CN1528783A - Bursopoietin extracting method and its use in disease treating and immune - Google Patents

Abstract

The invention relates to a bursin extracting method and its application to curing disease and immunity, having important value in application in the aspects of heightening organismal immunity and acting as immunoenhancer, heightening effect of vaccine, etc., and able to heighten body fluid and cell immune functions of mammal at the same time. It can be used to prevent and cure infectious diseases and young animal diseases singly or together with other drugs such as antivirus and antibacterial drugs or immunomodulators, also be applied to animal vaccine as adjuvant or immunoenhancer to strengthen the disease-resistant ability and immunoresponse ability to peculiar antigens, thus heightening the immune effect.

Description

A kind of extracting method of bursopoietin and the application in disease treatment and immunity thereof

One, technical field:

The present invention relates to a kind of immunological reagent that is used for livestock and poultry, especially relate to a kind of extracting method and the application in disease treatment and immunity thereof of bursopoietin.

Two, background technology:

Transmissible disease especially viral infectious be an important factor that influences animal husbandry development, after most of pathogenic micro-organisms are invaded body, can cause the damage of immune organ, make body's immunity low, disease resistance weakens, and eqpidemic disease is further developed, simultaneously, cause secondary infection and immuning failure, bring about great losses to aquaculture.The simple immuning failure that relies on vaccine immunity to cause owing to multiple reason on the one hand; on the other hand because the restriction of the immune protective rate of vaccine own; be difficult to prevent fully the generation of transmissible disease; thereby; seek immunostimulant; the anti-system method of protection, enhance immunity power and active and effective virus disease has become the focus that countries in the world veterinary work person makes great efforts.

The more immunostimulant of research mainly contains microbiology class, chemical agent, biotechnological formulation and Chinese medicine preparation 4 classes at present.Wherein biotechnological formulation mainly comprises thymosin, transfer factor, gamma globulin and Interferon, rabbit (table 1).

The immunostimulant that table 1 is commonly used

Specific name source mechanism of action

Microbiology class bcg bacteria attenuation bovine tuberculosis activating macrophage, immunological adjuvant

CBP seedling stub bacterium inactivated vaccine activating macrophage, immunological adjuvant

The chemosynthesis of chemical agent LEVAMISOLE HCL improves scavenger cell and T cell function

Isoprinosine chemosynthesis activating macrophage and T cell

Differentiation of biotechnological formulation thymosin animal thymus inducing T cell and propagation

(biological because of transfer factor lymphocyte transfer T cellular immunization information

Son) antiviral, antitumor, the adjusting immunity of interferon-' alpha ' Mo/ scavenger cell

Interferon-T cell is regulated immunity, MHC expresses, strengthens the NK cytoactive

IL-2 T cell amplification T cell clone

Chinese medicine class krestin ^*Rainbow conk (polyporaceae) promotes scavenger cell and T cell function

* similarly also have system tumor growths such as Pachymose, ganoderan, polyporusum bellatus, lentinan.

Above-mentionedImmunoenhancement result is good, but because High and difficult preservation of costAnd limited its application in veterinary clinic greatly.Therefore, reduce cost or seek with low cost, the respond well immunostimulant person's that become the veterinary work a vital task.

Bursopoietin (Bursin, BS) be the bioactive molecules that unique a kind of chemical constitution has been determined in the fabricius bursa tissue extract till settled the present, equal to organize the extract purifying and name from chicken bursa first in 1986 by T.Audhya, its chemical constitution is tripeptides, i.e. Lys-Hls-Gly-NH2.Studies have shown that BS exists Body OutwardHave the function of inducing the differentiation of bone-marrow-derived lymphocyte system, and the bone-marrow-derived lymphocyte inhibitor is had antagonistic action, can improve cAMP and cGMP level in the human B cell system-Duadi clone.

Three, summary of the invention:

The present invention is in order to solve the weak point in the above-mentioned background technology, a kind of extracting method and the application in disease treatment and immunity thereof of bursopoietin are provided, it is in enhance immunity power and as immunostimulant, having important use at aspects such as improving vaccine effect is worth, simultaneously can improve mammiferous body fluid and cellular immune function, and cost is low, easily preserves.

For achieving the above object, the technical solution used in the present invention is:

A kind of extracting method of bursopoietin, its special character is to comprise following operation steps: get healthy fabricius bursa 80g, add an amount of 1N Glacial acetic acid and use liquid, 20 000r/min homogenate 3min make it become pasty state.1.5h～the 2h that vibrates on horizontal shaker in 4 ℃ of centrifugal 60min of 4500r/min, gets supernatant, acts on 20min in the boiling water bath, and is once centrifugal again, gets supernatant and filters.Filtered liquid is evaporated to about 40ml between 60 ℃～70 ℃ with Rotary Evaporators, and concentrated solution is through 20 000g, high speed centrifugation 30min under 4 ℃ of conditions, get the about 20ml of supernatant liquor, be crude extract, get crude extract and cross the G-50 sephadex column, 1N acetic acid wash-out is collected CuSO ₄The reacting positive part, the NaHCO with 7.5% ₃Adjust pH is tissue extract-bursopoietin to neutral.

The application of a kind of bursopoietin in disease treatment, its special character is: bursopoietin is applied to the prevention and the treatment of communicable disease and young animal disease separately.

Above-mentioned bursopoietin cooperates prevention and the treatment that is used for communicable disease and young animal disease with antiviral.

Above-mentioned bursopoietin cooperates prevention and the treatment that is used for communicable disease and young animal disease with antibacterials.

The application of a kind of bursopoietin in immunity, its special special character is: described bursopoietin and animal vaccine are used, and are used to improve the animal disease resistant ability and to the immunne response ability of specific antigen and the immune efficacy of vaccine.

Above-mentioned bursopoietin is applied to improve the animal disease resistant ability as the animal vaccine adjuvant and to the immunne response ability of specific antigen and the immune efficacy of vaccine.

Above-mentioned bursopoietin is applied to improve the animal disease resistant ability as animal vaccine immunopotentiator and to the immunne response ability of specific antigen and the immune efficacy of vaccine.

Above-mentioned bursopoietin is directly as vaccine diluent.

The application of a kind of bursopoietin in immunity, its special character is: described bursopoietin is as additive for farm animal feed.

Compared with prior art, the advantage and the effect that have of the present invention is as follows:

1, the present invention has important use at aspects such as improving vaccine effect and is worth in enhance immunity power and as immunostimulant, and bursopoietin (BS) can improve mammiferous body fluid and cellular immune function simultaneously.

