CN106854647B - Duck viral hepatitis bivalent yolk antibody and preparation method and application thereof - Google Patents

Abstract

The invention provides a duck virus hepatitis bivalent yolk antibody and a preparation method thereof, wherein the bivalent yolk antibody comprises duck virus hepatitis DHAV-1 type and duck virus hepatitis DHAV-3 type yolk antibodies, and the preparation method comprises the following steps: (1) respectively inoculating a DHAV-1 strain and a DHAV-3 strain of duck virus hepatitis to SPF (specific pathogen free) chicken embryos and susceptible duck embryos, then harvesting allantoic fluid, mixing the harvested virus fluid in proportion, inactivating formaldehyde and preparing a vaccine; (2) immunizing laying hens by using the vaccine, sampling and measuring the neutralizing titer of anti-DHAV-1 and anti-DHAV-3 antigen antibodies in the hyperimmune egg yolk of the laying hens after immunization to be more than or equal to 1: 8192, and then collecting the hyperimmune eggs of the laying hens; (3) sterilizing the egg shell, collecting yolk, adding equal volume of distilled water, stirring, and inactivating at low temperature; purifying by an acidification distilled water method and an octanoic acid method; and (4) performing microfiltration and ultrafiltration. The bivalent egg yolk antibody provided by the invention has low cost and high titer, can effectively control duck virus hepatitis caused by DHAV-1 and DHAV-3, and can obtain remarkable social benefit.

Description

Duck viral hepatitis bivalent yolk antibody and preparation method and application thereof

The application is a divisional application of patent application 201210530208.5, and the original invention name is duck viral hepatitis bivalent yolk antibody and a preparation method and application thereof, and the application date is 12 months and 11 days 2012.

Technical Field

The invention relates to a high-immunity yolk antibody, in particular to a yolk antibody for duck virus hepatitis.

Background

Duck Viral Hepatitis (DVH) is an acute, highly lethal, virulent infectious disease of ducklings caused by Duck Hepatitis Virus (DHV) of enterovirus genus of picornaviridae family. The disease occurs all year round, and only ducklings within 3 weeks of age are infected in a natural state, so that great economic loss is caused, and the healthy development of the duck breeding industry is threatened. The world animal health Organization (OIE) classified duck viral hepatitis as a B-type animal disease.

Duck hepatitis virus has historically been classified as serotype i, ii and iii, with no antigenic correlation between the three. Wherein the type I duck viral hepatitis is distributed worldwide. Type II duck viral hepatitis was discovered in the United kingdom in 1965, and since the outbreak in the middle of the 80's 20 th century, the disease was not seen in the region. Type III was discovered in 1969 in the long island region of new york, usa. Type II and type III have been reclassified as members of the genus Duck astrovirus, respectively designated Duck astrovirus 1 (DAstV-1) and Duck astrovirus 2 (DAstV-2). In 2009, The International Committee for viral Classification (The International Committee on Taxolomy of Viruses, ICTV) has established The genus Avihepatoviruses among The picornaviridae, The Duck Hepatitis A Virus (DHAV) to which The classical serum I duck hepatitis virus belongs, known as DHAV-1, and includes two additional serotypes DHAV-2, DHAV-3, DHAV-2 and DHAV-3, which are serum-free cross-neutralizing with DHAV-1, respectively. Wang L et al (2008) were called type A (DHAV-A), type B (DHAV-B) and type C (DHAV-C) by genotyping. Studies have shown that genotypes distinguished by capsid protein coding sequences correspond to serotypes distinguished by neutralization assays.

Scholars at home and abroad have studied the prevalence of DHV serotypes. In 2007, Tseng isolated pathogen from diseased duck in Taiwan region 1989-1990, which has the viral hepatitis of duck, proved to be a new type of duck hepatitis virus through neutralization test, N-DHV (can be called as DHAV-2), which is only found in Taiwan region at present. In 2007, Kim et al in Korea isolated a virus from diseased ducks that developed viral hepatitis in ducks in 2003 and 2004, which was called DHV-AP (also called DHAV-3) unlike the serotype of DHV-I.

The main epidemic viruses in China in the past are duck hepatitis virus type I, and the traditional attenuated vaccine and the hyperimmune egg yolk antibody are adopted to prevent and treat DHV type I. However, in recent years, there has been a suspected duck hepatitis outbreak in which an ineffective duck group is prevented or treated by using a duck hepatitis type I attenuated vaccine or a hyperimmune yolk antibody, and a novel duck hepatitis is suspected. In recent years, Korean New type (DHAV-3) has been distributed widely in our country. Sujing and the like (separation and preliminary identification of novel DHV, Chinese veterinary science and technology, 2002, 32 (1): 15-16) separate small RNA viruses which have no serological cross immune reaction with DHV-I, DHV-III through pathogen separation identification and duck embryo serum neutralization test, and are called novel duck hepatitis viruses; huang' anguo et al (separation and preliminary identification of novel duck hepatitis virus, Guangxi animal husbandry veterinarian, 2003, 19 (5): 198-199) also separated novel duck hepatitis virus from diseased duck groups similar to duck viral hepatitis type I which can not be cured or prevented by duck viral hepatitis type I hyperimmune yolk antibody or duck hepatitis type I attenuated vaccine. Then Liujian and the like (novel DHV epidemiological investigation and immune prevention and treatment test, China veterinary journal, 2006, 42 (2): 3-6) separate 9 virus strains from duckling livers which are inspected in Beijing, Hebei, Shandong, Guangxi and the like and have obvious liver bleeding lesion characteristics, and through serological identification, 7 of the virus strains are found to be novel duck hepatitis viruses, and 2 of the virus strains are I-type duck hepatitis viruses. These results revealed that the treatment with hyperimmune serum or hyperimmune egg yolk antibody was ineffective in some areas in recent years after the occurrence of duck viral hepatitis.

Research results show that DHAV-1 and DHAV-3 are popular in a plurality of regions in China at the same time, cross protection is lacked between the DHAV-1 and the DHAV-3, and effective control measures for DVH caused by the DHAV-3 are also lacked.

Disclosure of Invention

In order to meet the urgent need of effective DVH prevention and treatment technology in production and control the generation of DVH in China in time, the invention prepares the bivalent egg yolk antibody by using the traditional I type (DHAV-1) duck hepatitis virus and the novel (DHAV-3) duck hepatitis virus.

Therefore, the invention provides a duck viral hepatitis virus strain, wherein the sequence of the coded amino acid of the VP1 gene of the virus strain comprises three amino acid substitution sites of 49-T, 196-N and 207-E and three consensus sites of 197-Q, 198-S and 199-D as shown in figure six.

