CN101081299B - Yelk immune globulin products for preventing and treating bainite cryptosporidiosis and application thereof - Google Patents

Abstract

The present invention provides yolk immunoglobulin preparation for preventing and treating cryptosporidiosis and health product of yolk immunoglobulin for strengthening immunity. The yolk immunoglobulin is prepared through the following steps: gathering cryptosporidium egg capsule from positive cryptosporidium excrement, separating, sucrose gradient centrifuging to purify cryptosporidium egg capsule and prepare cryptosporidium antigen, immunizing female laying fowl with the cryptosporidium antigen, collecting laid fowl egg, collecting yolk, diluting with distilled water, centrifuging to obtain supernatant, water diluting, and salting out with ammonium sulfate to obtain yolk immunoglobulin. The present invention is acted on the pathogen of cryptosporidiosis to reach specific inhibition.

Description

A kind of Yolk immunoglobulin product and application that prevents and treats the Cryptosporidium baileyi disease

Technical field

The invention belongs to biological technical field, relate to the aquaculture of aquatic animal technical field, specially refer to a kind of control to the Yolk immunoglobulin (IgY) of Cryptosporidium baileyi disease and use, the biological preparation of the protopathy that this kind IgY can cause as the control Cryptosporidium baileyi and the health product of raise immunity.

Background technology

1907, Tyzzer found first that in the section of mice mucosal tissue Cryptosporidium (Cryptosporidium spp.), cryptosporidiosis (Cryptosporidiosis) become a kind of universal people beast and suffer from parasitic disease altogether.This protozoon extensively parasitizes the humans and animals more than 180 kinds such as mammals, birds, reptiles and amphibian, can cause human diarrhoea, the especially fatefulue diarrhoea of infant and immunologic hypofunction or handicapped.Cause livestock and poultry diarrhea and respiratory symptom, cause fertility performance to descend or death.In recent years, cryptosporidiosis research has been become the focus of medical circle, the research concern of veterinary educational circles.

The Yolk immunoglobulin of chicken mainly is IgG, so be called Yolk immunoglobulin IgG (Yolk Immunobudin G), abbreviates IgY as.IgY is similar on molecular structure to mammiferous IgG, but its again some distinctive biological characteristics: IgY of tool the Fc district not with people or antibacterial Fc receptors bind, IgY does not activate mammal and people's complement system, IgY not with human serum in the antibody response of resisting mammal antibody, as people's anti-IgG antibody, IgY does not combine with SP (SPA) or streptococcus protein G (SPG) etc.; The IgY that is produced by certain specific pathogen antigenic stimulus is a species specific immunoglobulin, pointed to this cause of disease, thereby can be used for specific treatment, and because of it derives from egg, do not have toxic and side effects, do not have drug residue, do not have environmental pollution, its stable performance in addition, heat-resisting, acid and alkali-resistance and height ooze environment, be convenient to store and processing,, have very big advantage undoubtedly for manufacture of therapeutic medicine (as oral capsule) and green functional food.Not good for medication effect especially, perhaps not can be used for the pathogenicity disease for the treatment of, can produce the IgY biological preparation with the method and carry out at treatment.

Cryptosporidiosis does not still have the specific treatment medicine at present, though worldwide done a large amount of drug screening work, up to the present, does not still enter the bibliographical information of clinical practice.Research about anti-Cryptosporidium IgY preparation method for antibody, domestic and international rare report, this research is by development anti-Cryptosporidium baileyi (C.baileyi) IgY antibody, to preparation immunizing antigen, design immune programme for children, the dynamic law that produces by IgY antibody and serum antibody, the persistency of antibody response, established best preparation method, for preparing a large amount of Cryptosporidium specific IgY antibody from now on, or other weak immunogenicity cause of disease specific IgY antibody provides most basic technology.