2, chemical structure of the present invention is simple, stable in properties, and intramuscular injection or oral all being easy to absorb, and can not reduce activity with preservation under the normal temperature condition.

3, drug residue free of the present invention can promote humoral immunity of organism and cellullar immunologic response level simultaneously, can promote to damage the reparation of the fabricius bursa to bird, improves the immune effect of vaccine.

4, the present invention studies through animal experiment, directly injects 1-2 titre of immune antibody level raising of vaccine simultaneously as vaccine diluent or with vaccine.

5, the present invention is used for chick or young animal injection, the maternal antibody level is prolonged hold time.

6, the present invention and specific antibody fit applications can significantly improve result of treatment, and the curative ratio of newcastle disease and bursa of Fabricius improves 1 times.

7, the present invention injects separately or uses as fodder additives and can significantly improve lamb and weaned piglet surviving rate.

8, the present invention and antibiotic, antiviral fit applications (oral or intramuscular injection) can significantly improve curative ratio.

Four, description of drawings:

Fig. 1 is the influence of bursopoietin to mouse boosting cell PFC number.

Fig. 2 is bursopoietin and the LCGF influence to mouse PFC number.

Fig. 3 is haemolysis spectrophotometry result.

Fig. 4 is the variation that chicken group maternal antibody is tired behind the injection bursopoietin.

Five, embodiment:

Leaching process of the present invention is:

Get healthy fabricius bursa 80g, add an amount of 1N Glacial acetic acid and use liquid, 20 000r/min homogenate 3min make it become pasty state.1.5h～the 2h that vibrates on horizontal shaker in 4 ℃ of centrifugal 60min of 4500r/min, gets supernatant, acts on 20min in the boiling water bath, and is once centrifugal again, gets supernatant and filters.Filtered liquid is evaporated to about 40ml between 60 ℃～70 ℃ with Rotary Evaporators.Concentrated solution is through 20 000g, and high speed centrifugation 30min under 4 ℃ of conditions gets the about 20ml of supernatant liquor, is crude extract.Get crude extract and cross the G-50 sephadex column, 1N acetic acid wash-out is collected CuSO ₄The reacting positive part, the NaHCO with 7.5% ₃Adjust pH is tissue extract-bursopoietin to neutral.

The present invention is mainly used in:

1. use separately or cooperate prevention and the treatment that is used for communicable disease and young animal disease with other drug such as antiviral, antibacterials or immunological reagent.

2. be applied to animal vaccine as adjuvant or immunostimulant, to improve the animal disease resistant ability and, to improve the immune efficacy of vaccine to the immunne response ability of specific antigen.

3. directly as vaccine diluent.

4. can be used as additive for farm animal feed.

Effect of the present invention mainly shows:

1.BS in enhance immunity power and as immunostimulant, have important use at aspects such as improving vaccine effect and be worth, BS can improve mammiferous body fluid and cellular immune function simultaneously.

2.BS prepare into the curative effect that pulvis, injection or oral preparation all can significantly improve medicine with antiviral and antibacterials.

3. BS is used as fodder additives, can significantly improve the immunizing power of animal (livestock and poultry).

4. the BS in the tissue of the BS of synthetic and extraction has identical biologic activity, and can preserve and not reduce activity down with normal temperature condition.

Representative formula:

Anti-virus formulation: bursopoietin ribavirin

Antibiotic preparation: bursopoietin antibiotic

1. main research contents of the present invention and research method:

1.1 the research of bursopoietin

1.1.1 the preparation of bursopoietin:

Use respectively that the acetic acid extraction method is extracted, the purifying bursopoietin in the healthy chicken fabricius bursa and chicken bursa cell culture supernatant, the applied chemistry synthesis method has been synthesized bursopoietin and analogue thereof simultaneously, the single step purification of advancing of going forward side by side, its purity of applying high voltage liquid phase and stratographic analysis and content.

1.1.2 bursopoietin activity identification:

Use the activity that extraction and synthetic bursopoietin and analogue thereof are measured in hemolytic plaque test and cell transformation test respectively, determine activity unit.

1.1.3 the immunologic competence of bursopoietin is measured:

Bursopoietin is to the influence of chicken maternal antibody: with newcastle disease (ND) maternal antibody is index, uses β-micromethod, measures antibody growth and decline rule behind the chick injection bursopoietin.

Bursopoietin is to the influence of newcastle disease vaccine immunity: use β-micromethod, measure the influence that bursopoietin is just exempted from back antibody growth and decline rule and tired chick, further measure the chicken group and suffering from infectious bursal disease, after with the rehabilitation of fabricius bursa extract for treating, to the influence of newcastle disease vaccine second immunisation.

1.1.4 bursopoietin is to the therapeutic action of newcastle disease and bursa of Fabricius:

At first use bursopoietin artificial infection chicken group is carried out therapeutic test, determine the therapeutic action and the consumption thereof of bursopoietin, afterwards, it is cooperated with high immunity yolk antibody or serum antibody make preparation, carry out application test, the observed and recorded curative ratio is analyzed result of treatment.

2. main achievement in research:

2.1 determined the method for extracting bursopoietin from the healthy chicken fabricius bursa, simple, can be used for a large amount of preparations; Also finding in fabricius bursa cell culture supernatant has the existence of bursopoietin, and proves that it has identical immunocompetence with the bursopoietin that extracts.

Use Peptide synthesizer and artificial synthesis respectively and synthesized bursopoietin and two kinds of analogues (isomers) thereof, purity all reaches more than 75%.

2.2 use the formation that the prepared bursopoietin of hemolytic plaque test proof all can promote mouse anti sheep red blood cell (SRBC) hemolysis plaque (PFC), its activity does not have significant difference between the bursopoietin in synthetic and extraction and fabricius bursa culture supernatant, but three's activity is significantly higher than two kinds of analogues of synthetic.Each preparation is proportionate in range of doses to promoter action and the dosage that PFC forms.

Also obtained and above-mentioned consistent result by lymphocyte transformation test.

2.3 give the period of 1 Japanese instar chickling intramuscular injection bursopoietin energy significant prolongation maternal antibody existence, make its time lengthening that keeps high-level maternal antibody, its mechanism it be unclear that.

2.4 at the preceding 1d of ND, IBD vaccine immunity, simultaneously or immunity back 1d, the BS of intramuscular injection doses or its analogue, all the immunity to these two kinds of vaccines has promoter action.Test-results proves, can make the chicken group newcastle epidemic disease antibody rising 1.5-2 titre of tiring, and the fine jade of the anti-IBDV antibody of chicken group expanded tire and ELISA detects to tire and raises 2-4 respectively doubly.Both potent antibodies titre extended periods are prolonged.