Preferably, the duck viral hepatitis virus strain is a duck viral hepatitis virus SD1strain, the SD1strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO.V201225, and the preservation address is Wuhan-Wuhan university in China.

The present invention provides a DNA sequence substantially comprising the nucleotide sequence of seq No.1, wherein the term "substantially comprising" in the present invention means that the nucleotide sequence of SEQ ID NO.1 may have a mutation such as substitution, insertion or deletion within a range that retains the function of the nucleotide sequence of the present invention.

The invention provides a VP1 antigen protein, wherein the VP1 antigen protein comprises a duck viral hepatitis virus VP1 consensus sequence which is shown in figure six and has three amino acid substitution sites of 49-T, 196-N and 207-E and three consensus sites of 197-Q, 198-S and 199-D.

The invention mainly aims to provide a duck viral hepatitis divalent yolk antibody which comprises duck viral hepatitis DHAV-1 and duck viral hepatitis DHAV-3 yolk antibodies. The antibody has high titer and low cost, and can effectively control the duck virus hepatitis of different serotypes currently prevalent in China.

The invention also aims to provide a preparation method of the duck viral hepatitis bivalent yolk antibody, which is safe and easy to use, and comprises the following steps:

(1) respectively inoculating a 9-day SPF chicken embryo and a 10-day susceptible duck embryo by using a duck virus hepatitis DHAV-1 strain and a DHAV-3 strain, then harvesting allantoic fluid, mixing the harvested virus fluid in proportion, inactivating formaldehyde and preparing a vaccine;

(2) immunizing a laying hen by using the prepared vaccine, sampling and measuring the neutralizing titer of anti-DHAV-1 and anti-DHAV-3 antigen antibodies in a hyperimmune yolk of the hen to be more than or equal to 1: 8192 after immunization, and then collecting hyperimmune eggs of the hen;

(3) sterilizing the high-immunity egg shells, collecting egg yolks, adding distilled water with the same volume as the egg yolks, uniformly stirring and inactivating the egg yolks by low-temperature pasteurization; purifying by an acidification distilled water method and an octanoic acid method; and (4) performing microfiltration and ultrafiltration.

Preferably, the DHAV-1 strain of the duck viral hepatitis is a DRL-62 strain, and the DHAV-3 strain is SD 1.

Preferably, the immunization program is: the laying hens are subjected to intramuscular injection of 1 ml/egg at the age of 120 days, are subjected to intramuscular injection of equal dose after 14 days, are subjected to intramuscular injection of equal dose 10 days later for tertiary immunization, and are subjected to intramuscular injection of equal dose 10 days later for fourth intensified immunization.

Preferably, the low-temperature pasteurization condition is heating inactivation at 62.5 ℃ for 30 min;

preferably, the purification step by the acidified distilled water method comprises the following steps: adding sterilized distilled water with the volume 6 times that of the yolk into an interlayer reaction tank, wherein the pH value of the distilled water is 4.2, cooling to 4 ℃, adding the inactivated yolk liquid, stirring, standing at 4 ℃ for 4 hours, and centrifugally separating supernatant;

the method for purifying the caprylic acid comprises the following steps: adding caprylic acid according to 0.2% of the total volume of the supernatant, stirring, standing at room temperature for 4h, filtering to be clear, adding a saturated formaldehyde solution according to 0.1% of the total volume of the filtrate, stirring, and standing at room temperature for 24 h.

Preferably, the microfiltration is performed using a 0.22 μm microporous filter cartridge for sterile filtration, and the ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cut-off of 1000KDa to remove viruses.

The invention also aims to provide the application of the duck virus hepatitis bivalent yolk antibody in preparing the medicine for preventing and treating the duck virus hepatitis.

The invention also aims to provide a vaccine composition for preventing and treating duck viral hepatitis, which comprises an immunizing dose of antigen prepared from a duck viral hepatitis strain which is shown in figure six and has three amino acid substitution sites of 49-T, 196-N and 207-E and three common sites of 197-Q, 198-S and 199-D and a pharmaceutically acceptable carrier, wherein the gene sequence of the amino acid coded by the VP1 gene comprises the antigen.

Preferably, the duck viral hepatitis virus is strain SD 1.

The vaccine composition for preventing and treating duck viral hepatitis is prepared by using a conventional method in the field.

The invention also provides application of the vaccine composition in preparation of a medicine for preventing and treating duck viral hepatitis.

Technical effects

1. The invention adopts multiple inactivation technologies of physics, chemistry and the like, combines the acidification water dilution method with modern biological technologies of caprylic acid method, high-speed centrifugation, ultrafiltration and the like, effectively separates and purifies the immune globulin in the yolk, the yolk antibody does not contain any harmful substance and exogenous pollution, the safety is high, the immune duck group is injected without influencing the carcass quality, and the method can be industrially produced.

2. The duck virus hepatitis virus strain obtained by the invention has good immunogenicity, the produced inactivated vaccine has high antibody production speed, high antibody titer and good safety, and the prepared yolk antibody can effectively prevent and control infection of DHAV-3 virus, is convenient to store, transport (2-8 ℃) and use, has stable use effect, and effectively prevents and controls the occurrence of duck virus hepatitis.

3. When the inventor researches the VP1 gene sequence of the obtained duck viral hepatitis strain SD1strain, the inventor unexpectedly finds that the VP1 gene coding amino acid gene sequence contains three amino acid substitution sites of 49-T, 196-N and 207-E and three consensus sites of 197-Q, 198-S and 199-D which are shown in figure six and have important effects on the immunogenicity of the duck viral hepatitis strain. The subsequent examples prove that the VP1 gene sequences of other duck viral hepatitis strains have corresponding same mutation sites and also have the same function.

4. The bivalent egg yolk antibody provided by the invention has low cost and high titer, and animal experiments show that the bivalent egg yolk antibody can effectively control duck viral hepatitis caused by DHAV-1 and DHAV-3, and can obtain remarkable social benefits.

Drawings

FIG. 1 shows the results of RT-PCR detection of VP1 from DRL-62 strain and SD1strain using DHV type I standard strain primer;

FIG. 2 shows the results of RT-PCR detection of DRL-62 and SD1 VP1 using DHV Korean strain primer;

FIG. 3 shows the nucleotide sequence homology analysis of the SD1 VP1 gene and the DHV reference strain;

FIG. 4 shows the amino acid sequence homology analysis of the SD1 VP1 gene and DHV reference strain;

FIG. 5 is a nucleotide sequence evolutionary tree of the SD1 VP1 gene and DHV reference strain;

FIG. 6 is the amino acid sequence alignment analysis of the SD1 VP1 gene and DHV reference strain;

FIG. 7 shows the analysis of the hydrophilicity, antigen index and surface accessibility of SD1 VP1 protein.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

The preparation method of the duck viral hepatitis bivalent yolk antibody comprises the following steps: breeding duck hepatitis virus DHAV-1 and DHAV-3, preparing vaccine for immunization, immunizing laying hens and extracting yolk antibody. The DHAV-1 strain selected in the embodiment of the invention is a DRL-62 strain, the DHAV-3 strain is an SD1strain, and the specific method comprises the following steps:

1. the source of the virus seed is duck hepatitis virus DRL-62 strain (ATCC deposit with the number VR-1313); the SD1strain was isolated by Poechor bioengineering GmbH (CCTCC preservation, preservation number: CCTCC NO. V201225).

