CN104258376A - Application of soluble DHV-I (duck hepatitis virus) 3D protein in preparation of immunopotentiator of DHV-I vaccine - Google Patents

Abstract

The invention discloses an application of a soluble DHV-I (duck hepatitis virus) 3D protein in preparation of an immunopotentiator of a DHV-I vaccine. After the soluble DHV-I 3D protein is independently used for immunizing a duck or is used for immunizing the duck together with the DHV-I vaccine, the cellular immunity and the body fluid immunity of the immunized duck are enhanced, the lesion rate and the lethality rate of the immunized duck after the immunized duck is infected are reduced, and the application has great significance for the immunoprophylaxis of the I-type duck virus hepatitis.

Description

The application of soluble Type I DHV 3D albumen in the immunostimulant of preparation I type duck virus hepatitis vaccine

Technical field

The invention belongs to biochemical field, be specifically related to the application of soluble Type I DHV 3D albumen in the immunostimulant of preparation I type duck virus hepatitis vaccine.

Background technology

Duck viral hepatitis (DVH) is the duckling infectious disease caused by DHV (DHV), with liver hemorrhagic inflammation for feature, shows as acute, high degree in contact and lethal, reaches more than 90% at the mortality rate of new epidemic area.Clinical symptoms main manifestations is opisthotonus (nervous symptoms), liver enlargement see ecchymosis not of uniform size.DHV belongs to Picornaviridae (Picornaviridae) fowl hepatitis virus and belongs to (Avihepatovirus), this virus has three serotypes, be respectively DHAV-1, DHAV-2 and DHAV-3, without immunological cross-reaction or immunological cross-reaction weak.In China, Major Epidemic 1 type duck viral hepatitis.

At present mainly vaccination is relied on to the control of DHV.Although these vaccines serve great effect in control duck viral hepatitis, yet there is many aspects needing to be improved and improve, be therefore necessary the frontier opening up vaccine research and prevention and control.Wherein namely the research of immunostimulant be one of important directions.

The material of so-called immunostimulant to be a class by non-specific approach the improve former or microorganism specific reaction of body fight.From nineteen twenty-five France immunologist hold concurrently veterinary Gaston Ramon find to add in vaccine some material had no truck with can specifically enhancing body to since the opposing reaction of diphtheria and tetanus toxin, in medical science and veterinary field, many countries have carried out the research of this respect all to some extent.Especially in technique for gene engineering develop rapidly, today that recombinant vaccine is continually developed and living standard improves day by day, one side recombinant vaccine needs to add adjuvant and improves its protective rate; Public health consciousness strengthens day by day on the other hand, and the effect of immunostimulant in medical treatment, health care also more and more comes into one's own, and thus immunostimulant becomes the focus of ImmunoL Today research again.

Immunostimulant can separately or use with antigen simultaneously, can enhancing human body immunity response material, by specificity and the nonspecific immune response of different model of action enhancing body.At present, immunostimulant of a great variety, mainly contains 5 classes: microbe-derived medicine (as bacillus calmette-guerin vaccine etc.), biotic factor class medicine (as thymosin, IFN-α etc.), synthetic class medicine (as levamisole), microelement kind (as selenium, zinc etc.), natural class medicine (as astragalus polysaccharides etc.).There are five standards in World Health Organization (WHO) (WTO) to immunostimulant: (1) specific chemical components; (2) degraded is easy to; (3) stimulation is moderate; (4) without carcinogenic and mutagenic action; (5) avirulence and untoward reaction.

The special immunostimulant for duck at present, the immunostimulant report particularly for I type duck liver inflammation is less.

Summary of the invention

In view of this, an object of the present invention is to provide the application of soluble Type I DHV 3D albumen in the immunostimulant preparing I type duck virus hepatitis vaccine; Two of object of the present invention is to provide the application of soluble Type I DHV 3D albumen in preparation prevention I type duck viral hepatitis immune formulation.

For achieving the above object, the invention provides following technical scheme:

1, the application of soluble Type I DHV 3D albumen in the immunostimulant of preparation I type duck viral hepatitis.