2.5 therapeutic test shows that bursopoietin all has certain treatment and prophylactic effect to ND and IBD, cooperate with corresponding yolk antibody or antiserum(antisera), morbidity have the good curing effect in early days, curative ratio can reach respectively more than 85% and 90%.Can promote the reparation of chicken bursa fabricic morbidity back fabricius bursa damage, improve the antibody horizontal of morbidity back ND vaccine second immunisation, thereby effectively prevent the ND vaccine immunity failure that causes because of fabricius bursa damage.

Clinical application test on Ningxia centre halfback, Tianjin and Shaanxi and other places shows, bursopoietin is as immunological adjuvant, can significantly improve the immune effect of ND and IBD vaccine, polyinfection to chicken group ND and IBD has the good curing effect, and is early stage in morbidity, uses bursopoietin and specific antibody partner treatment, can be in back about 9 hours of injection, morbidity chicken group mental status is clearly better, and the chicken group reaches basicly stable behind the 3d, and clinical cure rate reaches more than 85%.

The scientific meaning and the application prospect of research:

This project system has been studied bursopoietin activity identification, immunologic competence and to the therapeutic action of chicken main diseases viral disease, the conclusion that obtains provide experimental basis for the further immunology and the effect in immunity of organism is regulated thereof of research bursopoietin, have important scientific meaning at aspects such as immunostimulant and new generation vaccine researchs.In application facet, set up the preparation method and the artificial synthesis of bursopoietin, make its mass production become possibility.This shows that this research has broad application prospects as immunostimulant and with aspect such as specific antibody fit applications treatment virus disease using bursopoietin.

Three, the preparation of bursopoietin

The tissue extraction of I bursopoietin

1. material and method:

The healthy chicken fabricius bursa: pick up from local fryer slaughterhouse, part is provided-20 ℃ of preservations by the moving inspection in Qingdao.

Main agents:

Glacial acetic acid (analytical pure, Xi'an chemical reagent factory); (electrophoresis is pure, Shanghai milk cow company integrated plant, lot number: 890642) for bovine serum albumin; Sephadex G-50 (the magnificent company in Xi'an);

The extraction of bursopoietin:

With reference to the method for Audhya etc., and improve.Main process is: get healthy fabricius bursa 80g, add an amount of 1N Glacial acetic acid and use liquid, 20 000r/min homogenate 3min make it become pasty state.1.5h～the 2h that vibrates on horizontal shaker in 4 ℃ of centrifugal 60min of 4500r/min, gets supernatant, acts on 20min in the boiling water bath, and is once centrifugal again, gets supernatant and filters.Filtered liquid is evaporated to about 40ml between 60 ℃～70 ℃ with Rotary Evaporators.Concentrated solution is through 20 000g, and high speed centrifugation 30min under 4 ℃ of conditions gets the about 20ml of supernatant liquor, is crude extract.Get crude extract and cross the G-50 sephadex column, 1N acetic acid wash-out is collected CuSO ₄The reacting positive part, the NaHCO with 7.5% ₃Adjust pH is tissue extract-bursopoietin to neutral.

The qualitative detection of extract:

Use Coomassie brilliant blue and 1% copper sulfate respectively to the extract detection of finalizing the design, and detect the existence of bursopoietin with the ELISA method.

The mensuration of content of peptides in the extract:

Utilize biuret method, the 540nm place surveys absorbancy (OD) value on 721 spectrophotometers, makes typical curve with bovine serum albumin, calculates the content of polypeptide in the extract.

2. result

2.1 the typing detected result of extract:

Obtain the 100mL extract altogether with the 160g fabricius bursa, this extract can not make the Coomassie brilliant blue variable color, and makes copper-bath metachromasia occur, shows that the composition in the extract mainly is a polypeptide.

Extract is done the continuous gradient dilution, detect with ELISA, the ELISA that is presented at bursopoietin in the extract detects and tires is 1: 10 ⁴, prove the bursopoietin that contains high density in the extract.

2.2 the content of polypeptide in the extract:

Measurement result shows that content of peptides is 2g/L in the extract.

3. discuss

Since Audhya announced the extracting method of its bursopoietin in 1986 at the Science magazine after, other investigators continued to use its method substantially and extract.Its main process is: 1. with the mixed homogenate by 1: 10 of the fabricius bursa and 1N acetic acid, 4 ℃ are spent the night; 2. under 4 ℃ of conditions, with the centrifugal 60min of 14000g, get supernatant, repeated centrifugation is got supernatant freeze-drying in freeze drier; 3. dried frozen aquatic products is dissolved in again 1N acetum (fully dissolving 1h under the room temperature), in 12000g, centrifugal 20min under 4 ℃ of conditions; 4. supernatant liquor is crossed the G-50 sephadex column, and it is dry to collect the liquid cooling freeze-drying; 5. xeraphium is with 50mM ammonium hydrogencarbonate dissolving, and the 7000g 5min that tears by dissension and discords gets supernatant, i.e. the bursopoietin extract.This method operation steps is many, and to the temperature requirement height, the length that expends time in needs expensive instruments such as freeze drier in addition, is unsuitable for applying.

Consider that the tripeptides bursopoietin is comparatively stable about 100 ℃, thereby after the fabricius bursa tentatively extracts, use boiling water bath method removal macromolecule protein wherein rapidly, leaching process is simplified, use Rotary Evaporators and replace freeze drier, this process is simplified, the time spent reduces.Audhya etc. think that the total content of bursopoietin is 0.06% in the fabricius bursa, and the rate of recovery of its extraction is 45%, and use the method in this test, and its rate of recovery obviously improves (64%).Show that the method after the improvement is better than former method.Because it is simple, portion needs specific apparatus, can be used for a large amount of preparations.

Qualitative test is the result prove, the material that this test is extracted is mainly polypeptide, and the ELISA method detects and to show and wherein mainly contain bursopoietin.Prove that simultaneously it has kept the characteristic with anti-bursopoietin antibodies.The follow-up test of this research proves that also the leaching process after the improvement does not influence the biological activity of bursopoietin.

The preparation of II fabricius bursa cells and supernatant

The cultivation of the fabricius bursa primary cell of chick and chicken embryo has been carried out in this experiment, and (Enzyme-Linked Immuno-adsorption Assay ELISA) contains tripeptides capsule element in the proof culture supernatant to use enzyme-linked immunosorbent assay.