2. And (3) diluting the duck hepatitis virus DRL-62 virus seeds with a sterilized PBS solution at a ratio of 1: 100, and inoculating 0.1ml of SPF (specific pathogen free) chick embryos of 9-11 days old through an allantoic cavity. And (3) continuously incubating at 37 ℃, discarding dead embryos within 48 hours, harvesting dead chicken embryos within 48-96 hours, and cooling at 2-8 ℃ for 12-24 hours. Mixing the sterile embryo solutions, packaging, and freezing. The date of harvest, the number of generations of poison seeds, etc. are noted.

Diluting duck hepatitis virus SD1strain virus seeds with sterilized PBS solution at a ratio of 1: 100, and inoculating susceptible duck embryos of 10-12 days old, 0.2ml per embryo, via allantoic cavity. And (3) continuously incubating at 37 ℃, discarding dead embryos within 48 hours, harvesting dead duck embryos within 48-96 hours, and cooling at 2-8 ℃ for 12-24 hours. Mixing the sterilized duck embryo solutions, packaging, and freezing. The date of harvest, the number of generations of poison seeds, etc. are noted.

3. Preparation of inactivated vaccine for immunization:

3.1 harvesting of viral fluid the harvested viral fluid is centrifuged at 3500 rpm for 30 minutes, the supernatant is taken up in a sterile container, 10% formaldehyde solution is added and shaken with the addition to mix thoroughly, the final concentration of the formaldehyde solution is 0.15%, the container is sealed and placed in a 37 ℃ incubator for 24 hours (shaking 3-4 times).

3.2 preparation of oil phase of inactivated vaccine for immunization: mixing 94 parts of white oil for injection with 806 parts of span-aluminum stearate, heating while stirring until the mixture is transparent, and autoclaving for later use to obtain an oil phase. Preparing a water phase: mixing DRL-62 strain antigen and SD1strain antigen at a ratio of 1:1, adding sterilized Tween-80 at 4% of antigen, and shaking to dissolve Tween-80 completely to obtain water phase. Emulsification: placing 2 parts of oil phase into an oil phase tank, starting a motor to stir, slowly adding 1 part of water phase, and emulsifying at 3400 r/min for 10 minutes. 1% thimerosal was added to a final concentration of 0.01% before the emulsification was terminated. The samples were centrifuged at 3000 rpm for 15 minutes without separation. Subpackaging: quantitatively subpackaging, and sealing with cover.

4. Immunization

4.1 immunization program about 120 days old laying hens are injected with 1ml of inactivated oil vaccine, 1ml of inactivated vaccine is injected with muscle after 14 days, 1ml of inactivated vaccine is injected with muscle after 10 days of second immunization, and 1ml of inactivated vaccine is injected with muscle after 10 days of third immunization to carry out fourth strengthening immunization.

4.2 sampling and measuring the neutralizing titer of DRL-62 antibody and SD1 antibody in the high-immunity egg yolk of the chicken every 10 days after the eggs are collected, and storing the high-immunity eggs at 4 ℃ for later use.

5. Preparation of egg yolk antibody

5.1 harvesting and inactivation of egg yolk the eggshells are disinfected and either manually or mechanically beaten. The egg white, blastoderm and frenulum were removed sufficiently and the egg yolk was collected. Stirring thoroughly to make yolk into uniform paste, adding equal volume of distilled water sterilized and cooled at 121 deg.C for 30min, stirring, and inactivating at 62.5 deg.C for 30 min.

5.2 purification of antibody by acidified distilled water method sterilized distilled water with pH 4.2, which is 6 times volume of original yolk, is added into a reaction tank with a separation layer, and the temperature is reduced to 4 ℃. Then, the inactivated egg yolk solution was added with stirring, and allowed to stand at 4 ℃ for 4 hours. The supernatant was centrifuged by a tubular low temperature continuous centrifuge at 14000rpm and transferred to another reaction tank.

5.3 purification of antibody by caprylic acid method caprylic acid was added in an amount of 0.2% (by volume) of the total amount, stirred well and left at room temperature for 4 hours. Filtering with filter cloth, filtering with K-type multi-layer plate frame to clarify, adding saturated formaldehyde solution 0.1% of the total amount into the filtrate, stirring, standing at room temperature for 24 hr while shaking for several times.

5.4 Filter-Sterilization, 0.22 μm microporous filter elements were used for filter sterilization. Filtering with ultrafiltration membrane with molecular weight cutoff of 1000KDa to remove virus.

5.5 antibody titer determination neutralization assay

DHV DRL-62 strain was diluted to 200ELD per 0.1ml₅₀The mixture was mixed with an equal amount of the antibody to be tested, which was serially diluted 2 times, and the mixture was allowed to act at 37 ℃ for 1 hour while shaking several times. Inoculating 5 SPF (specific pathogen free) chick embryos of 9 days old at each dilution, wherein each chick is 0.2ml, additionally arranging 5 virus control groups and 5 PBS blank control groups, wherein virus liquid and PBS are mixed in equal amount for sterilization, culturing at 37 ℃ for 168 hours, recording the death number of the chick embryos for 48-168 hours, and judging the result. The blank control group should be fully viable and the virus control group should be fully dead.

DHV SD1strain was diluted to 200ELD per 0.1ml₅₀The mixture was mixed with an equal amount of the antibody to be tested, which was serially diluted 2 times, and the mixture was allowed to act at 37 ℃ for 1 hour while shaking several times. Inoculating 5 susceptible duck embryos of 10 days old to each dilution, each embryo is 0.2ml, and virus solution and the like are added5 virus control groups and PBS blank control groups mixed with sterilized PBS are cultured at 37 ℃ for 168 hours respectively, the death number of the duck embryos in 48-168 hours is recorded, and the result is judged. The blank control group should be fully viable and the virus control group should be fully dead.

The highest dilution factor of the antibody which can prevent 50 percent of chick (duck) embryos from dying is the neutralization titer of the antibody.