Publication number is for the CN1569229 name is called the patent application of anti-oral cavity and upper respiratory tract infection IgY product and application thereof, discloses to relate to multiple technology of preparing with chicken yolk immune globulin IgY of specificity infection function, be used for caries prevention, treatment periodontal disease and diseases of oral mucosa, prevention and treatment influenza, treatment pharyngolaryngitis.Publication number is called the patent application of a kind of Yolk immunoglobulin of preventing and treating prawn virus disease and its production and application for the CN1583792 name, disclose and related to the aquaculture of aquatic animal technical field, specially referred to a kind of Yolk immunoglobulin (IgY) of preventing and treating prawn virus disease and preparation method thereof.It is characterized in that former single antigen or the complex antigen made of one or more deactivation strains/attenuation strain/gene subunit immunogen/nucleic acid immunization with the white spot syndrome virus (WSSV) class, with the birds of laying eggs as bioreactor, immunity obtains anti-prawn white spot syndrome specific IgY, and this IgY can be applied to prawn disease as the active component of feed additive, medicinal dipping bath agent or shrimp pond dousing agent separately or with Peptidoglycan, probiotics and Chinese herbal medicine combination jointly.Effect of the present invention and benefit are that this IgY plays a role by passive immunity mechanism, but directly kill the virus, and strengthen shrimp body immunity, prevention and treatment prawn white spot syndrome have effect significantly, safety, environmental protection, advantage such as easy to use.Publication number discloses the production method of the powdered egg that contains chicken yolk immunoglobulin for the CN1602720 name is called the patent application of the production method of the powdered egg that contains chicken yolk immunoglobulin, at first selects to contain the sterilization of chicken yolk immunoglobulin egg and cleans; Eggshell is separated with egg liquid, egg yolk, the egg white solution of collecting mixed make egg liquid, egg liquid is removed by filter scrambled egg shell and other foreign material in the egg liquid; Then, egg liquid is adopted the pasteurize sterilization, adopt centrifugal spray drying method or pressure spray dryer method that egg liquid is carried out drying again, dried powdered egg sieves by the screen cloth that per inch 24 holes are housed, and removes big particle, packs to get final product after weighing again.Publication number is called a kind of ice-cream patent application of caries prevention for the CN1620886 name, disclose a kind of ice cream that is used for kind of caries prevention, formed by popsicle ice cream 1000 weight portions, dental caries Yolk immunoglobulin (Igy) 0.1-50 weight portion, dental caries Yolk immunoglobulin stabilizing agent and cryoprotective agent 0.5-35 weight portion; This ice-cream preparation method is: after the popsicle icecream mix, before freezing, the IgY powder that desire is added dissolves with small amount of cold boiled water earlier, then with stabilizing agent, protective agent adds mixing and be thread under stirring movement to be poured in the popsicle icecream mix, stir, freezing by the temperature and time of general making popsicle ice cream regulation, promptly get dental caries cold drink ice cream popsicle; Publication number discloses a kind of special yolk immunoglobulin oral administration lyophilized formulations and preparation method and purposes for the CN1621090 name is called the patent application that special yolk immunoglobulin oral administration lyophilized formulations and preparation method thereof reaches purposes.The component of this lyophilized formulations comprises solute in specific immune globulin, excipient and the solvent.Its preparation method comprises preparation specific pathogen antigen; This specific pathogen antigen is added the fully emulsified immunogen of doing of the incomplete freund adjuvant of equivalent; With this immunogen the poultry of laying eggs is carried out reinforced immunological; Laying eggs, the poultry of laying eggs behind reinforced immunological extracts the special yolk immunoglobulin; Special yolk immunoglobulin and excipient, solvent are obtained lyophilizing stock solution; Lyophilizing stock solution is made special yolk immunoglobulin oral administration lyophilized formulations through freeze-drying.Its special yolk immunoglobulin of Yolk immunoglobulin preparation of the present invention's preparation is changeableness not, can keep natural activity, and being dissolved in water gets final product.This lyophilized formulations can be used in the medicine of the digestive tract infection disease that preparation causes by pathogenic microorganism or food, the health product.Publication number is used to prevent and treat the IgY preparation method of oral disease and based on the patent application of the toothpaste of this IgY for the CN1646565 name is called, a kind of preparation method that is used to prevent and treat the Yolk immunoglobulin of multiple oral disease is disclosed, and be the safety toothpaste of primary raw material.When preparation IgY, according to the main pathogens that causes dental caries, periodontal disease, halitosis, adopt conventional method to turn out main several pathogenic bacterium respectively, make complex antigen with the strain of being cultivated, use described complex antigen that laying poultry (comprising chicken, duck, turkey etc.) is injected then, the egg yolk of the immune egg of getting poultry again and being given birth to is made required IgY.Utilize described IgY to make toothpaste.Publication number is called the patent application of anti-rotavirus chicken yolk immunoglobulin product and application and activity detection for the CN1201693 name, it is (anti--ARVIgY) product to disclose the anti-A group rotavirus chicken yolk immunoglobulin that contains antibody activity, its preparation method adopts the antigen subcutaneous injection immunity chicken that contains the A group rotavirus to collect its yolk of laying eggs after 10 days, by known extraction anti--method of ARVIgY crude product obtains crude product, to slightly carry anti--ARVIgY through following step purification: a. again and cross DEAE-SephadexA ↓ [50] gel chromatographic columns, add buffer solution elution, desalination, lyophilization is dissolved again; B. cross SephadexG ↓ [200] gel chromatographic columns, add buffer solution elution, desalination, lyophilization is resisted-the pure product of ARVIgY.Publication number discloses a kind of immunoglobulin for the CN1526735 name is called the patent application of a kind of immunoglobulin and its production and application, adopts the no-special pathogen bird inlay to carry out Enterovirus 70 type and CA 24 type reinforced immunologicals respectively; Adopt aseptic phosphate buffer homogenize immunity ovum gallinaceum yolk, add polyethylene glycol 6000,, collect supernatant through centrifugal 15～20 minutes, add polyethylene glycol 6000, centrifugal 20 minutes, collecting precipitation was with aseptic phosphate buffer dissolution precipitation, add polyethylene glycol 6000, centrifugal 20 minutes, finish defat; Collecting precipitation carries out the desalination of PEGDEAE-Sephacel column chromatography gel filtration, chromatography, adopts phosphate buffer eluting destination protein; 50% saturation ammonium sulfate concentrates destination protein; 4 ℃～8 ℃ dialysis down, obtain the specificity ovum gallinaceum antibody of purification, promptly required immunoglobulin with aseptic phosphate buffer.Other also have publication number be CN1435260, CN1439281, CN1250056, patent application, though disclose the method for making and the application of many Yolk immunoglobulins, not at the Yolk immunoglobulin (IgY) and the application thereof of control to the Cryptosporidium baileyi disease.

Summary of the invention

The purpose of this invention is to provide a kind of Yolk immunoglobulin biological preparation of the protopathy that Cryptosporidium baileyi causes and Yolk immunoglobulin health product of raise immunity prevented and treated.

The objective of the invention is to realize by following technical solution:

1, a kind of Yolk immunoglobulin product of preventing and treating the Cryptosporidium baileyi disease, this product can obtain from following steps, at first from the positive feces of Cryptosporidium baileyi, collect egg capsule and separate back sucrose gradient centrifugation purification egg capsule, and be prepared into Cryptosporidium baileyi antigen, with the female laying fowl of Cryptosporidium baileyi antigen immune, collect it then and produce the fowl ovum, from the fowl ovum, collect egg yolk again, with distilled water diluting, the centrifuging and taking supernatant obtains Yolk immunoglobulin through water dilute sulphuric acid ammonium salting out method purification.

2, the described egg capsule of collecting from the positive feces of Cryptosporidium baileyi is meant the just middle egg capsule of the positive animal manure of collection, egg capsule is long oval, recording the egg capsule size is 6.5 μ m x4.7 μ m (5.4～7.8 μ m x4.1～5.5 μ m) shape indexs 1.31, removes the chicken coccidial oocyst that is mixed with through 3 age in days Carnis Coturnicis japonicae blind passages.Set up animal infection modal and the analysis of 18sRNA sequence alignment through this laboratory, be defined as Cryptosporidium baileyi.With the amplification of going down to posterity of 3 Japanese instar chicklings, to obtain a large amount of antigens.

3, describedly be meant, 1. prepare sucrose stock solution with sucrose gradient centrifugation purification egg capsule: sucrose 500g, after the distilled water 320mL dissolving, autoclaving; 2. prepare PBS buffer: NaCL8.0g, KCL0.2g, KH2PO40.2g, NaHPO412H2O2.9g, distilled water 1000mL transfers pH value 7.4 back sterilizations; 3. will be 1. sucrose stock solution and 2. PBS buffer be formulated as a, 1 part of sucrose stock solution to add after 1 portion of PBS buffer sucrose concentration (W/W%) be 30.4 to be called the 1:1 sucrose solution; It is 25 to be called the 1:2 sucrose solution that b, 1 part of sucrose stock solution add after 2 portions of PBS buffer sucrose concentration (W/W%); It is 18.7 to be called the 1:3 sucrose solution that c, 1 part of sucrose stock solution add after 3 portions of PBS buffer sucrose concentration (W/W%); It is 16.8 to be called the 1:3.5 sucrose solution that d, 2 parts of sucrose stock solutions add after 7 portions of PBS buffer sucrose concentration (W/W%); It is 12.5 to be called the 1:5 sucrose solution that e, 1 part of sucrose stock solution add after 5 portions of PBS buffer sucrose concentration (W/W%); 4. egg capsule is purified with the glass centrifuge tube of 10mL diameter 1.2cm, and program is as follows: 2 parts of a, addings earlier slowly add 1 part of egg capsule suspension, 3000rmin along glass tube walls again ^-1, 10min with suction pipe sucking-off egg capsule layer, adds 2 times of volume PBS buffer centrifuge washings 2 times, and precipitation is suspended in an amount of PBS buffer; 2 parts in b, adding 1:2 sucrose water slowly add 1 part of a step along glass tube walls again and precipitate the egg capsule suspension that is suspended in an amount of PBS buffer, 3000rmin ^-1, 10min with suction pipe sucking-off egg capsule layer suspension, adds 2 times of volume PBS buffer centrifuge washings 2 times, and precipitation is suspended in an amount of PBS buffer; 2 parts in c, adding 1:3 sucrose water, slowly add 1 part of b step along glass tube walls again and precipitate the egg capsule suspension that is suspended in an amount of PBS buffer, 3000rmin-1,10min, with suction pipe sucking-off egg capsule layer suspension, add 2 times of volume PBS buffer centrifuge washings 2 times, precipitation is suspended in an amount of PBS buffer, repeats once again; 1 part in d, adding 1:3.5 sucrose water, be added in the centrifuge tube along tube wall, slowly add 1 part of 1:5 sucrose liquid along tube wall then, the superiors add 1 part of 1:3 sucrose extract egg capsule suspension, slowly add 1 part of c step repeated washing along glass tube walls again and precipitate the egg capsule suspension that is suspended in an amount of PBS buffer, 3000rmin ^-1, 10min reclaims egg capsule layer suspension; 5. use PBS buffer dilution back with 5 μ m filter membrane microfiltration degerming, concentrating back adding final concentration is the two anti-of 1000U/mL, 4 ℃ of preservations.