Preferably, described immunostimulant is cellular immunization reinforcing agent or humoral immunization reinforcing agent.

2, the application of soluble Type I DHV 3D albumen in preparation prevention I type duck viral hepatitis immune formulation.

In the present invention, solubility 3D albumen is prepared by the following method: comprise the steps: that 9th ~ 1376 nucleotide by sequence shown in SEQ ID NO.3 are connected to the polyclone enzyme action site of pET-32 (a)+carrier, then with escherichia coli Rosetta, BL21 or BL21 (DE3) PLYS for Host Strains, in the LB culture medium of Amp resistance after activation, IPTG final concentration be 0.2 ~ 1.0mmol/L, temperature be 20 ~ 39 DEG C of conditions under abduction delivering 4 ~ 12 hours.Wherein Host Strains is preferably e. coli bl21 (DE3) PLYS; The optimum condition of abduction delivering is IPTG final concentration is 0.8mmol/L, temperature is abduction delivering 6 hours under 25 DEG C of conditions; The concrete steps of activation are: be inoculated in by expression strain in the LB culture medium of Amp resistance, 37 DEG C, incubated overnight under 120r/min condition, are then that 1:100 amplification culture is to OD by volume by the LB culture medium of culture fluid and Amp resistance ₆₀₀be 0.6.

Also purification step is comprised after expression, specific as follows: to collect bacterium liquid, thalline is collected after centrifugal 10min under 12000r/min condition, thalline 20mmol/L, pH of collecting are the Tris-HCl suspension of 8.0, then ultrasonic under ice bath, by the thalline after fragmentation at 4 DEG C, centrifugal 10min under 12000r/min condition, collect supernatant, supernatant Ni ²⁺-NTA agarose gel column purification, dialysis, obtains the soluble Type I DHV 3D albumen of purification with the ultrafiltration through membranes of 0.45 μm.

Under ice bath, ultrasonic optimum condition is ultrasonication 6 times under condition of ice bath, 30sec/ time, interval 30sec between each.

Beneficial effect of the present invention is: the application of soluble Type I DHV 3D albumen in preparation I type duck virus hepatitis vaccine immunostimulant, by soluble Type I DHV 3D albumen separately immunity or with I type duck virus hepatitis vaccine altogether immune duck after can increase CD8+ T lymphocyte quantity, and improve IL-2, the content of the cytokines such as IL-4 and IFN-γ, show cellular immunization and the humoral immunity that can strengthen immune duck, simultaneously can also draw the pathological changes after can also reducing immune duck counteracting toxic substances after immune soluble Type I DHV 3D albumen and fatality rate through protest test and dissection and analysis, lay a good foundation for immunoprophylaxis I type DHV infects.

Accompanying drawing explanation

In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing:

Fig. 1 is the 3D gene PCR amplified production electrophoresis result (M:DL 2000Marker, 1:3D gene PCR amplified production) of I type DHV strain DHAV-H.

Fig. 2 is PCR qualification and enzyme action qualification result (qualification of A:pJET-1.2+/DHAV-H-3D plasmid PCR, M:DL2000Marker, 1:PCR product in the 3D genophore building process of I type DHV strain DHAV-H; B:pJET-1.2+/DHAV-H-3D plasmid enzyme restriction is identified, M:DL15000Marker, 1:Kpn I/Xho I double digestion band, 2:Xho I single endonuclease digestion band; The PCR qualification of C:pET-32 (a) +/DHAV-H-3D plasmid, M:DL2000Marker, 1:PCR product; The enzyme action qualification of D:pET32a+/DHAV-H-3D plasmid, 1:Kpn I/Xho I double digestion result, 2:Xho I single endonuclease digestion band).