Materials and methods

Chicken embryo instar chicken embryo on the 17th～18 or 1～3 Japanese instar chickling, the brooder house provides by the Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology test farm;

Main agents and material

Trypsin GIBCO, 1: 250), collagenase IV type (SIGMA), MEM substratum (GIBCO), M199 substratum (Japan), RPMI1640 (Hyclone), new-born calve serum (NCS, self-control), 100mL Tissue Culture Flask (FALCON), 24 porocyte culture plates (NUNC)

Fabricius bursa cell cultures

Get instar chicken embryo on the 17th～18 or 1～3 Japanese instar chickling (brooder house, Northwest Agricultural University test farm provides), the aseptic fabricius bursa of winning, place the plate that fills 15mL Hank ' s liquid or PBS fully to rinse and wash, reject the blood stains and fabricius bursa reticular tissue on every side, move in penicillin bottle and cut to 1mm ³Size adds Hank ' s liquid washing 2～3 times, with pasteur pipet sucking-off precipitation tissue, put one and be added with in the triangular flask of bead, add about 10 times of amount 0.25% pancreatin, digest 20～30min in 37 ℃ of water-baths behind the mixing, staticly settle, inhale and abandon trypsin solution, add Hank ' the s liquid that contains 10%NCS in right amount, firmly jolting, break up tissue block, leave standstill, treat indigested tissue block post precipitation, the sucking-off cell suspension, with 400 order nylon net filters, get cell suspension, add capacity Hank ' s liquid or nutrient solution, 3 (1000r/min of centrifuge washing, 10min), last centrifugal after, add an amount of nutrient solution, sedimentation cell is dispelled, make into uniform suspension.The adding platform that takes a morsel is expected blue dyeing counting, adds and contains 10%NCS, and the 2mM glutamine, the 1mM Sodium.alpha.-ketopropionate, the cell culture fluid of 10mM HEPES is adjusted cell concn, is sub-packed in 100mL Tissue Culture Flask or 24 well culture plates, puts 37 ℃, 5%CO ₂, cultivate under the saturated humidity condition, behind the 24h, observed and recorded cell growing state day by day, wait to grow up to 90% above monolayer cell after, the Fractional Collections culture supernatant, concentrating under reduced pressure, the centrifugal 1h of 20 000r/min gets supernatant liquor, adjust pH is to neutral ,-20 ℃ of preservations are standby.

The detection of tripeptides capsule element in the culture supernatant

Use the ELISA method, by using operation instructions to carry out.

2. result

2.1 nutrient solution is to the influence of fabricius bursa cell cultures:

3 kinds of nutrient solutions have been used in test respectively, cultivate under identical conditions, and the result shows, except that RPMI1640, and no significant difference between two kinds of nutrient solutions of MEM and M199.Simultaneously, also no significant difference between tassel culturing bottle and the culture plate.This test later stage is all used the MEM nutrient solution.

2.2 cell growth condition:

Cultivate 24h, cell becomes greatly gradually, still keeps circle, begins adherent growth to the 48h cell, and 84h is paved into individual layer substantially.Cellular form mainly is spindle shape, Polygons or asterism shape, mixes distribution.In 24 well culture plates, as seen in some hole, with a certain cell showed increased.Be cultured to 84h or the longer time, cell begins to come off, the cell rounding that comes off, in be the cavity shape, nucleus is cracked.

2.3 the influence that chicken embryo and chick pair cell are cultivated:

From the cell attachment situation, be followed successively by instar chicken embryo＞19 day instar chicken embryo ≌ 1 Japanese instar chickling on the 18th.But obtain from cell and to take temperature, then antithesis, and the 1 Japanese instar chickling fabricius bursa is bigger, is easy to separate.In addition, 1 age in days that can select for use the brooder house to eliminate is public young, can reduce cost greatly.

2.4 the influence of Digestive system:

The viable cell quantity that application collagenase IV digestion is obtained is higher than trypsinase, to the chicken embryo source fabricius bursa of 18 and 19 ages in days, is tryptic 3 times but use the collagenase digesting time especially.

When using tryptic digestion, the time is advisable with 12～15min, overlong time, and then fiery cell quantity reduces, and the cell quantity of too short then results is few.The application cell motility rate that tryptic digestion obtained is all greater than 90% in this test.

2.5 the detection of bursopoietin:

No matter detect through the ELISA method, be the culture supernatant of 48h, or change behind the liquid in 24h, 48h or the culture supernatant of longer time, all can detect bursopoietin, shows that the fabricius bursa emiocytosis bursopoietin of vitro culture continues.

3. discuss

Because it is big than the viral harvest yield of other cell to use fabricius bursa primary cell culture IBDV, thereby at present used mainly is the Ji Peifashi bladder cell, and because of its fabricius bursa volume is little, the harvested cell amount is few, and the cost of viral proliferation is improved.This evidence, the public young bird of using 1 age in days carries out fabricius bursa cell cultures, and is close substantially with the result who uses the Ji Peifashi capsule, because of the cell concentration of its acquisition is big, and be easy to get, economy, provide experimental basis for further reducing the vaccine manufacturing cost from now on.

Up to the present, natural bursopoietin mainly is to extract from the fabricius bursa tissue of healthy chicken, because the restriction in fabricius bursa source, the research and extension that has restricted bursopoietin is used.This evidence, in fabricius bursa cells and supernatant, contain bursopoietin, this on the one hand, secreting law and influence factor for the research bursopoietin have practical significance, on the other hand, enlarged the acquiring way of bursopoietin,, effectively utilized culture supernatant to have reference value in utilizing fabricius bursa primary cell culture propagative viruses process.

Studies show that of pair cell digestion and culture condition used trypsinase and digested, and can satisfy the requirement of cellular segregation fully, and its key is the concentration and the digestion time of the good Digestive system of GPRS.Can use MEM or M199 nutrient solution in the cultivation.

Four, the immunocompetence of bursopoietin detects

Materials and methods

Experimental animal

60 LACA mouse (18g～20g, male and female are not limit), 3 adult cavys, more than all available from Xian Medical Univ's Experimental Animal Center.Healthy sheep has Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology experimental animal center to provide.

Main agents

RPMI1640 substratum (Nihon Pharmaceutical Co., Ltd.), calf serum (self-control), (electrophoresis is pure, the East Sea, Shanghai pharmaceutical factory, lot number: 890624) for gelose

The agar plate hemolytic plaque test is carried out according to a conventional method.Main process is:

The preparation aseptic technique of SRBC, from the blood sampling of sheep jugular vein, granulated glass sphere takes off fine anti-freezing, uses the PBS liquid of pH7.2 to wash 3 times, each centrifugal 5min of 2000r/min, red corpuscle is amassed in pressure, with an amount of PBS dilution, counting, is 30 * 10 with PBS accent cell concn ⁸/ mL is for using the same day.