5.6 freeze-drying the purified egg yolk antibody, adding a freeze-drying protective agent into the purified and qualified egg yolk antibody liquid, subpackaging the purified and qualified egg yolk antibody liquid in a penicillin bottle, putting the penicillin bottle into a freeze-drying box, reducing the temperature to-40 ℃ at the lowest temperature in the freeze-drying process, keeping the temperature for 4 hours at the temperature, heating the product to 18-20 hours at the speed of 1.0 ℃/hour until the product is basically dried, heating to 25 ℃ at the speed of 4-6 ℃/hour until the product is completely freeze-dried, plugging and taking the product out of the box, and storing the product at.

Example 1 molecular biological identification of DHV SD1 isolate

1. The duck hepatitis virus SD1strain virus is a newly separated and identified virus and has the following characteristics:

1.1 the clinical symptoms caused by DHAV-3 and DHAV-1 are very similar, mainly expressed as: the duckling suffers from sudden onset of disease, obvious neurological symptoms appear quickly, convulsion occurs, and death occurs quickly, and the duckling is in the posture of opisthotonus after death. The pathological changes are manifested as hepatomegaly, with a large number of bleeding spots and plaques on the surface of the liver, and with the prolongation of death time, the bleeding is more obvious.

1.2 multiple pairs of specific primers were designed based on the published DHV genome sequence in GenBank, and VP1 gene was amplified by conventional RT-PCR method, and VP1 gene of isolate SD1 was subjected to sequence determination analysis. The results showed that the VP1 gene of SD1 has the highest similarity to VP1 sequence of DHAV-3 in China and Korea, and has lower similarity to DHAV-1 and DHAV-2 in Taiwan. The analysis results of nucleotide sequence and evolutionary relationship show that the genetic distance between the isolated virus and DHAV-3 in China and Korea is the closest, and the genetic distance between the isolated virus and DHAV-1 and DHAV-2 in Taiwan is larger.

1.3 VP1 amino acid sequence comparison analysis and hydrophilicity, antigen index and surface accessibility analysis show that the change of amino acid sites between DHV SD1 isolate and other domestic DHAV-3 are located in a hydrophilic region and an accessibility region, and the difference with DHAV-1 indicates that completely different antigen epitopes may exist on DHV SD1 isolate VPl protein and are reflected in immunoprotection.

1.4 serum Cross-neutralization assay showed no cross-protection between the DHV SD1 isolate and DHAV-1, which were classified into different serotypes. The SD1 isolate and the domestic DHAV-3 strain have cross protection, and the SD1strain has better neutralization protection than the contrast strain.

1.5 physical and chemical property identification results show that the strain can resist the treatment of chloroform, ether, heat, pancreatin and acid.

The virus strain has good immunogenicity, and the neutralizing titer of DRL-62 antibody in the hyperimmune egg yolk of the chicken is determined by sampling 10 days after the quadruplicate immunization at a ratio of 1:17620, and the neutralizing titer of SD1 antibody is determined at a ratio of 1: 13932; the treatment experiment shows that the bivalent yolk antibody can effectively control the occurrence of duck virus hepatitis of different serotypes currently prevalent in China, and the protection rate reaches 80-100 percent.

The duck viral hepatitis SD1strain is preserved in China center for type culture Collection in 2012 at 06-08. The preservation unit is abbreviated as: CCTCC, preservation number: CCTCC No. v201225, classification name: duck viral hepatitis virus, having the english name of Duck hepatitis virus SD1Strain, having the latin name of tarpeiapuli, and having the deposit unit address: wuhan, Wuhan university in China.

2.RT-PCR

RNA was extracted from diseased duck liver by virus isolation, and RT-PCR was performed on VP1 sequence using the following primers based on the complete genome sequences of DHAV-1 strain DRL-62 strain and Korean DHAV-3 strain N-DHV strain published by GenBank (see method of reverse transcription kit of great Lianbao bioengineering Co., Ltd.):

DHAV-1 type upstream primer: 5'-GGTGATTCTAACCAGTTGG-3' the flow of the air in the air conditioner,

a downstream primer: 5'-TTCAATTTCCAGATTGAGT-3', respectively;

② DHAV-3 type upstream primer: 5'-CAGATGGCCGCCAATGATCAG-3' the flow of the air in the air conditioner,

a downstream primer: 5'-GTCTCTGACATTTCGAAATTGGTATGA-3' are provided.

The positive amplification product was sent to Weijie funding (Shanghai) trade company Limited for sequencing.

3. Analysis of Gene sequences

The nucleotide sequence of the VP1 gene of the isolated strain was subjected to homology comparison analysis using DNAStar7.1 analysis software with the nucleotide sequences of the DHV type I standard strain DRL-62 and other DHV strains logged in GenBank (see Table 1), and phylogenetic trees were drawn using the method in Megalign for genetic evolution analysis.

TABLE 1 reference strains and GenBank accession numbers thereof

4. Molecular biological identification results

4.1 RT-PCR results

Results referring to FIGS. 1 and 2, RT-PCR was performed on DRL-62 and isolates using DHV VP1 specific primers, wherein in FIG. 1, lane M is DL2000Marker, lane 1 is RT-PCR result of DRL-62 using DHAV-1 type standard primer, lane 2 is RT-PCR result of SD1 isolate using DHAV-1 type standard primer, and lane 3 is negative control;

in FIG. 2, lane M is DL2000Marker, lane 1 is the result of RT-PCR using DHAV-3 type primer for SD1 isolate, lane 2 is the result of RT-PCR using DHAV-3 type primer for DRL-62 isolate, and lane 3 is the negative control.

And (4) analyzing results: by using DHAV-1 type standard strain primers, the DRL-62 strain can amplify a specific band with the size of about 700 bp; using DHAV-3 type primer amplification, it was shown that the isolate showed a specific band at about 750 bp.

4.2 VP1 sequence analysis results

4.2.1 sequencing of the DHV SD1 VP1 Gene see sequence SEQ 1:

4.2.2 DHV SD1strain VP1 gene nucleotide and coded amino acid sequence homology analysis thereof

Referring to FIGS. 3 and 4, sequence analysis shows that the nucleotide similarity of the DHV SD1 isolate and the DHV type I standard strain DRL-62 strain and the DHV type I MY strain isolated in China (DHAV-1) is only about 69%, and the amino acid similarity is 77.3% and 76.5% respectively; the nucleotide similarity of DHAV-3 strains isolated from China, such as DHV FS strains, C-YDF strains and the like, is respectively 93.1-97.9 percent, and the amino acid similarity reaches 97.5-99.2 percent; the nucleotide sequence similarity with Korean New DHV (DHAV-3) is 93.1%, and the amino acid similarity is 92.5% and 92.9% respectively; the nucleotide sequence similarity with the Taiwan novel DHV (DHAV-2) is only 72.7%, and the amino acid similarity is 80.5% and 80.1%, respectively.