5, the described Cryptosporidium baileyi antigen that is prepared into is meant the Cryptosporidium baileyi egg capsule after the purification degerming is suspended in the PBS buffer (10 ⁸/ mL), ice-bath ultrasonic ripple (80HZ) 2min * 5 broken egg capsules, multigelation 5 times (37 ℃ of liquid nitrogen and water-baths) again, 4 ℃, 10,000rmin ^-1Centrifugal egg capsule treatment fluid 15min, supernatant is used antigen as detecting, and ultraviolet spectrophotometry is surveyed protein concentration, and-20 ℃ of preservations are standby; The precipitation part adds an amount of PBS buffer, with the complete or non-Freund's complete adjuvant emulsifying of Fu Shi of 2 times of volumes, and as immunity antigen, 4 ℃ of preservations of vaccine that emulsifying is good.

6, the described immunity that is meant the chicken group with the female laying fowl of Cryptosporidium baileyi antigen immune, exempt from altogether six times, each 15～20d at interval, the adjuvant mixing of antigen and 2 times of volumes is made oil emulsion, exempt from the Freund's complete adjuvant except that one, all the other all use incomplete Freund, and each 1mL/, antigenic content is respectively one and exempts from 1 * 10 ⁷Individual/only, two exempt from 2 * 10 ⁷Individual/only, three exempt from 3 * 10 ⁷Individual/only, four exempt from 3 * 10 ⁷Individual/only, five exempt from 5 * 10 ⁷Individual/only, six exempt from 5 * 10 ⁷Individual/only, the inoculation mode is under the chicken wing and the intramuscular injection of femoribus internus muscle multiple spot.

7, described collection its produce the fowl ovum, from the fowl ovum, collect egg yolk again, with distilled water diluting, the centrifuging and taking supernatant is meant that getting the immunity back height that produces tires after the clean sterilization of Ovum Gallus domesticus album, breaks eggshell, abandons eggshell, Ovum Gallus domesticus album and yolk clothing film, obtain yolk liquid (about 10mL/ piece) and press the 1:9 dilution with the single water (D.W) that steams of sterilization, fully stirring and evenly mixing is transferred pH value to 5.1～5.4, put 4 ℃, 6h or spend the night, the filter paper filtering supernatant, remove the buoyant yolk lipid of the superiors after, centrifugal (10,000rmin ^-1, 4 ℃ of 10min), abandon precipitation, obtain supernatant;

8, describedly be meant two step of water dilution ammonium sulfate salting-out process,, add 50% saturated ammonium sulfate, stir the pretreatment supernatant through water dilute sulphuric acid ammonium salting out method purification, more than 4 ℃ of effect 2h, centrifugal (10,000rmin ^-1, 4 ℃ of 10min), abandon supernatant, the PBS dissolution precipitation with an amount of volume adds 333% saturated ammonium sulfate, stirs, more than 4 ℃ of effect 2h, centrifugal (10,000rmin ^-1, 4 ℃ of 10min), obtain precipitation and be dissolved among the PBS of ovulum yellow liquor volume, put in the bag filter, the flowing water dialysed overnight, stir dialysis with the PBS buffer more than 1000 times next day, changed liquid once in per about 3 hours, dialysed 3 days, obtain antibody protein, add the two anti-of 1000U/mL ,-20 ℃ of cold preservations.

9, the purposes of the Yolk immunoglobulin product of described control Cryptosporidium baileyi disease is the biological preparation that is used to prevent and treat the protopathy that Cryptosporidium baileyi causes.

10, the purposes of the Yolk immunoglobulin product of described control Cryptosporidium baileyi disease is the health product as raise immunity.

Effect of the present invention and benefit are, the specific IgY of the anti-Cryptosporidium baileyi disease that the application the technical program is prepared directly acts on the pathogen of Cryptosporidium baileyi disease, the performance specific inhibitory effect, the use that overcomes other medicines is for the viral shortcoming that there is no killing action itself, and production cost is also very low.

The specific embodiment

Height is exempted from the preparation method of Cryptosporidium baileyi IgY antibody

One, the preparation of immunizing antigen

1, the amplification of Cryptosporidium baileyi egg capsule and purification are collected egg capsule in the positive feces in herding station, urban district, be long oval, recording the egg capsule size is 6.5 μ m x4.7 μ m (5.4～7.8 μ m x4.1～5.5 μ m) shape indexs 1.31, remove the chicken coccidial oocyst that is mixed with through 3 age in days Carnis Coturnicis japonicae blind passages, set up animal infection modal and the analysis of 18sRNA sequence alignment through this laboratory, after being defined as Cryptosporidium baileyi (C.baileyi), go down to posterity with 3 age in days healthy chicks, Cryptosporidium baileyi egg capsule in a large number increases.

With reference to methods such as Arrowood, improved with sucrose gradient centrifugation purification egg capsule.Sucrose stock solution is joined method: sucrose 500g, and after the distilled water 320mL dissolving, autoclaving is standby; PBS buffer: NaCL8.0g, KCL0.2g, KH ₂PO ₄0.2g, NaHPO ₄12H ₂O2.9g, distilled water 1000mL transfers pH value 7.4 back sterilizations standby.