Fig. 3 is that (M is low molecular weight protein standard substance for the optimization of solubility 3D protein expression condition; A: the screening expressing bacterium, 1 is pET-32 (a)+empty carrier expression product, and 2-4 is respectively the expression product of pET-32 (a) +/different expressive host bacterium of DHAV-H-3D recombinant plasmid transformed Rosetta, BL21 and BL21 (DE3) PLYS tri-kinds; B: the optimization of abduction delivering time, 1-5 are followed successively by BL21 (DE3) the PLYS Host Strains expression product of induction 12h, 10h, 8h, 6h and 4h; C: the optimization of abduction delivering temperature, 1-6 are respectively BL21 (DE3) the PLYS Host Strains expression product of 39 DEG C, 37 DEG C, 35 DEG C, 30 DEG C, 25 DEG C and 20 DEG C; D:IPTG concentration optimization, 1-5 is respectively the expression product of the IPTG induction of 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L and 1.0mmol/L.

Fig. 4 be solubility 3D albumen Western-blot detect and purifying protein electrophoresis (A is the immune-blotting method result of 3D albumen, and M is protein Marker, and 1 is 3D Western blot.B is that SDS-PAGE detects the 3D albumen of purification, and M is low molecular weight protein Marker, and 1 is the 3D albumen of purification).

Fig. 5 is duck and the liver (A: the significant opisthotonus posture of the dead duck of counteracting toxic substances of counteracting toxic substances death; B: the dirty characteristic of the dead duck liver of counteracting toxic substances is hemorrhage).

Detailed description of the invention

Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, usually conveniently condition, the such as condition described in Molecular Cloning: A Laboratory guide (third edition, the work such as J. Pehanorm Brooker), or according to the condition that manufacturer advises.

The 3D gene of embodiment 1, clone I type DHV strain DHAV-H

I type DHV strain H (DHAV-H) (GenBank:JQ301467), E.coli DH5 α strain and pET-32 (a)+carrier are preserved by Sichuan Agricultural University's poultry disease prevention and control research center and provide.Various molecular biology reagents is purchased from biological reagent company.

According to the primer of genome sequence (GenBank:JQ301467) the design amplification 3D gene of DHAV-H, concrete primer is as follows:

P1:5 '-gg ggtaccgatcaagggaaagtagtgagcaag-3 ' (SEQ ID NO.1), underscore represents Kpn I restriction enzyme site;

P2:5 '-acgc ctcgagtcagatcatcatgcaagctgt-3 ' (SEQ ID NO.2), underscore represents Xho I restriction enzyme site; Then the primer of design is synthesized by precious biological engineering (Dalian) company limited.

The DHAV-H virus liquid deposited of going bail for does 5 times of dilutions with sterilizing PBS, add after dual anti-(final concentration of penicillin, streptomycin is respectively 100IU/mL and 100 μ g/mL) of 1/100 volume in 37 DEG C of incubation 1h, then inoculate 9 ages in days to physically well develop, without the duck embryo of maternal antibody, discard dead embryo in 24h, collect allantoic fluid and the idiosome of the dead embryo of 24 ~ 72h, extract viral RNA according to Trizol test kit description.

By extract reverse transcription of viral RNA synthesis cDNA, be then that template carries out pcr amplification with cDNA, reverse transcription and PCR amplification system as shown in table 1.

Table 1 reverse transcription and PCR reaction system

Reverse transcription program is: 30 DEG C of 10min, 42 DEG C of 15min, 95 DEG C of 5min, 4 DEG C of 5min, circulation primary; PCR program is: 94 DEG C of denaturation 5min; 94 DEG C of degeneration 40sec, 56 DEG C of annealing 40sec, extend 30sec after 72 DEG C, 30 circulations, prolong raw 10min after last 72 DEG C.Pcr amplification product is carried out agarose gel electrophoresis, and result as shown in Figure 1.Result shows; amplification obtains the fragment (do not comprise restriction enzyme site and protectiveness base is 1368bp) that length is about 1386bp; its nucleotide sequence is as shown in SEQ ID NO.3; identical with expection clip size; therefore reclaim test kit description according to the glue of TIANGEN Biotech (Beijing) Co., Ltd. and reclaim object band, reclaim the fragment called after DHAV-H-3D obtained.