Referring to FIG. 5, the results of the evolutionary relationship analysis further showed that the DHV SD1 isolate has the closest genetic distance to DHAV-3 strain in China and Korean strains in one branch, and has a great difference to DHAV-1 and DHAV-2 strains. Meanwhile, the DHV SD1 isolate and domestic DHAV-3 and Korean DHAV-3 are respectively shown on two small branches, and the DHV SD1 isolate and other domestic DHAV-3 logged in GenBank are respectively shown in two branches.

4.2.3 alignment analysis of the amino acid sequence of VP1 and analysis of hydrophilicity, antigenic index and surface accessibility

VP1 is the main antigenic gene of small RNA virus, most of the protein is exposed on the surface of the virus, is the main structural protein, contains multiple antigen epitopes capable of inducing T, B lymphocyte reaction, and can induce the body to produce protective neutralizing antibody. Because VP1 is located on the surface, the selection pressure is large, the variation rate is high, and the antigenic sites of certain amino acids are changed, thereby having influence on the variation of the antigenicity of the strains.

In the picornaviridae family, the structural protein VP1 has a conserved amino acid sequence RGD, which performs an important function of adsorbing cells to bind to cell receptors and also induces viruses to produce neutralizing antibodies. From the deduced amino acid sequences, it can be seen that the VP1 sequence of the DHAV-1 type strain does not have the RGD sequence, but is SGD, and the corresponding DHAV-3 is QSD. This change suggests that the binding of novel DHV to cellular receptors may differ from FMDV and the like.

Referring to FIG. 6, the comparison of DHV SD1 isolate and DHAV-1 shows that the isolate has 2 amino acids GG insertions at positions 143 and 144, and the mutation regions are mainly at positions 140-146, 180-200, and 212-219. A comparison of the DHV SD1 isolate with Korean novel DHV (NC-009750) revealed that amino acids at positions 18 were mutated, and the hypervariable region was mainly at positions 178-196. Comparison with the Taiwan DHAV-2 shows that 2 amino acids DG are deleted from the 51 and 52 position isolates, 3 amino acids GGG are inserted into the 143-145 positions, and 1 amino acid L is inserted into the 185 positions. Comparison of the DHV SD1 isolate with other DHAV-3 strains (10 strains) in China shows that point mutation mainly exists, wherein the 49 th amino acid of the SD1strain is T, and other strains are G or S. The amino acid at position 196 is N, and 7 strains in the comparison strain are D. Amino acid 207 is E, and 4 of the control strains are K.

Referring to FIG. 7, the VP1 gene was analyzed by the Protean program. The hydrophilic region analysis was performed by Kyte-Doolittle method, the amino acid position on the surface of the molecule analysis was performed by Emini scheme, and the antigen index analysis was performed by Jameson-Wolf method, respectively. The result shows that the VP1 protein has wide distribution of regions with high hydrophilicity, similar to the regions with high antigenic index, wherein the antigenic index, the hydrophilicity peak value and the surface accessibility value of the three segments of 131-141, 195-205 and 210-224 are high, and the distribution of the three segments is consistent with the distribution of the main flexible region, and the segment of the region is supposed to be the main antigenic peptide segment of VP 1. The amino acid site changes of the DHV SD1 isolate and other domestic DHAV-3 are located in a hydrophilic region and an accessibility region, and the difference with DHAV-1 indicates that completely different antigen epitopes possibly exist on the DHV SD1 isolate VPl protein and are reflected in immunoprotection. The research finds that the strain with the three amino acid substitution sites of 49-T, 196-N and 207-E and the three consensus sites of 197-Q, 198-S and 199-D has good immunogenicity on the duck viral hepatitis.

Example 2 serum Cross neutralization assay

Virus diluted serum was fixed, and DHV DRL-62 strain and SD1 isolate positive serum were serially diluted 2-fold with physiological saline, respectively. The virus liquid containing 200ELD50/0.2ml DRL-62 strain and SD1 isolate was mixed with positive serum of different dilutions of each strain in an equal amount of 1.0ml, and the mixture was allowed to react at 37 ℃ for 1 hour. At the same time, virus and physiological saline controls were set. 5 duck embryos are inoculated to each neutralization group and the control group through an allantoic cavity, the embryo concentration is 0.2m 1/embryo, the embryos are cultured at 37 ℃, the observation is carried out for 7 days, and the number of dead embryos in each group is recorded. The highest serum dilution for 50% duck embryo protection was taken as the neutralization titer of the serum.

The neutralization test result shows that the isolate can be completely neutralized by the positive serum of the isolate, can not be neutralized and protected by the positive serum of DHV standard type I duck hepatitis, and all test groups die; after the DRL-62 positive serum acts on the isolate, the neutralization titer is 1: 16; after the SD1 positive serum and the DRL-62 strain act, the neutralization titer<1:8, showing that the separated strain and the DRL-62 strain have no serum cross protection effect, and the result is shown in a table 2. Calculating the genetic relationship between different strains R: (

Wherein R1 is the ratio of the neutralization titer of serum 1 to virus 2 to the neutralization titer of serum l to virus 1; r2 is the ratio of the neutralization titer of serum 2 to virus i to the neutralization titer of serum 2 to virus 2. ) According to the affinity value (R), distinguishing standards for distinguishing serotypes and subtypes according to foot-and-mouth disease viruses (when the R value is more than 70%, the subtypes of the two strains are the same; when the R value is 32-70%, the two strains are different subtypes; r value is less than 10%, strains are different types), R value between DRL-62 strain and isolate is less than 10%<0.74%) that does not belong to the same serotype, is a novel serotype.

TABLE 2 serum Cross-neutralization test results

Example 3 serum Passive Immunoprotection assay 1

The 230 susceptible ducklings of 5 days old are randomly divided into 5 groups, 50 groups of 1, 2, 3 and 4 groups, and 10 groups of 5, 6 and 7 groups. SD1 and GD strain (national veterinary microorganism culture collection management center preservation number: CVCC AV321) positive serum (self-made) are respectively diluted to have the neutralization titer: 1:32, 1:64, 1:128, 1:256, 1: 512. 1.2 groups of ducklings are injected with SD1 positive serum 1.0m1 per muscle, and 10 ducklings are injected per titer; 3. GD strain positive serum 1.0m1 was injected into each muscle of 4 groups of ducklings, and each titer was injectedShoot 10. After 24 hours, each duckling in 1, 3 and 5 groups is injected with 100LD subcutaneously₅₀SD1strain 0.2ml, 2, 4, 6 groups of ducklings each intramuscular injection 100LD₅₀GD strain 0.2ml, group 7 ducklings did not make any injection as blank control. The animals were kept separately and observed for 10 days, and the number of deaths in each group was recorded.