Egg capsule is purified with the glass centrifuge tube of 10mL diameter 1.2cm, and program is as follows: add 2 parts of sucrose solutions of 1:1 (about 6mL) earlier, slowly add 1 part of egg capsule outstanding (about 3mL), 3000rmin-along glass tube walls again ¹, 10min is with suction pipe sucking-off egg capsule layer, add 2 times of volume PBS liquid centrifuge washings 2 times, precipitation is suspended among an amount of PB liquid S, uses the 1:2 sucrose solution then successively, 1:3 sucrose solution (twice), the same method is centrifugal, collects each egg capsule layer, to suspend behind the PBS liquid centrifuge washing, with 1:3.5 sucrose solution 3mL, be added in the centrifuge tube for the last time, slowly add 1:5 sucrose solution 3mL along tube wall then along tube wall, the superiors add 1:3 sucrose extract egg capsule suspension 3mL, 3000rmin- ¹, 10min reclaims the egg capsule layer.The purity that sample after each centrifugal takes out each layer of microscopic examination sample respectively, PBS liquid dilution back is with 5 μ m filter membrane microfiltration degerming, and blood cell counting plate is counted, and it is that the two of 1000U/mL resist that concentrated back adds final concentration, and 4 ℃ of preservations are standby.

The kind of table 1 sucrose solution and concentration

2, the C.baileyi egg capsule of antigenic preparation after with the purification degerming is suspended in the PBS liquid (10 ⁸Individual/mL), ice-bath ultrasonic ripple (80HZ) 2min * 5 broken egg capsules, multigelation 5 times (37 ℃ of liquid nitrogen and water-baths) again.4 ℃, 10,000rmin- ¹Centrifugal egg capsule treatment fluid 15min, supernatant is used antigen as detecting, and ultraviolet spectrophotometry is surveyed protein concentration, and-20 ℃ of preservations are standby; The precipitation part adds the complete or non-Freund's complete adjuvant emulsifying of Fu Shi of an amount of PBS liquid and 2 times of volumes, as immunity antigen, and the preparation of freund adjuvant reference literature method, 4 ℃ of preservations of vaccine that emulsifying is good are standby.

Two, chicken group immunity

1, different experimental grouies is set, determines immune programme for children

Experimental animal provides 150 ages in days by laying hen field, suburbs, just opens and produces 40 of extra large blue laying hens, is divided into 5 groups, 8 every group.The immune situation of dividing into groups is as follows.

First group: exempt from secondary altogether, one exempts from, and antigenic content is 5 * 10 ⁷Individual egg capsule/2mL.- ¹, Freund's complete adjuvant; Exempt from six all backs two at interval, and antigenic content is 5 * 10 ⁷Individual egg capsule/2mL.- ¹, incomplete Freund, the inoculation mode is under the chicken wing and the intramuscular injection of femoribus internus muscle multiple spot.

Second group: exempt from altogether six times, each 15～20d at interval, antigenic content is respectively one and exempts from 1 * 10 ⁷Individual/only, two exempt from 2 * 10 ⁷Individual/only, three exempt from 3 * 10 ⁷Individual/only, four exempt from 3 * 10 ⁷Individual/only, five exempt from 5 * 10 ⁷Individual/only, six exempt from 5 * 10 ⁷Individual/as only, to exempt from the Freund's complete adjuvant except that one, all the other all use incomplete Freund, and the inoculation mode is the same.

The 3rd group: exempt from altogether four times, each 15～20d at interval whenever exempts from antigenic content and is 1 * 10 ⁷Individual/as only, one to exempt from, two exempt from outside the Freund's complete adjuvant, three exempt from, four exempt to use incomplete Freund, the inoculation mode is the same.

The 4th group: egg capsule counting 1 * 10 ⁷Individual. only- ¹, the egg capsule per os of living is irritated and is fed 165 ages in days sea blue grey laying hen.

The 5th group: with blank vaccine is that (0.01M pH7.4) makes oil emulsion with the adjuvant mixing of 2 times of volumes to PBS solution, and each 2mL/ only, exempt from six times, each 15～20d at interval, one exempts from two exempts from outside the Freund's complete adjuvant, all the other all use incomplete Freund, and the inoculation mode is the same.

2, set up indirect ELISA method, detect antibody horizontal with each vaccinated flock group of monitoring

Set up indirect elisa method and measure serum and the yolk antibody level of respectively organizing the chicken group: operating procedure is carried out on the poly-third ethylene micro plate routinely.Get aforementioned soluble antigen, be diluted to debita spissitudo with coating buffer (0.05M, pH9.6 carbonate buffer solution), every hole 100 μ L, 4 ℃ are spent the night, with PBST (0.01M, pH7.4PBS add 0.05% tween 20, and be as follows) washing 6 times; 1%BSA-PBST seals 37 ℃ of 1h, washing; Adding is with the 1%BSA-PBST suitably serum to be checked or the yolk liquid of dilution, and makes negative control, washing with known negative serum or yolk liquid (collected specimens rather); It is anti-to add the anti-chicken IgG of HRP-rabbit labelling two, hatches after scouring for 37 ℃; With tetramethyl benzidine (TMB) substrate solution, color development at room temperature, 2M sulphuric acid cessation reaction.Enzyme-linked immunosorbent assay instrument is measured light absorption value under the 450nm wavelength, observed result.

Five experimental group serum antibody titers of table 2 relatively

Exempt from serum and the egg that 15d gathers each immune group chicken since one, every interval 15d once sampling is exempted to finish until six, uses serum antibody and yolk antibody level that indirect ELISA method detects each group.By table 2 as seen, exempt from back 15d one for one, two, three group and all can measure low-level antibody, but it is remarkable respectively to organize antibody ascending water adjustment heteropole two after exempting from.Exempt from the back antibody horizontal for first group one and slowly rise, two of 6 weeks exempt from antibody horizontal is significantly improved at interval; The 3rd group of antibody horizontal kept low-level state all the time; The 4th group of oocyst infection group alive and the 5th group of blank immune group all do not detect antibody; Second group is this antibody horizontal experimental group preferably that rises, and the immune programme for children that the gradient of immunizing antigen increases progressively stimulates to produce high-caliber specific antibody, reaches peak region six after exempting from, and can continue about 3 months.Therefore we select second group immune programme for children.Second group serum antibody and the horizontal growth and decline rule of yolk antibody are compared, as seen from Figure 1, serum antibody rises gradually after second group of hen immunity, four exempt from the back increasing degree more remarkable, exempt from back 15d six, the serum antibody level reaches peak region (1:180000～1:213000), continue to descend gradually about 8 weeks.The growth and decline trend of yolk antibody and the basically identical of serum antibody, but its antibody horizontal is apparently higher than the serum antibody level same period, and the two difference is (P＜0.01) extremely significantly.This may be the immunizing antigen immune hen, stimulating in the serum antibody that produces mainly is IgG, a small amount of IgM, IgA are also arranged, indirect ELISA detection method mainly can measure the IgG in the serum, and on the other hand, blood flow is when ovary, because there is a large amount of IgG receptors on the vitellinae membrana surface, make IgG in ovum, accumulate, form concentrating of yolk antibody IgY, thereby cause the indirect ELISA detection of yolk antibody to be tired apparently higher than serum titer.

