacterium; Shigella dysenteriae; Diplococcus pneumopniae; Sore blister rod bacillus; Streptococcus sanguis; Streptococcus salivarius; Streptococcus mutans; Streptococcusagalactiae.

4. the method for claim 1, wherein ovum product oral administration administration.

5. the method for claim 1, wherein the ovum product comprises the fraction of whole egg.

6. method as claimed in claim 5, wherein said fraction comprise that size is or less than 3000 daltonian fractions approximately.

7. method as claimed in claim 6, wherein the effective dose of ovum product at about 0.1 μ g/mL to the scope of about 2000mg/mL.

8. method as claimed in claim 7, wherein the effective dose of ovum product at about 0.2 μ g/mL to the scope of about 200 μ g/mL.

9. method as claimed in claim 8, wherein the effective dose of ovum product at about 0.5 μ g/mL to the scope of about 20 μ g/mL.

10. the method for claim 1, wherein birds comprise the poultry of raising and train.

11. by the ovum product of described animals administer effective dose being strengthened the method for the immune t-cell in the animal.

12. method as claimed in claim 11, wherein said ovum product comprise ovum or the egg-development stage branch that derives from birds, wherein said birds adopt at least a immunogene to carry out hyperimmune.

13. method as claimed in claim 12, wherein said immunogene is selected from:

The imitation staphylococcus; Staphylococcus epidermidis; Streptococcus pyrogenes; Colon bacillus; The intestines salmonella; Pseudomonas aeruginosa; Friedlander; Salmonella typhimurium; Hemophilus influenzae; Streptococcus mitis; Common sex change bacterium; Shigella dysenteriae; Diplococcus pneumopniae; Sore blister rod bacillus; Streptococcus sanguis; Streptococcus salivarius; Streptococcus mutans; Streptococcusagalactiae.

14. method as claimed in claim 11, wherein ovum product oral administration administration.

15. method as claimed in claim 11, wherein the ovum product comprises the fraction of whole egg.

16. method as claimed in claim 15, wherein said fraction comprise that size is or less than 3000 daltonian fractions approximately.

17. method as claimed in claim 16, wherein the effective dose of ovum product at about 0.1 μ g/mL to the scope of about 2000mg/mL.

18. method as claimed in claim 17, wherein the effective dose of ovum product at about 0.2 μ g/mL to the scope of about 200 μ g/mL.

19. method as claimed in claim 18, wherein the effective dose of ovum product at about 0.5 μ g/mL to the scope of about 20 μ g/mL.

20. method as claimed in claim 11, wherein birds comprise the poultry of raising and train.

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