The results show that both SD1strain and GD strain can be protected by respective sera; after the duckling is passively immunized when the positive serum antibody titer of SD1 is 1:128, 7/10 protection can be generated by the attack on the GD strain, and 10/10 protection can be generated when the positive serum antibody titer of SD1 is 1: 256; after passive immunization of ducklings at a GD positive serum antibody titer of 1:256, a challenge with SD1strain resulted in 5/10 protection, and when SD1 titer was 1:512, 9/10 protection. It can be seen that the protective effect of the SD1 serum on GD strain is better than that of the GD serum on SD1strain, and the results are shown in Table 3.

TABLE 3 Passive Immunoprotective test results 1

Note: the ratio in the table is the survival rate of the ducklings, and less than 10 groups of test animals are caused by nonspecific death.

Example 4 serum Passive Immunoprotection assay 2

5 pairs of specific primers are designed and synthesized according to the whole genome sequence of the DHAV-3 GD strain, and the DHV whole genome cDNA is amplified in 5 sections by using an RT-PCR method. The amplified cDNA overlapping fragments were directionally cloned into the vector pBluescript II KS (+) to obtain the DHAV-3 GD full-length genomic cDNA plasmid. The plasmid was sequenced and sent to Shanghai bioengineering company to design mutation primers, and PCR-mediated overlap extension mutagenesis was used to perform site-directed mutagenesis of codon 49 GGT → ACT, codon 196 GAT → AAT, and codon 207 AAG → GAG of VP 1. Through sequencing identification, the site-directed mutagenesis is accurate, and other sequences are not changed. The fragments were ligated to obtain a full-length genomic cDNA clone of DHAV-3 GD. The full-length cDNA plasmid was linearized with NruI and transcribed in vitro using the SP6 RNA polymerase system to obtain viral RNA. The method of phenol/chloroform extraction and isopropanol precipitation is used to remove the impurities such as enzyme in the reverse transcription system, thereby improving the transfection efficiency. When the BHK-21 cell density in the cell culture plate was 80%, transfection was performed according to the DMRI-C transfection reagent instructions. After 72h, the transfected virus RNA cell is frozen and dissolved for 1 time and then inoculated with BHK-21 cell, thereby saving the virus GD-BHK. After the virus is rescued and inoculated to 10-day-old duck embryos, the duck embryos can be killed. Seroneutralization assays indicate that rescued viruses can be neutralized by GD positive sera. Sequencing results show that the rescued virus and the parental virus have 8 amino acid differences, and reports that the 8 amino acids are related to the virus virulence are not found.

Preparing positive serum by using virus-rescued immune SPF chicken, and respectively diluting the neutralization titer to: 1:32, 1:64, 1:128, 1:256, 1: 512. 1.2 groups of ducklings were injected intramuscularly with GD rescue virus positive serum 1.0m1, 10 per titer. After 24 hours, the ducklings of 1 and 2 groups use 100LD respectively₅₀SD1strain and GD strain, 0.2 ml/strain. Additionally, there were SD1strain, DG strain challenge control and blank control group without any injection. Each group was kept separately, observed for 10 days, and the death of the ducklings was recorded.

The result shows that the GD rescue strain can generate protection of more than 9/10 on SD1 when the neutralization titer is more than or equal to 1:64, and can generate protection of more than 9/10 on GD when the neutralization titer is more than or equal to 1:256, and the result is similar to the protection effect of SD1 positive serum in the passive immune protection test 1, which indicates that the immunogenicity of the GD rescue strain is changed to a certain extent by mutation, so that the GD rescue strain obtains the toxicity attack protection effect similar to that of the SD1strain, and has the immunogenicity equivalent to that of the SD1strain, and the mutation of the GD rescue strain is the same as that of the SD1strain, and is the same as that the mutation of the GD rescue strain generates the same change on three amino acid substitution sites of 49-T, 196-N and 207-E and three common sites of 197-Q, 198-S and 199-D of the structural protein VP 1. Therefore, the mutant site has important influence on the immunogenicity of the duck viral hepatitis virus, the immunogenicity of the SD1strain is enhanced due to the mutation of the corresponding site, and the immunogenicity of the GD strain is correspondingly changed after the corresponding site is mutated. Thus, the strains with the three amino acid substitution sites of 49-T, 196-N and 207-E and the three consensus sites of 197-Q, 198-S and 199-D have good immunogenicity.

TABLE 4 Passive Immunoprotective test results 2

Note: the ratio in the table is the survival rate of the ducklings, and less than 10 groups of test animals are caused by nonspecific death.

Example 5 identification of physicochemical Properties of DHV SD1Strain

1. The harvested duck embryo liquid is taken for centrifugation at 3000 rpm for 15min in an ether sensitivity test, and larger particles are removed. 1.6ml of the supernatant was aspirated and transferred into 2 sterilized vials, each containing 0.8ml of ether, and 0.2ml of ether for anesthesia was added to one of the vials, and no ether was added to the other vial as a control. Both bottles were stoppered with rubber stoppers and then left at 4 ℃ for 24 hours with occasional shaking. A vial of ether was added, whereupon two clear layers separated: the upper layer is ether, and the lower layer is virus liquid. The virus fluid was then aspirated by a capillary pipette, transferred to another vial and blown appropriately to evaporate the residual ether. And finally, respectively carrying out 10-time serial dilution on the two virus solutions, inoculating 10 non-immune duck embryos of 10 days old to each dilution, observing for 168 hours, and recording the death condition of the duck embryos. The virus content of the ether-treated virus fluid and the control virus fluid were compared.

2. The chloroform sensitivity test takes the harvested duck embryo liquid, centrifuges for 15min at 3000 r/min, and removes larger particles. Chloroform (analytical grade) was added to the virus solution to give a final concentration of 4.8%, and the mixture was stirred at 4 ℃ for 10 minutes, followed by centrifugation at 500 rpm for 5 minutes, and the supernatant liquid was aspirated to measure the virus content. The virus content was determined by adding an amount of sterilized normal saline equal to the amount of chloroform to the control virus solution and treating the same. The virus content of the chloroform-treated virus fluid and the control virus fluid were compared.

3. The acid resistance test takes the harvested duck embryo liquid, centrifuges for 15 minutes at 3000 r/min, and removes larger particles. The supernatant was aliquoted into 2 vials. The virus solution in 1 vial was adjusted to pH 3.0 with 0.1mol/L HCl, and sterile physiological saline equivalent to the amount of acid was added to the virus solution in another 1 vial as a control, and after exposure to 37 ℃ for 2 hours, the pH was adjusted to about 7.2 with 5.6% NaHCO3 solution. The control was supplemented with sterile normal saline equivalent to the amount of base. And (3) serially diluting two bottles of virus solution by 10 times, inoculating 10 non-immune duck embryos of 10 days old to each dilution, observing for 168 hours, and recording the death condition of the duck embryos. The virus content of the acid-treated virus fluid and the control virus fluid were compared.