Four, the collection purification and the mensuration of yolk antibody (IgY)

1, the collection of IgY antibody and pretreatment with acidifying water dilution-ammonium sulfate salting-out process, are collected the IgY antibody in the purification yolk with reference to pertinent literature.The pretreatment of yolk: get the immunity back height that produces and tire after the clean sterilization of Ovum Gallus domesticus album, break eggshell, abandon eggshell, Ovum Gallus domesticus album and yolk clothing film, obtain yolk liquid (about 10mL/ piece) and press the 1:9 dilution with the single water that steams of sterilization, abundant stirring and evenly mixing, transfer pH value to 5.1～5.4, put 4 ℃, 6h or spend the night, the filter paper filtering supernatant, after removing the buoyant yolk lipid of the superiors, centrifugal (10,000rmin- ¹, 4 ℃ of 10min), abandon precipitation, obtain supernatant.

2, the above-mentioned pretreatment supernatant of the purification of IgY antibody adds the saturated ammonium sulfate of 50% saturation, stirs, and more than 4 ℃ of effect 2h, centrifugal (10,000rmin- ¹, 4 ℃ of 10min), abandon supernatant, with the PBS dissolution precipitation of an amount of volume, add the saturated ammonium sulfate of 33.3% saturation, the same centrifugal, obtain the PBS liquid that precipitation is dissolved in ovulum yellow liquor volume (0.01M, pH7.4) in, put in the bag filter that conventional treatment crosses and dialyse, the flowing water dialysed overnight, next day, (0.01M pH7.4) stirred dialysis with the PBS liquid more than 1000 times, change liquid once about 3 hours, dialysed 3 days, obtain antibody protein, add the two anti-of 1000U/mL ,-20 ℃ of cold preservations are standby.

3, the purity testing of IgY antibody adopts the SDS-PAGE method to carry out, and reference literature is prepared 8% separation gel, 5% respectively and concentrated glue, the electrophresis apparatus of packing into, concentrate glue voltage 80V, after the dyestuff forward position enters separation gel, voltage is brought up to 140V, powered-down when the dyestuff forward position arrives apart from separation gel bottom 1cm, put fixative, examine in the bright blue R-250 dyeing liquor of Ma Shi and dye, room temperature 4 hours or spend the night, go in the destaining solution and decolour, transparent to the gel back of the body end, protein band is clear, and sentence read result is also taken pictures.

4, the protein content determination of IgY antibody adopts UV spectrophotometer measuring to carry the protein content of IgY antibody, calculates the response rate of purifying protein.

5, result

With the IgY antibody in the acidifying water dilution-ammonium sulfate salting-out process collection purification yolk.Analyze the purity that antibody purification was reached with SDS-PAGE, present clear protein band more than 11 when not purifying, the IgY antibody behind the purification mainly contains 2 protein bands, i.e. the heavy chain of 60～67KD, the light chain of 31～40KD.The IgY antibody that this experiment obtains is 9.44mg/mL yolk liquid through the rough content of UV spectrophotometer measuring albumen, illustrates that the method has good purification recovering effect.

Four, the specific assay of yolk antibody (IgY)

1, the specificity of determination of immunofluorescence method IgY

The Immunofluorescence Reactions program:

1) the Cryptosporidium baileyi egg capsule suspension after will purifying drips in the reacting hole on microscope slide, and every hole 7～10 μ l do not overflow for good to fill it up with, and are air-dry.Put in the methanol in 4 ℃ of fixing 10min, take out air-dry, with PBS liquid (0.01M, pH7.4) soaking and washing is 3 times, each 3min uses the distilled water immersion desalination at last, air-dry back is as the fluorescent antibody staining specimen,

Positive IgY that 2) will purify and negative IgY7～10 μ L drip respectively in the antigen specimen hole, put in the wet box in 37 ℃ of effect 60min.With PBS rinsing 3 times, each 5min, vibration is air-dry for the last time gently.

3) add suitable dilution FITC labelling goat-anti chicken IgY, put in the wet box in 37 ℃ of effect 60min.

4) with PBS rinsing 3 times, each 5min, distilled water rinsing desalination 5min is used in vibration at last gently, and is air-dry, observes, takes a picture down in fluorescence microscope.

Immunofluorescence Reactions the results are shown in Figure 3, and positive IgY can discern the C.baileyi egg capsule, present the fluorescence reaction of diffusivity viride nitens, and negative IgY can not discern the C.baileyi egg capsule, illustrates that the IgY antibody of this acquisition has anti-C.bailey specificity.

2, indirect elisa method is measured the specificity of IgY

The anti-C.baileyi yolk of positive chicken liquid is done 1:100～1:1600 dilution, each dilution factor adds the dilution C.baileyi soluble antigen of equivalent, 37 ℃ hatch the blocking-up 30min after, carrying out ELISA again measures, establish corresponding contrast simultaneously, the results are shown in Figure 4, the C.baileyi soluble antigen is when carrying out blocking test with the anti-C.baileyi yolk of positive chicken liquid, each dilution positive yolk antibody is tired and is obviously reduced, and its OD value and negative control are approaching.

Artificial challenge's experimental animal therapeutic test of anti-Cryptosporidium baileyi specific IgY antibody

Cryptosporidiosis (Cryptosporidiosis) is that a kind of global people beast suffers from parasitic disease altogether.Cause livestock and poultry diarrhea and respiratory symptom, and the mankind, to immunologic hypofunction or handicapped, especially infant and AIDS patient cause fatefulue diarrhoea, even threat to life.Cryptosporidiosis has been listed in and has caused one of human modal 6 kinds of diarrhea diseases.2003, this disease was listed in one of two important parasite diseases of China's palpus guard key.In recent years, the research of cryptosporidiosis becomes a new focus.At present, both at home and abroad, kind of medicine is used for the treatment of the infection of cryptosporidiosis surplus in the of existing 120 in laboratory and clinical trial, and generally acknowledged specific drug and Therapeutic Method are not arranged so far as yet, and screening and development more and more cause people's attention.Anti-Cryptosporidium baileyi (C.baileyi) the specific IgY antibody that this research will prepare, animal artificial challenge therapeutic test experimentizes.

One, materials and methods

1, preparation before the test

1) Sheather sucrose stock solution: sucrose 500g, after the distilled water 320mL dissolving, autoclaving is standby.

2) PBS buffer: NaCL8.0g, KCL g, KH ₂PO ₄0.2g, NaHPO ₄12H ₂O2.9g, distilled water is settled to 1000mL, transfers pH value 7.2～7.4 back sterilizations standby.

3) peacock green dyeing liquor: peacock green dyestuff 1.6g, dodecyl sodium sulfate 1.0g, distilled water 100mL dissolves mixing.

4) anti-Cryptosporidium baileyi (C.baileyi) specific IgY Antibody Preparation is aforementioned.

5) extraction of negative control IgY and specific IgY, purification are aforementioned.Adopt the acidifying water dilution slightly to carry IgY, then respectively with the saturated ammonium sulfate solution of the 50% and 33% saturation purification of saltouing, and with the protein concentration of the ultraviolet spectrophotometer survey IgY antibody of carrying.

6) the IgY activity mensuration of tiring adopts indirect elisa method to measure, and is aforementioned.