Mouse immune is got 30 small white mouses (LACA) and is divided into 5 groups at random, and 6 every group, wherein one group is the blank group, only injects the 0.4mL cell culture fluid, and all the other 4 groups are carried out sheep red blood cell (SRBC) (SRBC) immunity, and dosage is 3 * 10 ⁷Individual/0.2mL only, wherein one group as SRBC immunity control group, remains 3 groups, injects the bursopoietin of various dose more simultaneously, 3 groups dosage only is respectively 0.5mg/, 1.0mg/ is only and 2.0mg/.Other gets 30 mouse, and it is the same to divide into groups, and test group is injected bursopoietin and its analogue respectively.

The mouse boosting cell suspension is prepared in immunity back 4d, take off neck and put to death mouse, get spleen, scrape extracting spleen cell with slide, cross 200 orders and 400 mesh filter screens successively, with Hank ' the s liquid washing that contains 5%NCS 2 times, the RPMI1640 nutrient solution that contains 5%NCS washs 1 time, and, expect blue dyeing counting with platform with this nutrient solution diluting cells, adjust viable cell concentrations to 1 * 10 ⁶Individual/mL, require living cell rate greater than 95%.

Hemolytic plaque test

The dull and stereotyped gelose of using Hank ' s liquid preparation 1.4% of preparation bottom agar-agar, heating is fully dissolved it, when treating that temperature is reduced to 50 ℃ of left and right sides, is poured in the plate of 75mm, and every ware 2～3mL makes its uniform distribution on horizontal stand.

Preparation top layer agar-agar flat board is got the splenocyte suspension 0.1mL and the 0.1mL SRBC suspension (3 * 10 of preparation ⁸Individual/as mL), to add simultaneously in 2mL 0.7% agarose of 48 ℃～50 ℃ of pre-temperature, mixing, impouring is covered with in the plate of bottom-layer agar sugar, evenly spreads out, and every mouse is cooked 3 repetitions, in 37 ℃, 5%CO ₂, hatch 1.5h under the temperature of saturation condition.

Add the flat board taking-up that complement will be hatched, every plate adds 2mL through the fresh cavy pooled serum that dilutes at 1: 5 that SRBC absorbs, and puts and continues to hatch 30min in the incubator.

The result takes out after observing and hatching end, uses stereomicroscope observation, counts the plaque number in each plate, calculates the mean number that contains the plaque founder cell in per 1,000,000 splenocytes.

The haemolysis spectrophotometry

Get the mouse boosting cell suspension of preparation, be made into 2 * 10 ⁷Individual/mL, getting its 0.5mL adds in the small test tube, add 1% SRBC 0.5mL again, 1: 10 guinea pig serum 0.5mL, fully mixing is put 37 ℃ of incubation 1h, take out, 3000r/min, centrifugal 5min surveys the hemoglobin content (with the OD value representation) that erythrocyte splitting discharges in its supernatant liquor with microplate reader (DG5030) at 410nm.

2. result

2.1 the bursopoietin of various dose is to the influence of mouse plaque-forming bacteria number (PFCs): the results are shown in Figure 1, as seen from Figure 1, bursopoietin can obviously promote mouse plaque-forming bacteria number, be PFCs, test of significance shows, inequality heteropole remarkable (P＜0.01) between each test group and the immune control group.The bursopoietin of various dose all has significant difference (P＜0.01) to the influence of PFC.Dosage is when 1.0mg/mL, and promoter action is the strongest.Show that its promoter action to immunity of organism has dose-dependently.

2.2 bursopoietin and analogue thereof are to the influence of PFC:

Bursopoietin and different analogue thereof are seen Fig. 2 to the influence of PFC, as seen from Figure 2, bursopoietin and pHGF all can significantly improve plaque-forming bacteria's number (P＜0.01) of mouse, difference not significantly (P＞0.05) between the two, show that bursopoietin and pHGF have similar immunologic enhancement.The bursopoietin group is compared difference extremely significantly (P＜0.01) with its isometry analogue, illustrate that its effect is closely related with molecular structure.

The haemolysis spectrophotometry is (see figure 3) as a result, and Fig. 3 shows that the result that the application spectrophotometry records is consistent with application hemolytic plaque test gained result.Further specify bursopoietin and analogue thereof promoter action to B cellular immunization.

3. discuss

BS is unique a kind of active polypeptide that can promote fabricius bursa growth and humoral immunity of organism of up to the present organizing purifying from chicken bursa.Audhya etc. think that BS can accelerate the speed that the interior DNA of B cell is transcribed into mRNA, promotes the synthetic of intracellular protein, thus the ability reinforcement that makes B emiocytosis and produce antibody.In this test, hemolysis barren spot experiment and haemolysis spectrophotometry prove that all synthetic BS can promote the mouse internal antibody to form the quantity (P＜0.01 or P＜0.05) of cell.With resulting result such as Baba-it can improve lymphocytic transformation efficiency, promote the differentiation of B cell-consistent.

LCGF is a kind of active kyrine from the separation and purification of mouse liver organization, and its composition is identical with BS, but the amino acid order is inconsistent.Audhya proved once that the LCGF of high dosage had the effect that similarly promotes generation of B cell and secretory antibody with BS.When this evidence was approximately 1000 times of left and right sides of BS when the dosage of LCGF, the two did not have significant difference at the celliferous quantitative aspects of raising mouse antibodies.Whether this shows that mammiferous liver has the effect of similar bird bursal, remains further to be studied from now on.In addition, liver is organized easily than the fabricius bursa and is obtained, and the further confirmation of LCGF immunization will provide experimental basis for its application at veterinary clinic.

Five, bursopoietin is to the influence of chicken immune

(Hewcastle disease, ND) immunization experiment has tentatively been inquired into the effect of tripeptides capsule element to the chick immunity by chicken Newcastle disease.

Materials and methods

1.1 laboratory animal

200 of the young cocks of 1 age in days are available from Northwest Agricultural University stock-farms kind chicken house brooder house.

The weak malicious seedling of ND IV system:

The Lasota strain, the biological medical instruments in Nanjing factory produces.The strong poison of the fabricius bursa is provided by my school herding Experiment on Microbiology.The IBD-ELISA quick diagnosis reagent kit, the Guangxi veterinary institute is produced, lot number 9811.Anti-bursal disease virus hyper-immune serum is provided by my domestic animal reproductive endocrine laboratory, school, and fine jade expands tires 2 ⁶

Main agents

Synthetic bursopoietin structural formula is Lys-His-Gly-NH2, molecular weight (MW): 339, and purity 70% is made into respective concentration with the phosphate buffered saline buffer (PBS) of 0.05M pH7.2, and 4 ℃ of preservations are standby.0.05M the phosphate buffered saline buffer of pH7.2 (PBS).