4. The heat resistance test takes the harvested duck embryo liquid, centrifuges for 15 minutes at 3000 r/min, and removes larger particles. The supernatant was aliquoted into 2 vials, 1 vial was placed in a 50 ℃ water bath for 30 minutes, and the other 1 vial was left untreated as a control. And (3) serially diluting two bottles of virus solution by 10 times, inoculating 10 non-immune duck embryos of 10 days old to each dilution, observing for 168 hours, and recording the death condition of the duck embryos. The virus content of the heat-treated virus fluid and the control virus fluid were compared.

5. The trypsin sensitivity test takes the harvested duck embryo liquid, centrifuges for 15 minutes at 3000 r/min, and removes larger particles. The supernatant was aliquoted into 2 vials, 1.0ml per vial, 1.0ml of 1% trypsin was added to 1 vial to give a final concentration of 0.5%, 1.0ml of 1640 medium was added to the other vial, the vial was closed with a rubber stopper, the vials were inverted several times to mix thoroughly, left to act at 37 ℃ for 1 hour, 8.0ml of inactivated fetal bovine serum was added immediately, and the mixture was mixed thoroughly to terminate the action of trypsin. And (3) serially diluting two bottles of virus solution by 10 times, inoculating 10 non-immune duck embryos of 10 days old to each dilution, observing for 168 hours, and recording the death condition of the duck embryos. The virus content of the trypsin-treated virus fluid and the control virus fluid were compared.

And physical and chemical property identification results are as follows:

referring to the above test methods, it can be seen that DHV SD1 isolate was treated with ether, chloroform, acid, heat, and pancreatin without significant reduction in toxicity value, and the results are shown in tables 5, 6, 7, and 8.

TABLE 5 results of lipid solvent sensitivity test

TABLE 6 acid resistance test results

TABLE 7 Heat resistance test results

TABLE 8 results of trypsin sensitivity test

Example 6: preparation of duck virus hepatitis bivalent yolk antibody

1ml of laying hens of 120 days old are injected into the bigeminy inactivated oil vaccine prepared from strains DRL-62 and SD1 intramuscularly, and second immunization is carried out after two weeks, third immunization is carried out after 10 days, fourth boosting immunization is carried out after 10 days after third immunization, and the immunization dose of each time is the same as that of first immunization. And (3) sampling 10 days after the four-time immunization to determine the neutralizing titer of the DRL-62 antibody in the high-immunity egg yolk of the chicken to be 1:17620 and the neutralizing titer of the SD1 antibody to be 1:13932, collecting eggs, and storing at 4 ℃ for later use. And (5) performing aseptic egg beating to collect egg yolks. Adding equal volume of distilled water sterilized and cooled at 121 deg.C for 30min, stirring, and inactivating at 62.5 deg.C for 30 min. Adding 6 volumes of sterilized distilled water (pH 4.2) into a reaction tank, cooling to 4 deg.C, slowly adding inactivated egg yolk liquid while stirring, and standing at 4 deg.C for 4 hr. The supernatant was centrifuged by a tubular low temperature continuous centrifuge at 14000rpm and transferred to another reaction tank. Adding octanoic acid according to 0.2% (volume ratio) of the total amount, stirring well, and standing at room temperature for 4 h. Filtering with filter cloth, filtering with K-type multi-layer plate frame to clarify, adding saturated formaldehyde solution 0.1%, stirring, standing at room temperature for 24 hr while shaking for several times. Filtering and sterilizing by using a 0.22 mu m microporous filter element. Filtering with ultrafiltration membrane with molecular weight cutoff of 1000KDa to remove virus.

Example 7 preventive test of Duck viral liver bivalent yolk antibody

Duck liver bivalent yolk antibody (group A), DHAV-1 antibody (group B) and DHAV-3 antibody (group C) are respectively injected intramuscularly with ducklings of 1 day age, 6 days age, 12 days age and 18 days age, each timeGroups were 10 per day of age. The ducklings of 1 day age are injected with 0.5ml of antibody per duck, and the ducklings of 6 day age, 12 day age and 18 day age are injected with 1.0ml of antibody per duck. 100LD for each duckling 24 hours after antibody injection₅₀DRL-62 and SD 1. The virus control and the blank control are respectively 10 for virus attack. The test groups were separately housed and observed for 10 days, and the number of dead ducklings was recorded.

The results show that the duck liver bivalent yolk antibody can protect all the ducklings at each age of days to be healthy, and the DHAV-1 antibody and the DHAV-3 antibody which are used alone can obviously not resist the attack of the DHAV-1 and the DHAV-3. The results are shown in Table 9.

TABLE 9 preventive test results

Note: the ratio in the table is the survival rate of the ducklings, and less than 10 groups of test animals are caused by nonspecific death.

Example 8 Duck viral hepatitis bivalent yolk antibody treatment test

8.1: the test was divided into A, B, C3 groups, each of which was randomly selected 60 4-day-old ducklings infected with DHAV-1 and DHAV-3 artificially in laboratory conditions. The ducklings in the group A are divided into 3 groups, each group comprises 20 ducklings, and duck virus hepatitis bivalent yolk antibodies are respectively injected into muscles 24 hours, 36 hours and 48 hours after infection, wherein each ducklings is 1.0 ml; group B and group C were performed as in group A, and were treated with DHAV-1 and DHAV-3 duck viral hepatitis antibodies, respectively. The experiment was carried out with 10 ducklings without injection as a blank control. 10 post-infection controls were included in each group. Feeding each component separately, observing for 10 days, and observing and recording the death condition of the ducklings.

The results show that all ducklings do not die within the observation period after being treated by the antibody 24 hours after infection, and the curative ratio of the ducklings is obviously reduced by using the antibody treatment group 48 hours after toxicity attack. Therefore, the earlier the duckling is infected with the antibody, the protection effect can be greatly improved. The results also show that effective protection was not achieved with DHAV-1 or DHAV-3 duck liver antibodies alone. The blank control was fully alive and the untreated group was fully dead after 10 days, and the results are shown in table 10.

TABLE 10 test results for treatment of Artificial infections

Note: the ratio in the table is the survival rate of the ducklings, and less than 10 groups of test animals are caused by nonspecific death.