7) the Cryptosporidium egg capsule is preserved the gu-shi chicken source Cryptosporidium baileyi egg capsule that goes down to posterity for this laboratory.Be made into the 1:2 sucrose solution with Sheather sucrose stock solution and PBS buffer and slightly extract, blood cell counting plate counting is suspended in the PBS buffer, adds two anti-1000IU/mL, and it is standby to be stored in 4 ℃ of refrigerators.

8) experimental animal provides 120 of 0 age in days Luo Man cockerels by suburbs hatching factory, and isolated rearing to 3 age in days after the excrement inspection parasite egg feminine gender, selects healthy active person to be used for test.

Two, test method

1, test grouping

Divide five tests, five groups of test chickling sub-cage rearings guarantee no cross-contamination between each group in no Cryptosporidium environment, the full price of normally feeding chicken feed, and 0.2% double play king freely drinks water.

First test group: infect matched group, infect C.baileyi egg capsule 3 * 10 through crop ⁶Individual ^-1, feedstuff drinking-water is normally fed.

Second test group: negative IgY treatment group, infect C.baileyi egg capsule 3 * 10 through crop ⁶Individual ^-1, infect back 11d, beginning is treated with negative IgY antibody, irritates every day and only feeds amount 2000 μ g/, uses 10d continuously, and feedstuff drinking-water is normally fed.

The 3rd test group: the heavy dose of treatment group of positive IgY, infect C.baileyi egg capsule 3 * 10 through crop ⁶Individual ^-1, infect back 11d, beginning is irritated and is only fed amount 2000 μ g/ with positive IgY Antybody therapy every day, uses 10d continuously, and feedstuff drinking-water is normally fed.

The 4th test group: dosage treatment group among the positive IgY, infect C.baileyi egg capsule 3 * 10 through crop ⁶Individual ^-1, infect back 11d, with the IgY Antybody therapy, irritate every day and only feed amount 1000 μ g/, use 10d continuously, feedstuff drinking-water is normally fed.

The 5th test group: the low dose of treatment group of positive IgY, infect C.baileyi egg capsule 3 * 10 through crop ⁶Individual ^-1, infect back 11d, with the IgY Antybody therapy, irritate every day and only feed amount 500 μ g/, use 10d continuously, feedstuff drinking-water is normally fed.

2, observation index

1) respectively organizes the Changing Pattern that the chicken group discharges egg capsule

Behind 24h behind the artificial challenge, every interval 1d collects fresh excreta, collect egg capsule and peacock green dyeing blood cell counting count board inspection counting with saturated sucrose water floating method, observe not ovulation rule on the same group, comprise latent period, peak period, duration, egg capsule intensity, and calculate the egg capsule number (OPG) of every gram feces.

1. saturated sucrose water floating method is collected egg capsule: collecting weighs respectively organizes feces 5g, adds the 50mL tap water and soaks, and Glass rod stirs, and removes the human excrement slag with 80 order screens, and filtrate is with 3000rmin ^-1Centrifugal 10min, supernatant discarded adds saturated sucrose solution in the precipitation of remainder, stir evenly 3000rmin ^-1Centrifugal 5min dips in the iron wire ring then and gets the top layer flotation fluid 6～8 times, places clear water, and so after the repetitive operation 2 times, centrifugal collection egg capsule is settled to 5mL with the PBS buffer.

2. peacock green dyeing blood cell counting count board is counted egg sac number: draw 0.2mL egg capsule suspension and 0.8mL peacock green dye liquor respectively with pipet, put abundant mixing in the 1mL delivery line, after placing 5min, inject hematimeter, counting is calculated as follows: egg capsule number (OPG)=[A/4 * 5 * 5 * 10 in every gram feces under 40 * 10 optical microscopes ⁴]/M (A is the egg capsule sum, and M is a stool weight).

2, the observation of clinical symptoms

Observe in the process of the test and write down and respectively organize clinical symptoms (appetite, drink desire, feces, the mental status and respiratory symptom), observe with following standard and judge: (-): no clinical unusual.(+): spirit is general, and appetite is not good enough, but does not have symptom of diarrhea.(++): spirit is relatively poor, and appetite is low, and the laxativeness symptom is arranged.(+++): spirit is depressed, no appetite, and moderate diarrhoea and respiratory symptom are arranged.(#): spirit is extremely depressed, and it is useless exhausted that appetite is tending towards, and the diarrhoea and the dyspnea of severe are arranged.

3, to the influence of weight gain

Before infecting and during off-test, each test group chicken is weighed, calculate average weight gain value of each group, to weigh in the therapeutic process influence weight gain.

Computing formula: counterpoise during average weight when each organizes average weight gain=off-test-on-test.

Three, result and analysis

1, each group ovulation capsule rule observed result

Duration of test, 5 test group latent periods are 3d.Infect matched group (first group) and appear at infection back the 11st～17d with negative IgY treatment group (second group) ovulation capsule peak period, two groups of groups do not have obvious poor (see figure 1) at latent period, peak period, duration, ovulation capsule intensity basically identical.Three various dose groups of negative IgY treatment group and positive IgY treatment compare (see figure 2), discovery 3 treatment groups on the ovulation capsule peak of 11d during the initial stage, 2d after crop gavages positive IgY antibody preparation, egg capsule significantly lasting decline of output, and descend fast more with the increase of therapeutic dose: the 3rd group of chicken group OPG value decline 67% (OPG; 31 * 10 ⁴Individual/g feces), ovulation capsule turn out cloudy (OPG:0) behind the 9d; The 4th group of chicken group OPG value decline 58% (OPG:40 * 10 ⁴Individual/g feces), the ovulation capsule is turned out cloudy behind the 13d; The 5th group of chicken group OPG value decline 41% (OPG:56 * 10 ⁴Individual/g feces), the ovulation capsule is turned out cloudy behind the 15d.

Three experimental grouies of positive IgY treatment are compared (see figure 2) with feminine gender treatment matched group, and the OPG value has descended 55～79%, and egg capsule is turned out cloudy and shifted to an earlier date 5～11d.On different therapeutic doses, heavy dose of treatment group (the 3rd group: 2000 μ g/ are day only) and middle dosage treatment group (the 4th group: 1000 μ g/ days) are relatively, the OPG value descends 36%, excrement ovulation capsule is turned out cloudy and has been shifted to an earlier date 3d, compare with low dose treatment group (the 5th group: 500 μ g/ are day only), the OPG value descends 53%, and excrement ovulation capsule is turned out cloudy and shifted to an earlier date 7d.Analysis result shows that the positive IgY of anti-C.baileyi specificity shows good curative effect in laboratory animal artificial challenge therapeutic test, can significantly reduce chicken group OPG value, and the size of IgY antibody dosage is directly proportional with therapeutic effect.