The preparation of 1% chicken red blood cell (PBC) suspension;

Aseptic collection grand duke's chicken wings venous blood, the Trisodium Citrate anti-freezing, the PBS liquid repetitive scrubbing of usefulness 0.05M pH7.2 three times, each centrifugal 15min of 1500r/min after the last washing, adds an amount of PBS liquid, is made into 1% concentration, and 4 ℃ of preservations are for using the same day.

NDV 4 unit poison;

With the NDIV attenuated vaccine with after the PBS dilution.From allantoic cavity inoculation instar chicken embryo (northwest agricultural university stock-farms kind chicken brooder house provides) on the 9th～11, get dead chicken embryo in 48h～96h, collect allantoic fluid, survey blood clotting valency (HA) in 96 hole blood-coagulation-boards, frozen standby, at every turn according to HA, venom (HA is above person at 1: 128) is received by institute be diluted to 4 unit poison, later on for using the same day.

Bursopoietin is to the influence of maternal antibody level

Chicken is divided into 4 groups at random, and 50 every group, during 3 ages in days, experimental group A, B, C group are injected the bursopoietin of various dose respectively, and control group D organizes intramuscular injection equal-volume physiological saline (table 2);

Table 2 test grouping sheet unit: μ g

First group (A) second group (B) the 3rd group (C) the 4th group (D)

Bursopoietin 0.3 0.6 1.2-

Physiological saline---0.5

In the blood sampling of 3,6,9,12,15 ages in days, measure newcastle disease maternal antibody level (HI).

1.7 bursopoietin is the influence of vaccine immunity to NDIV;

(maternal antibody reduces to 2 in 22 ages in days ¹About) time, all chicken collunarium eye droppings NDIV are each 50 μ L (1 plumage part) of vaccine, antibody horizontal (HI) is measured in 3d, 5d, 10d, 15d, 18d blood sampling after immunity.

1.8 use behind the fabricius bursa extract for treating chicken group IBD influence to ND vaccine second immunisation:

When chicken 34 ages in days, put at Ji Qunzhong and to suffer from the sick chicken of IBD, make chicken group natural infection morbidity, the IBD disease symptom appears in the chicken group and after, use bursopoietin and cooperate specific corrosioning anteserum to treat, establish simultaneously and do not treat group and compare.After the chicken group is all stable, respectively choose 15 healthy chickens from A, B, C group.Never choose anti-15 of the chickens that cross in the treatment group, when 42 ages in days, carry out secondary ND immunity, the scheme method is equal to 1.7.3d, 5d, 10d, 15d, 18d after immunity, the heart blood sampling, room temperature is separated out serum, measures ND blood clotting inhibition (HI) antibody horizontal in the serum with β-micromethod.

The result

2.1 Fa Shi is wrapped up in plain influence to maternal antibody (see Table 3, Fig. 4):

Chicken group source of parents HI antibody titer changes X ± Sd behind the table 3 injection bursopoietin

Fate (age in days) behind the injection bursopoietin

Group

3(6) 6(9) 9(12) 12(15) 15(18)

A 4.5±0.71 4.25±0.50 4.25±0.5 4.0±0.82 2.0±0.82

B 5.0±0.41 5.0±0.1 4.5±1.29 4.25±0.5 2.25±0.96

C 5.0±0 5.25±0.96. 4.75±0.5 4.5±0.71 2.5±0.58

D 3.5±0.96 4.0±0.82 3.25±1.5 3.0±0 1.5±0.58

As can be seen from Table 3, from 6 ages in days to 18 ages in days (injection BS after 3～15d), the experimental group antibody horizontal is than high 0.25～1.5 titre of control group, and especially heavy dose of group C group compares with control group that (P＜0.05=is at the extremely remarkable (P＜0.01=of 15 age in days differences at 9,12 age in days significant differences.B, C compare at 6 ages in days to 9 ages in days during this period of time for two groups, and B group antibody horizontal is higher than the C group, and C group antibody horizontal is higher than the B group after 9 ages in days, but two groups of differences are not remarkable.C group antibody horizontal peaks at 9 ages in days, reduces to minimum later on gradually.

As can be seen from Figure 4, from 6 ages in days to 18 ages in days, three groups of injection BS groups, antibody horizontal all is higher than control group, and C group is higher than the B group, and the B group is higher than the A group, but per two group differences of experimental group not significantly (P＞0.05), from then in the whole process mean value of all antibody horizontals as can be seen, three groups of A, B, the C of injection BS are respectively than high 0.75,1.15 and 1.35 titre of control group.

2.2 BS is the influence (table 4) that vaccine head exempts from effect to ND IV

As can be seen from Table 4, the experimental group antibody horizontal in this process all than control group (normal immune group) height, the the 3rd, the 6th day after immunity all than high 1 titre of control group, especially the immunity back is the 6th day, two groups of A, B compare significant difference (P＜0.05) with control group, the C group is compared difference extremely significantly (P＜0.01) with control group.After immunity the 6th day, antibody horizontal all rises to the highest, descend gradually later on, and the antibody change dynamics trend of A, B, C group is organized consistent with D.

The 12d of (promptly from 22 ages in days to 33 ages in days) from 3d after the immunity to 14d, the average antibody level of three groups of BS injection groups all is higher than normal immune group, but per two group differences not significantly (P＞0.05), A, B, the C group of injection BS are higher by 0.75 than normal immune group respectively, 1,1.2 titre.

Chicken group HI antibody changes after table 4 first immunisation

Fate after the first immunisation (d)

Group

3 6 9 14

A 5.5±1 6.5±0.58 5.5±1 5.0±0.82

B 5.75±0.82 7.0±0.82 5.75±0.96 5.0±0.82

C 6.0±0.82 7.0±0.82 6.0±0.82 5.0±0.82

D 4.5±0.82 5.25±0.5 5.0±0 4.75±0.96

3 BS are to infecting the influence (table 5) of NDIV immunity (two exempt from) effect behind the IBD

As can be seen from Table 5, the experimental group antibody horizontal in this process all than the control group height, especially heavy dose of group C group was compared difference extremely significantly (P＜0.01) on the the 3rd, 5,15,18 day with control group after immunity, 10d significant difference after immunity (P＜0.05=, A, B, three groups of antibody change dynamics of C trend basic one

Chicken group HI antibody changes X ± Sd behind table 5 second immunisation

Fate behind the second immunisation (d)

Group

3 5 10 15 18

A 1.5±0.58 3.0±0.82 4.25±1.25 4.75±0.5 4.25±0.5

B 2.0±0.82 3.25±0.5 5.0±0 5.0±0 4.5±0.58

C 2.75±0.5 3.5±0.58 6.0±1.4 5.5±0.58 5.0±0

D 1.0±0 1.75±0.5 3.75±0.5 3.75±0.96 4.0±0

Exempt from the 17d of back (45 ages in days are to 61 ages in days) two.The average antibody level of each BS injection group all is higher than control group, and C group＞B group＞A group, 3d after immunity, 10d, 18d, two groups of significant differences of A, C (P＜0.05), immunity back 5d, 15d difference is remarkable (P＞0.05) not, A group and B group, B group and C group be difference not significantly (P＞0.05) in whole process.