The expression vector of embodiment 2, construction expression I type DHV strain H 3D albumen

Connected by DHAV-H-3D and the pJET1.2 carrier reclaimed, coupled reaction is carried out with reference to pJET1.2 Cloning Kit description, and reaction system is as shown in table 2.

Table 2, connection DHAV-H-3D and pJET1.2 carrier

Carry out brief centrifugation by after the mixing of table 2 system, then under 20 DEG C of conditions, connect 15min.Connect product conversion DH5 α competent cell, and with the LB solid of Amp resistance and fluid medium screening, and be that primer carries out PCR detection by sequence shown in screening bacterium colony SEQ ID NO.1 and SEQ ID NO.2, amplified production carries out agarose gel electrophoresis, and result is as shown in A in Fig. 2.Result shows, and screening obtains positive colony.

In order to detect positive colony further, the bacterial strain of positive colony is extracted plasmid, then through Kpn I and Xho I double digestion and the qualification of Xho I single endonuclease digestion, enzyme action system is as shown in table 3.

Table 3, Kpn I and Xho I enzyme action identification reaction system

By brief centrifugation after the mixing of table 3 system, then in 37 DEG C of condition water-bath 3h, reaction terminates digestion products to carry out agarose gel electrophoresis detection, and result is as B in Fig. 2.Result shows, and DHAV-H-3D fragment is correctly connected in pJET1.2 carrier, and called after pJET-1.2+/DHAV-H-3D.Sent by pJET-1.2+/DHAV-H-3D Invitrogen company to check order, the sequence that screening does not suddenly change is for construction of expression vector.

Get correct pJET-1.2+/DHAV-H-3D and pET-32 (a)+carrier of order-checking and carry out double digestion with Kpn I and Xho I respectively, reclaim 3D gene and carrier framework, then connect by system shown in table 4.

Table 4,3D gene and carrier framework linked system

By brief centrifugation after the mixing of table 4 system, connection of then spending the night under 16 DEG C of conditions, obtains pET-32 (a) +/DHAV-H-3D plasmid.Connect product conversion DH5 α Host Strains, with Amp resistance LB solid and fluid medium screening, then carry out PCR qualification, result is as shown in C in Fig. 2.Then PCR is accredited as positive bacterial strain to be used for extracting plasmid, and plasmid Kpn I and Xho I is carried out double digestion and Xho I carries out single endonuclease digestion, digestion products carries out agarose gel electrophoresis, and result is as shown in D in Fig. 2.From C and D, 3D gene in Fig. 2 and carrier framework exact connect ion.

The expression of embodiment 3, I type DHV strain H solubility 3D albumen

Expressive host bacterium escherichia coli (Escherichia coli) Rosetta, e. coli bl21 and e. coli bl21 (DE3) PLYS strain are preserved by Sichuan Agricultural University's poultry disease prevention and control research center; Expression vector pET-32 (a) +/DHAV-H-3D containing I type DHV strain H 3D albumen builds by embodiment 2; Various molecular biology reagents is purchased from biological reagent company.

The pET-32 (a) embodiment 2 obtained +/DHAV-H-3D plasmid transforms BL21, Rosetta and BL21 (DE3) PLYS respectively and expresses bacterium, transformed bacteria is 37 DEG C, incubated overnight under 120r/min condition in the LB fluid medium of Amp resistance, the LB fluid medium of overnight culture and fresh Amp resistance is 1:100 amplification culture about 3 hours by next day by volume, bacterium liquid OD ₆₀₀adding IPTG when reaching 0.6 to final concentration is 0.2mmol/L, and 12h is induced at 37 DEG C, then collect bacterium liquid, by bacterium liquid under 12000r/min condition, centrifugal 10min, thalline 20mmol/L Tris-HCl (pH 8.0) suspends by 1:10 (V/V).Simultaneously using pET-32 (a)+vector corresponding expression bacterium as negative control.