2, the observed result of clinical symptoms

Five 7ds (first OPG peak value) of test group after infecting the C.baileyi egg capsule, each is organized the part chicken and slight dysentery symptom occurs, and spirit is poor slightly; Infect back 11d, three, the 4th and the 5th group of anti-C.baileyi specific IgY antibody with various dose is treated, doing contrast synchronously with negative IgY antibody for second group treats, observe each group clinically and begin to occur respiratory symptom, indivedual crows are hoarse, spirit is relatively poor, and appetite is low, and symptom of diarrhea is arranged; 13d observes first, second group clinically and begins to occur significant respiratory symptom, diarrhoea, and indivedual chickens stretch the neck hello sound of stridulating, cough, spirit, inappetence, three, the 4th and the 5th group rarely seen search for food, drink water not good enough, the laxativeness symptom, the 5th group than third and fourth the group spirit poor slightly; Infect back 19d, first, second group respiratory symptom remission, but lassitude, appetite is not good enough, the laxativeness symptom, the the 3rd, the 4th and the 5th group is not had clinical visible symptom, drink water, search for food normal (seeing Table 1).

Table 1 test group clinicing symptom observation

3, to the influence of weight gain

The situation of growth-weight gain before and after each group of observed and recorded infects.Body weight change saw Table 2 before and after each group infected.

Table 2 is respectively organized chicken and is infected front and back average weight situation of change table

Experimental result shows that chickling infects Cryptosporidium baileyi and influences growth-weight gain.Use anti-C.baileyi specific IgY treatment Cryptosporidium condition of disease and reduce the OPG value, alleviate clinical symptoms, improve the conversion ratio of feedstuff, analyzing reason may be that specific IgY may exist some mechanism that stops the Cryptosporidium zygoblast to pierce enterocyte, reduce polypide and colonize in the interior worm lotus number of intestinal epithelial cell, recover the enteric epithelium mucosa and absorb the nutrient function, positive IgY treatment group chicken weightening finish is higher than negative treatment group.

Recent two decades comes, and for the cryptosporidiosis control, successively has anticoccidial drug, antiprotozoal drug, vermifuge, broad ectrum antibiotic and sulfonamides to be used for the treatment of cryptosporidiosis, all can not effectively control the infection of Cryptosporidium.Both at home and abroad researcher is also used some biological preparation such as height and is exempted from colostrum, hyper-immune serum, anti-Cryptosporidium monoclonal antibody specific (McAb), cattle transfer factor, cleavage of peptide and interleukin or the like and carry out immunization therapy and obtain some curative effects, but There are many different versions of a story to its curative effect.Domestic researcher uses some Chinese herbal and crude drugs preparationses (garlicin preparation, Radix Sophorae Flavescentis mixture etc.) treatment cryptosporidiosis to obtain certain curative effect, but because of lacking strict clinical control group, can not be certain to its curative effect.

This research is carried out the cryptosporidiosis therapeutic test with the specific yolk antibody of anti-Cryptosporidium, infect the peak initial stage of Cryptosporidium baileyi ovulation capsule chickling and carry out oral medication, three experimental grouies of positive IgY treatment are compared with feminine gender treatment matched group, the OPG value has descended 55～79%, and the ovulation capsule has shifted to an earlier date 5～11d and turned out cloudy; Matched group begins to occur significant digestive tract and respiratory symptom (diarrhoea, indivedual chickens are stretched the neck hello sound of stridulating, cough, spirit, inappetence etc.) on the clinical symptoms, and three treatment groups do not have obviously visible clinical symptoms substantially, drink water, search for food normally; To the influence of weight gain, chickling infects Cryptosporidium baileyi and influences growth-weight gain, and treatment group chicken group mean increases weight apparently higher than matched group, and bibliographical information (Bao Jiaming etc., 2001 are arranged; Chen Zhaoguo etc., 1995) infect cryptosporidiosis with garlicin treatment chicken, the egg capsule discharge capacity is slightly reduced, but it is relatively poor to increase weight, clinical symptom remission is not obvious, no obvious treatment effect, analyzing reason may be that the garlicin abnormal smells from the patient is heavier, influences searching for food of chicken.And yolk antibody preparation is treated and is not had these problems, and not only mouthfeel is good for it, and not having bad abnormal smells from the patient stimulates, and in enhancing immunity, also has special at curative effect.

At present, research to yolk antibody deepens continuously, the IgY technology has become the new focus in biological technical field and the medical research, and yolk antibody (IgY) is a kind of immunoglobulin that contains in the birds egg yolk, and IgY antibody chemical property is stable, output height, cost are low, and the animal kind is the advantage of distance, is more suitable for being used for the production specific antibody, is developed as " the green medicine " of treatment disease, functional food, the health product of raise immunity.

Description of drawings

Fig. 1, second group of serum antibody and yolk antibody growth and decline rule;

Among Fig. 2, the figure:

M: low-molecular-weight standard protein

A: back chicken IgY purifies

B: the IgG of chicken

C: the IgY of Ti Chuning not

(8% separation gel 5% concentrates glue)

IgY purifying antibody effect

Fig. 3, indirect immunofluorescence reaction pair IgY antibody specificity are measured (10 * 40) negative IgY antibody;

Fig. 4, indirect immunofluorescence reaction pair IgY antibody specificity are measured (10 * 40) positive IgY antibody;

The blocking test ELISA of Fig. 5, IgY measures OD value curve chart;

Fig. 6, infection matched group and negative IgY treatment group are relatively;

Fig. 7, negative IgY treatment group and positive IgY treatment group are relatively.

Claims (10)

1. Yolk immunoglobulin product of preventing and treating the Cryptosporidium baileyi disease, this product can obtain from following steps, at first from the positive feces of Cryptosporidium baileyi, collect egg capsule and separate back sucrose gradient centrifugation purification egg capsule, and be prepared into Cryptosporidium baileyi antigen, with the female laying fowl of Cryptosporidium baileyi antigen immune, collect it then and produce the fowl ovum, from the fowl ovum, collect egg yolk again, with distilled water diluting, the centrifuging and taking supernatant obtains Yolk immunoglobulin through water dilute sulphuric acid ammonium salting out method purification.

2. the Yolk immunoglobulin product of control Cryptosporidium baileyi disease according to claim 1, it is characterized in that: from the positive feces of Cryptosporidium baileyi, collect egg capsule and be meant the just middle egg capsule of the positive animal manure of collection, egg capsule is long oval, recording the egg capsule size is 6.5 μ mx4.7 μ m shape indexs 1.31, removes the chicken coccidial oocyst that is mixed with through 3 age in days Carnis Coturnicis japonicae blind passages.

3. the Yolk immunoglobulin product of control Cryptosporidium baileyi disease according to claim 2, it is characterized in that: separate being meant with saturated sucrose solution floating method collection egg capsule, the sucrose solution of 27%W/V is slightly carried egg capsule, with the amplification of going down to posterity of 3 Japanese instar chicklings, set up animal infection modal and the analysis of 18sRNA sequence alignment, be defined as Cryptosporidium baileyi.