3. discuss

From above-mentioned chart in general, BS can make the maternal antibody level keep higher level, to NDIV is that vaccine immunity has immunologic enhancement, and it is relevant with dosage, suitably dosage can improve the HI level in the whole immunologic process, and heavy dose can make the HI level be elevated to a higher level in a short time rapidly, but this experiment to exempt from BS in the process at head be not clearly to the promoter action of immunity, this may be relevant with the physiological situation of chick, remains further to be tested.

Six, bursopoietin and analogue thereof are to the therapeutic action of chicken IBD

Central immune organ-fabricius bursa and other tissue of the infected chicken of infectious bursal disease virus (IBDV) energy grievous injury cause disease fowl immunologic hypofunction, bring about great losses to poultry husbandry.Thereby, especially promote the recovery of immunologic function after being ill of morbidity chicken to effective treatment of IBD, be veterinary work person institute extensive concern.(bursin BS) can optionally induce the differentiation of B cell, the normal development of Cu Jinfashi lens capsule tissue to be present in bursopoietin in the fabricius bursa tissue.And relevant synthetic BS and analogue-pHGF thereof (liver cell growth factor, LCGF) less at the report of aspects such as IBD treatment, this test is studied this, in the hope of seeking the effective methods of treatment to IBD.

1. material and method

1.1 chicken use in test: 1 Japanese instar chickling, available from Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology's stock-farms, isolated rearing to 21 age in days, treat that fabricius bursa maternal antibody detects feminine gender after, be used for testing;

Synthetic BS molecular formula is LYS-HIS-GLY-NH2, molecular weight 339.41, and purity 70% is with no Ca ⁺⁺, Mg ⁺⁺Hank ' s liquid (CMFS) is mixed with the application liquid of A liquid and two kinds of concentration of B liquid, Entkeimung, and 4 ℃ of preservations are stand-by; Synthetic LCGF molecular formula is GLY-HIS-LYS-NH2, and molecular weight is 340.39, and purity 80% is mixed with solution with CMFS, Entkeimung, and 4 ℃ of preservations are stand-by.Above-mentioned two kinds of reagent are U.S. connection bio tech ltd (Xi'an) product.(the fine jade expansion tires 2 to fabricius bursa high immunity yolk antibody ⁶) and strong poison provide by herding Microbiological Lab of Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology.

2.3 synthetic BS and LCGF are to the therapeutic test of IBD

With the artificial challenge fall ill chicken with wait to try chicken and mix the group, make its natural infection morbidity, sick chicken shows typical IBD clinical symptom, cuts open and picks up visible typical IBD pathology, gets tissue homogenate respectively such as the fabricius bursa, spleen and kidney, centrifugal, get supernatant liquor, detect (test kit is available from the Guangxi veterinary institute), performance IBDV strong positive reaction with the ELISA method, prove that it really is IBD, sickness rate reaches more than 80%.The chicken that will fall ill subsequently is divided into 7 groups at random, and 25 every group, carry out therapeutic test, observed and recorded is respectively organized chicken mass-sending symptom condition and death toll day by day therebetween, and the chicken that dies of illness is cutd open inspection, the record pathological change.After treating group resolution of symptoms, stochastic sampling is dissected, and observes the recovery situation of pathological tissues.Testing program sees Table 6.

BS and LCGF are to the therapeutic test of ND

Chicken group grouping and processing are all the same.

Sick chicken grouping of table 6 and processing unit: mL

Treatment group control group

1 2 3 4 5 6 7

BS(B) 0.3 - - - - - -

BS(A) - 0.3 - 0.3 - - -

LCGF - - 0.3 - 0.3 - -

High immunity yolk antibody 0.3 0.3 0.3--0.3.-

Physiological saline---0.3 0.3 0.3 0.6

Annotate: above medicine is all injected in chest muscle.

Result and analysis

The IBD treatment result

Clinical condition:

The chicken group falls ill in 27 ages in days, sickness rate 80%, and sick chickens' extract god is tired, and food consumption reduces, and feather is fluffy, and chilly is dozed off, the lethargic sleep of hanging one's head.Arrange white thickness and water sample loose stool.Cut open the sick chicken of inspection, visible leg flesh, chest muscle severe haemorrhage liver khaki color have the striated blutpunkte, fabricius bursa enlargement, and inner mucous membrane has hemorrhage and cheesy exudate in various degree; Spleen, kidney have in various degree enlargement and hemorrhage; Glandular stomach and muscular stomach intersection have the strip blutpunkte.Gather disease chicken bursa, spleen, kidney and add an amount of physiological saline and grind, centrifugal, supernatant liquor is strong positive with the detection of IBD-ELISA quick diagnosis reagent kit, and this reaction can be fabricius bursa positive serum and suppresses.

Treatment result:

Treatment back 2d, the most of spirit of treatment group chicken group begins to take a turn for the better, and the high immunity yolk antibody group partly begins to take a turn for the better; Behind the 3d, refreshing basic recovery of all treatment group survival chickens' extracts, drinking-water, feed recover normal.6d, each group survival chicken clinical symptom all disappears.

The chicken that dies of illness is cutd open inspection find, every pathological change is no significant difference between each group, cuts open inspection and changes consistent with dying of illness before the treatment.Treat and the survival chicken is cutd open inspection behind the 6d and show that each organizes chicken muscle and the visible old pathology of liver, no significant difference between each group; But fabricius bursa recovery situation has nothing in common with each other, and wherein the 2nd, 4 group of fabricius bursa observed normally substantially, big as broad bean, obviously respectively organizes 0.5～1.0 times greater than other; The obvious atrophy of the control group fabricius bursa, dry, still have cheesy thing in indivedual fabricius bursa.