In order to screen the expression bacterium giving expression to solubility 3D albumen, then by the ultrasonication 6 times under condition of ice bath of above-mentioned obtained suspension bacteria liquid, 30sec/ time, interval 30sec between each, then by the bacterium liquid after fragmentation at 4 DEG C, centrifugal 10min under 12000r/min condition, abandon precipitation (for insoluble inclusion body expressing protein), collect supernatant (for soluble express protein).Draw 80 μ L supernatants, add 20 μ L containing 5 × SDS loading buffer of beta-mercaptoethanol, to boil after 10min the centrifugal 5min of room temperature under 12000r/min condition, then carry out SDS-PAGE electrophoretic examinations, result is as shown in A in Fig. 3.Result shows, after recombiant plasmid pET-32 (a) +/DHAV-H-3D being transformed into three kinds of expression bacterium, object band has been there is at about 68kD place, and negative control is not containing this band, and the expressing quantity transforming BL21 (DE3) PLYS bacterial strain expression that is maximum, BL21 takes second place, so filtering out optimum expression bacterium according to expression is BL21 (DE3) PLYS.

The optimization of I type DHV strain H solubility 3D protein expression condition:

(1) optimization of IPTG concentration: by the bacterium liquid of BL21 (DE3) the PLYS bacterial strain containing pET-32 (a) +/DHAV-H-3D recombiant plasmid by volume for 1:100 is inoculated in 5 containing in the LB fluid medium test tube of Amp resistance, and in 37 DEG C of shaken cultivation to OD ₆₀₀about=0.6, add IPTG and be respectively 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L and 1.0mmol/L to final concentration, then inducing culture 12h under 37 DEG C of conditions, then detect the expression of solubility 3D albumen with SDS-PAGE, result is as shown in B in Fig. 3.Result shows, and IPTG final concentration is that under 0.8mmol/L condition, the expression of solubility 3D albumen is the highest, and namely IPTG final concentration is 0.8mmol/L is optimum concentration.

(2) abduction delivering temperature optimization: by the bacterium liquid of BL21 (DE3) the PLYS bacterial strain containing pET-32 (a) +/DHAV-H-3D recombiant plasmid by volume for 1:100 is inoculated in 6 containing in the LB fluid medium test tube of Amp resistance, and in 37 DEG C of shaken cultivation to OD ₆₀₀about=0.6, adding IPTG to final concentration is 0.8mmol/L, and be then induce 12h under 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 37 DEG C and 39 DEG C of conditions respectively at temperature, then detect the expression of solubility 3D albumen with SDS-PAGE, result is as shown in C in Fig. 3.Result shows, and the expression of temperature solubility 3D albumen under 25 DEG C of conditions is the highest, and namely inducing temperature is 25 DEG C is the suitableeest expression temperature.

(3) abduction delivering is time-optimized: by the bacterium liquid of BL21 (DE3) the PLYS bacterial strain containing pET-32 (a) +/DHAV-H-3D recombiant plasmid by volume for 1:100 is inoculated in 5 containing in the LB fluid medium test tube of Amp resistance, and in 37 DEG C of shaken cultivation to about OD600=0.6, adding IPTG to final concentration is 0.8mmol/L, then under temperature is 25 DEG C of conditions, 4h, 6h, 8h, 10h and 12h is induced, then detect the expression of solubility 3D albumen with SDS-PAGE, result is as shown in D in Fig. 3.Result shows, and the expression of temperature solubility 3D albumen after abduction delivering 6h is to reach the highest, and namely the abduction delivering time is 6h.

Through above-mentioned optimization, can find out that BL21 (DE3) the PLYS bacterium optimal expression condition containing pET-32 (a) +/DHAV-H-3D recombiant plasmid is 0.8mmol/L IPTG, 25 DEG C of induction 6h.Follow-uply according to this condition, great expression is carried out to 3D albumen.