4. according to the Yolk immunoglobulin product of claim 1 or 3 described control Cryptosporidium baileyi diseases, it is characterized in that: be meant with sucrose gradient centrifugation purification egg capsule, 1. prepare sucrose stock solution: sucrose 500g, after the distilled water 320mL dissolving, autoclaving; 2. prepare the PBS buffer: NaCl 8.0g, KCl 0 .2g, KH ₂PO ₄0.2g, NaHPO ₄12H ₂O 2.9g, distilled water 1000mL transfers pH value 7.4 back sterilizations; 3. will be 1. sucrose stock solution and 2. PBS buffer be formulated as a, 1 part of sucrose stock solution and add after 1 portion of PBS buffer sucrose concentration W/W% and 30.4 be called 1: 1 sucrose water; B, 1 part of sucrose stock solution add after 2 portions of PBS buffer sucrose concentration W/W% and 25 are called 1: 2 sucrose water; C, 1 part of sucrose stock solution add after 3 portions of PBS buffer sucrose concentration W/W% and 18.7 are called 1: 3 sucrose water; D, 2 parts of sucrose stock solutions add after 7 portions of PBS buffer sucrose concentration W/W% and 16.8 are called 1: 3.5 sucrose water; E, 1 part of sucrose stock solution add after 5 portions of PBS buffer sucrose concentration W/W% and 12.5 are called 1: 5 sucrose water; 4. egg capsule is purified with the glass centrifuge tube of 10mL diameter 1.2cm, and program is as follows: a, add 2 parts in 1: 1 sucrose water earlier, slowly add 1 part of egg capsule suspension, 3000rmin along glass tube walls again ^-1, 10min with suction pipe sucking-off egg capsule layer, adds 2 times of volume PBS buffer centrifuge washings 2 times, and precipitation is suspended in an amount of PBS buffer; B, add 2 parts in 1: 2 sucrose water, slowly add 1 part of a step precipitation along glass tube walls again and be suspended in egg capsule suspension in an amount of PBS buffer, 3000rmin ^-1, 10min with suction pipe sucking-off egg capsule layer suspension, adds 2 times of volume PBS buffer centrifuge washings 2 times, and precipitation is suspended in an amount of PBS buffer; C, add 2 parts in 1: 3 sucrose water, slowly add 1 part of b step precipitation along glass tube walls again and be suspended in egg capsule suspension in an amount of PBS buffer, 3000rmin ^-1, 10min with suction pipe sucking-off egg capsule layer suspension, adds 2 times of volume PBS buffer centrifuge washings 2 times, and precipitation is suspended in an amount of PBS buffer, repeats once again; D, add 1 part in 1: 3.5 sucrose water, be added in the centrifuge tube along tube wall, slowly add 1 part of 1: 5 sucrose liquid along tube wall then, the superiors add 1 part of 1: 3 sucrose extract egg capsule suspension, 3000rmin ^-1, 10min reclaims egg capsule layer suspension; 5. use PBS buffer dilution back with 5 μ m filter membrane microfiltration degerming, concentrating back adding final concentration is the two anti-of 1000U/mL, 4 ℃ of preservations.

5. the Yolk immunoglobulin product of control Cryptosporidium baileyi disease according to claim 4 is characterized in that: be prepared into Cryptosporidium baileyi antigen and be meant the Cryptosporidium baileyi egg capsule after the purification degerming is suspended in the PBS buffer 10 ⁸/ mL, ice-bath ultrasonic ripple 80HZ 2min * 5 broken egg capsules, 5 liquid nitrogen of multigelation and water-bath are 37 ℃ again, and 4 ℃, 10,000rmin ^-1Centrifugal egg capsule treatment fluid 15min, supernatant is used antigen as detecting, and ultraviolet spectrophotometry is surveyed protein concentration, and-20 ℃ of preservations are standby; The precipitation part adds an amount of PBS buffer, with the complete or non-Freund's complete adjuvant emulsifying of Fu Shi of 2 times of volumes, and as immunity antigen, 4 ℃ of preservations of vaccine that emulsifying is good.

6. the Yolk immunoglobulin product of control Cryptosporidium baileyi disease according to claim 5, it is characterized in that: be meant chicken group's immunity with the female laying fowl of Cryptosporidium baileyi antigen immune, immunity is six times altogether, each 15～20d at interval, the adjuvant mixing of antigen and 2 times of volumes is made oil emulsion, exempts from the Freund's complete adjuvant except that one, and all the other all use incomplete Freund, each 2ml/, antigenic content is respectively one and exempts from 1 * 10 ⁷Individual/only, two exempt from 2 * 10 ⁷Individual/only, three exempt from 3 * 10 ⁷Individual/only, four exempt from 3 * 10 ⁷Individual/only, five exempt from 5 * 10 ⁷Individual/only, six exempt from 5 * 10 ⁷Individual/only, the inoculation mode is under the chicken wing and the intramuscular injection of femoribus internus muscle multiple spot.

7. the Yolk immunoglobulin product of control Cryptosporidium baileyi disease according to claim 6, it is characterized in that: collect it and produce the fowl ovum, from the fowl ovum, collect egg yolk again, with distilled water diluting, the centrifuging and taking supernatant is meant that getting the immunity back height that produces tires after the clean sterilization of Ovum Gallus domesticus album, break eggshell, abandon eggshell, Ovum Gallus domesticus album and yolk clothing film obtain the single water D.W that steams of 10mL/ piece of yolk liquid and sterilization and diluted by 1: 9, abundant stirring and evenly mixing, transfer pH value to 5.1～5.4, put 4 ℃, 6h or spend the night, the filter paper filtering supernatant, after removing the buoyant yolk lipid of the superiors, centrifugal 10,000rmin- ¹, 4 ℃ of 10min abandon precipitation, obtain supernatant.

8. the Yolk immunoglobulin product of control Cryptosporidium baileyi disease according to claim 7, it is characterized in that: be meant two step of water dilution ammonium sulfate salting-out process through water dilute sulphuric acid ammonium salting out method purification, with the pretreatment supernatant, the saturated ammonium sulfate that adds 50% saturation, stir, more than 4 ℃ of effect 2h, centrifugal 10,000rmin ^-1, 4 ℃ of 10min abandon supernatant, with the PBS dissolution precipitation of an amount of volume, add the saturated ammonium sulfate of 33.3% saturation, the same centrifugal, obtain precipitation and be dissolved in the PBS liquid 0.01M of ovulum yellow liquor volume, among the pH7.4, put in the bag filter that conventional treatment crosses and dialyse, the flowing water dialysed overnight, next day, pH 7.4 stirred dialysis with 1: 1000 above PBS liquid 0.01M, change liquid once about 3 hours, dialysed 3 days, obtain antibody protein, add the two anti-of 1000U/mL ,-20 ℃ of cold preservations.

9. the purposes of the Yolk immunoglobulin product of control Cryptosporidium baileyi disease according to claim 8 is characterized in that: the biological preparation that is used to prepare the protopathy that the control Cryptosporidium baileyi causes.

10. the purposes of the Yolk immunoglobulin product of control Cryptosporidium baileyi disease according to claim 8 is characterized in that: the health product that are used to prepare raise immunity.

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