Each organizes chicken group survival rate

The survival rate of each treatment group chicken all is significantly higher than control group (P＜0.01); Yolk antibody treatment group is significantly higher than does not treat group (P＜0.05); Relatively, difference is not significantly (P＞0.05, table 7) all between each treatment group (1 ~ 5 group).Show that synthetic BS and LCGF cooperate high-immunity yolk or independent the application all IBD to be had the obvious treatment effect respectively; Use the surviving rate that high-immunity yolk also can significantly improve the disease chicken separately.

Table 7 is respectively organized chicken group's survival rate (%)

Treatment group control group

BS (A) BS (B) LCGF physiology

BS(A) LCGF Yab

+ Yab+Yab+Yab salt solution

Survival rate 84 80 80 76 72 40 12

Compare and deposit with group 6

44 40 40 36 32 - -

Motility rate improves

Compare and deposit with group 7

72 68 68 64 60 28 -

Motility rate improves

The ND treatment result

1 clinical symptom:

After infecting 2～3d, morbidity appears beginning in the chicken group, and gong sound, sick chicken runny nose are breathed, cough, occurred in performance, forms around the nostril to do scab.The crop deliquescing when holding, is flowed out the smelly liquid of water sample acid from mouth and nose.Cut open the visible glandular stomach myxedema of inspection, thelorrhagia, under the muscular stomach stratum corneum blutpunkte is arranged, cecal tonsil enlargement, hemorrhage, intestinal mucosa has blutpunkte and the necrosis region that differs in size.

2.3.2 treatment result:

Treatment back is clinical lapse to after performance and the IBD treatment consistent.

2.3.3 chicken group survival condition (seeing Table 8):

Table 8 ND morbidity chicken group treats the back survival condition

Treatment group control group

A B C D

Survive several 27,/50 38,/50 42,/50 4/50

Survival rate 54 76 84 8

By table 8 as seen, BS+Ab or BS use separately all has the obvious treatment effect to chicken ND, compares difference extremely significantly (P＜0.01) with control group.

3. discuss

BS is unique a kind of active polypeptide that can promote fabricius bursa growth and humoral immunity of organism of up to the present organizing purifying from chicken bursa.Audhya etc. ^[2]Think that BS can accelerate the speed that the interior DNA of B cell is transcribed into mRNA, promotes the synthetic of intracellular protein, thus the ability reinforcement that makes B emiocytosis and produce antibody.

LCGF is a kind of active kyrine from the separation and purification of mouse liver organization, and its composition is identical with BS, but the amino acid order is inconsistent ^[2]Audhya ^[2]Once prove that the LCGF of high dosage has the effect that similarly promotes generation of B cell and secretory antibody with BS.

For IBD, many at present employing specific antibodies and Chinese medicine preparation are treated, and have obtained certain effect.This test-results shows, though yolk antibody can significantly improve the surviving rate of IBD chicken, significantly is lower than BS or LCGF and the two and combines the effect of using (P＜0.01) with yolk antibody.Especially yolk antibody does not have tangible effect to the damage of the fabricius bursa, thereby can not fundamentally solve secondary infection and to problems such as other immune effect of vaccine exert an influence.BS has obtained more test-results support to the promoter action of immunity of organism and fabricius bursa growth.This evidence, synthetic BS and LCGF all have therapeutic action to IBD and combine application with high immunity yolk antibody, and curative ratio is higher than independent application, but statistic analysis result does not have significant difference, and this may be little relevant with antibody dosage.Though the curative ratio between the treatment group of application various dose BS does not have significant difference, but the fabricius bursa recovery of high dose group (A group) chicken obviously is better than low dose group, bursopoietin is similar to result of treatment and the IBD of chicken ND, the mechanism of pointing out its effect mainly is the recovery of Cu Jinfashi lens capsule tissue and the generation of body disease-resistant poison antibody, and its effect is relevant with dosage.

Above-mentioned test shows:

The bursopoietin of synthetic has identical immunologic competence with bursopoietin from healthy chicken fabricius bursa tissue extraction, also has bursopoietin in the fabricius bursa histocyte culture supernatant.

But above-mentioned various bursopoietin preparation is the period of significant prolongation chick maternal antibody existence all.

Above-mentioned various bursopoietin preparation all has significant promoter action to ND and IBD attenuated vaccine immunity.

Above-mentioned various bursopoietin preparation is used separately or cooperated with specific antibody has the obvious treatment effect to ND and IBD, especially can promote to damage the reparation of the fabricius bursa.

Claims (9)

1, a kind of extracting method of bursopoietin is characterized in that comprising following operation steps: get healthy fabricius bursa 80g, add an amount of 1N Glacial acetic acid and use liquid, 20000r/min homogenate 3min makes it become pasty state.1.5h～the 2h that vibrates on horizontal shaker in 4 ℃ of centrifugal 60min of 4500r/min, gets supernatant, acts on 20min in the boiling water bath, and is once centrifugal again, gets supernatant and filters.Filtered liquid is evaporated to about 40ml between 60 ℃～70 ℃ with Rotary Evaporators, and concentrated solution is through 20000g, high speed centrifugation 30min under 4 ℃ of conditions, get the about 20ml of supernatant liquor, be crude extract, get crude extract and cross the G-50 sephadex column, 1N acetic acid wash-out is collected CuSO ₄The reacting positive part, the NaHCO with 7.5% ₃Adjust pH is tissue extract-bursopoietin to neutral.

2, the application of a kind of bursopoietin in disease treatment is characterized in that: bursopoietin is applied to the prevention and the treatment of communicable disease and young animal disease separately.

3, the application of bursopoietin according to claim 2 in disease treatment is characterized in that: described bursopoietin cooperates prevention and the treatment that is used for communicable disease and young animal disease with antiviral.

4, the application of bursopoietin according to claim 2 in disease treatment is characterized in that: described bursopoietin cooperates prevention and the treatment that is used for communicable disease and young animal disease with antibacterials.

5, the application of a kind of bursopoietin in immunity is characterized in that: described bursopoietin and animal vaccine are used, and are used to improve the animal disease resistant ability and to the immunne response ability of specific antigen and the immune efficacy of vaccine.

6, the application of bursopoietin according to claim 5 in immunity is characterized in that: described bursopoietin is applied to improve the animal disease resistant ability as the animal vaccine adjuvant and to the immunne response ability of specific antigen and the immune efficacy of vaccine.

7, the application of bursopoietin according to claim 5 in immunity is characterized in that: described bursopoietin is applied to improve the animal disease resistant ability as animal vaccine immunopotentiator and to the immunne response ability of specific antigen and the immune efficacy of vaccine.

8, the application of bursopoietin according to claim 5 in immunity is characterized in that: described bursopoietin is directly as vaccine diluent.

9, the application of a kind of bursopoietin in immunity is characterized in that: described bursopoietin is as additive for farm animal feed.

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