I type DHV strain H solubility 3D albumen Western-blot detects, concrete steps are as follows: express solubility 3D albumen by optimal expression condition, then SDS-PAGE is carried out, subsequently the gel after electrophoresis is transferred on pvdf membrane, 80V transfer printing 90min, after transfer printing, pvdf membrane is taken out, 1h is hatched in 37 DEG C of shakes with 1%BSA, then take out after 1h is hatched in 37 DEG C of shakes with the IgG of the anti-DHAV of 1:100 dilution, 3 times are washed with TBS, each 2min, 1h is hatched in 37 DEG C of shakes subsequently with two anti-igg of the HRP labelling of 1:3000 dilution, to develop the color according to DAB colour reagent box description after TBS washing, with distilled water flushing color development stopping when object band is high-visible, result is as shown in A in Fig. 4, finally pvdf membrane drying is kept in Dark Place.

The purification of I type DHV strain H solubility 3D albumen: BL21 (DE3) PLYS containing recombiant plasmid pET-32 (a) +/DHAV-H-3D is expressed under optimal condition, then thalline is collected, ultrasonication 6 times under condition of ice bath after suspending with 20mmol/L Tris-HCl (pH 8.0), 30sec/ time, interval 30sec between each, then by the bacterium liquid after fragmentation at 4 DEG C, centrifugal 10min under 12000r/min condition, collect supernatant (for soluble express protein); Then by Ni that the supernatant of collection provides in Bio-rad company ²⁺-NTA agarose gel column purification (refined solution and purification process are undertaken by test kit description), with the ultrafiltration through membranes of 0.45 μm after being dialysed by the purifying protein liquid of collection, is finally placed in 4 DEG C ,-20 DEG C ,-70 DEG C or lyophilizing preservation.Solubility 3D albumen after purification is carried out SDS-PAGE electrophoresis, and result is as shown in B in Fig. 4.The 3D albumen of result Explicit Expression is through Ni ²⁺after-NTA affinity purification, obtaining the albumen that purity, concentration are all higher, is 1.5mg/mL through nucleic acid-protein analysis-e/or determining protein concentration.

Embodiment 4, solubility 3D albumen are to the cellular immunization increasing action of I type DHV immune duck

Duck is divided into 3D protein groups (P group), 3D albumen+vaccine group (P+V group), vaccine group (V group) and matched group (C group), then carries out immunity and injection according to the immunizing dose of table 5 and approach.

Table 5, animal grouping and immunity

Immunity blood sampling in latter 7 days, measure CD8+ T cell subgroup and immune cell factor, detection method is carried out according to test kit description, and result is as shown in table 6.

Table 6, CD8+ T cell subgroup and cytokines measurement result

Result shows, and the cytokine levels of immunity latter 7 days each experimental grouies rises all to some extent.

Due to the micromolecule polypeptide that cytokine is secreted by various kinds of cell, have and regulate Growth of Cells differentiation, immunologic function, participate in the effects such as inflammation generation and wound healing.Cytokine can be divided into Th1 type and Th2 type according to type of immune response.Thl type has IL-2, IFN-γ, tumor necrosis factor (TNF) and lymphotoxin (LT), produces primarily of cell-mediated immune response process.Th2 cytokine is produced by the activation process of B cell, comprises IL-4, IL-5, IL-6, IL-10 and IL-l3.

IL-2, IL-4 and IFN-γ is all important immune indexes.In immunne response process, IL-2 is the initial factor of cellular immunization, and induction produces IFN-γ, and IFN-γ, also known as immune interferon, is an important indicator of T cell immunity.IL-2 stimulates Thp cell to Th0 cell differentiation, Th0 emiocytosis IL-2, IL-4, IL-5 and IFN-γ, usually represent humoral immunity level with IL-4 again, IFN-γ represents cellular immune level, and the ratio of IL-4/IFN-γ can react the poised state of Th2/Th1 cell.IL-2, IFN-α still participates in the important cytokine of body disease-resistant poison immunologic mechanism.IL-2 can maintain sustainable existence and the propagation of immunocyte, and IFN-α directly can suppress virus replication.CD8+ T lymphocyte quantity is also the index embodying reaction immune level, and the content of CD4+ and CD8+ two kinds of cells also can the sequencing of reacting antigen submission, the mechanism of reaction immune stimulating.Therefore the present embodiment is to CD8+ T lymphocyte quantity, the cytokines such as IL-2, IL-4 and IFN-γ measure, to obtain the master data of 3D albumen to duckling immunological enhancement.

The above results can be found out, the level of CD8+ presents the variation tendency similar to cytokine, shows after immunity, and the cellular immunization of body obtains and excites.The level rising of these factors raises with ELISA antibody horizontal and conforms to.The level of P group IFN-γ is high compared with its excess-three group, shows that 3D albumen can stimulate body to produce stronger cellular immunization.Contrast P+V group and V group, infer the immune level of entirety from the level of IL-2, IL-4,3D albumen can strengthen the immunocompetence of vaccine, and the amplitude that IL-4 rises is little, and hint 3D stimulates body generation cellular immunization to be better than humoral immunization.To sum up, the 3D albumen of preparation can stimulate duckling to strengthen cellular immunization and humoral immunization, and cellular immunization is better than humoral immunization.

Embodiment 5, solubility 3D albumen are to the humoral immunization increasing action of I type DHV immune duck

Animal is divided into 3D protein groups (P group), 3D albumen+vaccine group (P+V group), vaccine group (V group) and matched group (C group), carries out immunity and injection according to the immunizing dose of table 7 and approach.

Table 7, animal grouping and immunity

Get the blood serum sample of the rear 3d of immunity, according to 3D albumen, the DHAV ELISA method set up, detect 3D protein antibodies, the duck hepatitis A virus (HAV) antibody horizontal in serum respectively.Result is as table 8.Result display 3D protein antibodies can detect in immunity for latter 3 days, and matched group is feminine gender.

Totivirus antibody titer all can detect for the 3rd day after immunity, and matched group does not all detect antibody.

Table 8 3D albumen and totivirus antibody horizontal detect

The above results indicates the humoral immunization effect that solubility 3D albumen can increase I type DHV immune duck.

Embodiment 6, solubility 3D albumen are to the immanoprotection action of I type DHV infected duck

Animal is divided into 3D protein groups (P group), 3D albumen+vaccine group (P+V group), vaccine group (V group) and matched group (C group), carries out immunity and injection according to the immunizing dose in table 9 and immunization route.

Table 9, animal grouping and immunity

After immunity, 11d is strong malicious according to 10000ELD with I type DHV ₅₀dosage is to three experimental grouies and matched group 5 duckling counteracting toxic substances, and matched group remains 5 duck isolated rearings.After counteracting toxic substances, record infection symptoms, statistics protective rate, result is as shown in table 10.To all utensils, feces sterilization before and after counteracting toxic substances, every day three times, ensure the accurate of counteracting toxic substances dosage and bio-safety.

Table 10, counteracting toxic substances protective rate

Result shows, and after counteracting toxic substances, dead 7 ducks of P group, protective rate is that 30%, P+V group and V group are all survived, and protective rate is 100%, and matched group non-counteracting toxic substances person all survive, and 5 of counteracting toxic substances all dead.The duck of counteracting toxic substances death is carried out dissection and analysis, and as shown in Figure 5, the duck of result display counteracting toxic substances death all presents typical opisthotonus posture to result, and sees distinctive liver ecchymosis.Therefore, solubility 3D albumen can increase immunization of cell and the humoral immunization effect of duck and hepatitis A virus (HAV) immune duck, thus opposing hepatitis A virus (HAV) is to the pathogenic change effect of duck and lethal.

What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.

Claims (3)

1. the application of soluble Type I DHV 3D albumen in the immunostimulant of preparation I type duck virus hepatitis vaccine.

2. application according to claim 1, is characterized in that: described immunostimulant is cellular immunization reinforcing agent or humoral immunization reinforcing agent.

3. the application of soluble Type I DHV 3D albumen in the immune formulation of preparation prevention I type duck viral hepatitis.