8.2: the disease of 6-day-old ducklings in Henan Xinyang duck farms is caused, the ducklings show lassitude, anorexia, necking down and lying down, death begins to occur at the age of 7 days, leg spasm and angle bow reversal occur before death, the ducklings are sent to the Xinyang animal epidemic disease prevention and control center for inspection, the autopsy finds that liver swelling, bleeding, kidney swelling, congestion and spleen swelling, liver tissues are collected for RT-PCR detection, and the result is that DHAV-1 and DHAV-3 duck virus hepatitis is positive. At 8 days of age, the control treatment groups 1 and 2 were treated with a bivalent yolk antibody subcutaneously and DHAV-1 and DHAV-3 duck liver antibodies, respectively, for 10 days. The results showed that 1622 ducklings did not die from day 5 after treatment, 1339 ducklings were alive at the same time, the protection rate was 81.6%, and the protection rates of the control treatment groups 1 and 2 were only 6.3% and 57.8%, respectively, and the results are shown in table 11. Therefore, the bivalent antibody can play a relatively ideal protective effect on the prior clinical multiple DHAV-1 and DHAV-3 duck viral hepatitis, and the DHAV-1 or DHAV-3 antibody alone cannot effectively control the occurrence of different serotype duck viral hepatitis.

TABLE 11 treatment of natural onset of disease test results

Example 9 preparation of DHAV-3 SD1 yolk antibody and test of protective Effect

Preparation of DHAV-3 SD1 yolk antibody

1.1 preparation of antigen Duck hepatitis virus SD1strain virus seed is diluted 1: 100 with sterilized PBS solution, and 10-day-old susceptible Duck embryo, 0.2ml per embryo, is inoculated via allantoic cavity. And (3) continuously incubating at 37 ℃, discarding dead embryos within 48 hours, harvesting dead duck embryos within 48-96 hours, mixing the duck embryo solutions for sterile inspection, centrifuging at 3500 rpm for 30 minutes, sucking the supernatant into a sterilization container, adding 10% formaldehyde solution, shaking with the addition of the formaldehyde solution to fully mix the duck embryo solutions, wherein the final concentration of the formaldehyde solution is 0.15%, sealing, and then placing in a 37 ℃ incubator for 24 hours (shaking for 3-4 times).

1.2 preparation of oil phase of inactivated vaccine for immunization: mixing 94 parts of white oil for injection with 806 parts of span-aluminum stearate, heating while stirring until the mixture is transparent, and autoclaving for later use to obtain an oil phase. Preparing a water phase: adding sterilized Tween-80 in an amount of 4% of the antigen, and shaking to dissolve Tween-80 completely to obtain water phase. Emulsification: placing 2 parts of oil phase into an oil phase tank, starting a motor to stir, slowly adding 1 part of water phase, and emulsifying at 3400 r/min for 10 minutes. 1% thimerosal was added to a final concentration of 0.01% before the emulsification was terminated. The samples were centrifuged at 3000 rpm for 15 minutes without separation. Subpackaging: quantitatively subpackaging, and sealing with cover.

1.3 immune layer chicken about 120 days old is injected with 1ml of inactivated oil vaccine, 14 days later is injected with 1ml of inactivated vaccine, 10 days later for secondary immune layer and 10 days later for tertiary immune layer are injected with 1ml of inactivated vaccine. When the neutralization titer of the SD1 antibody in the egg yolk is more than or equal to 1: 8192, egg collection is carried out, and the egg is stored at 4 ℃ for standby.

1.4 preparation of yolk antibody eggshells were disinfected and the yolk collected. Stirring thoroughly to make yolk into uniform paste, adding equal volume of distilled water sterilized and cooled at 121 deg.C for 30min, stirring, and inactivating at 62.5 deg.C for 30 min. Sterilized distilled water with the pH value of 4.2 and the volume which is 6 times of the volume of the original yolk is added into an interlayer reaction tank, and the temperature is reduced to 4 ℃. Then, the inactivated egg yolk solution was added with stirring, and allowed to stand at 4 ℃ for 4 hours. The supernatant was centrifuged by a tubular low temperature continuous centrifuge at 14000rpm and transferred to another reaction tank. Adding octanoic acid according to 0.2% (volume ratio) of the total amount, stirring well, and standing at room temperature for 4 h. Filtering with filter cloth, filtering with K-type multi-layer plate frame to clarify, adding saturated formaldehyde solution 0.1% of the total amount into the filtrate, stirring, standing at room temperature for 24 hr while shaking for several times. Filtering and sterilizing by using a 0.22 mu m microporous filter element. Filtering with ultrafiltration membrane with molecular weight cutoff of 1000KDa to remove virus. Thus obtaining the SD1 egg yolk antibody.

DHV SD1 yolk antibody toxic counteracting and protecting effects

The 4-day-old ducklings are divided into 1 group, 2 groups and 3 groups, wherein each group of 1 group and 2 groups comprises 10 ducklings, and each group of 3 groups comprises 5 ducklings. The antibody is injected into each duck in 0.5 ml/group 1, and 100LD is used for each duckling in groups 1 and 224 hours after the injection of the antibody₅₀SD1 virulent attack. Group 3 is a blank control group. The test groups were separately housed and observed for 10 days, and the number of dead ducklings was recorded.

The results show that the SD1 antibody can protect the ducklings to completely survive after the immune group is attacked, the virus control group to completely die, and the blank control group to completely survive. The results are shown in Table 12.

TABLE 12 SD1 antibody challenge protection test results

Group of	Immunization group	Virus control group	Blank control group
				Survival rate of ducklings	10/10	0/10	5/5

Example 10 DHAV-3 SD1 VP1 protein immune Effect test

Extracting virus RNA from the virus liquid harvested from the duck embryo according to the instructions of the Trizol virus RNA extraction kit. The DHAV-3 SD1 VP1 gene sequence was amplified by RT-PCR. Cloning the VP1 gene into an expression vector pET-32a (+), screening a prokaryotic expression vector pET-32a-VP1, transforming Escherichia coli BL21 cells for mass expression, and cracking, washing, purifying, renaturing and concentrating the inclusion body protein to obtain the DHAV-3 SD1 VP1 protein. The protein concentration was quantified by UV absorption and the resulting protein was diluted to 850. mu.g/ml and vaccinated as described in 1.2 of example 9, immunizing 10 SPF-chickens with 1.0 ml/ml of protein injected subcutaneously. Blood is collected 10 days after the three-immunization, serum is separated, and inactivation is carried out at 56 ℃. The 4-day-old ducklings are divided into 4 groups of 10 ducklings. Group 1 injection of recombinant protein (VP1) antiserum, 0.5 ml/mouse; group 2 injections of SD1 antiserum, 0.5 ml/mouse; group 3 was non-immunized group without any serum injection; the ducklings of 1, 2 and 3 groups on the next day were attacked with SD1 virulent strain, and 4 groups were used as blank control groups. Each group was kept in isolation for 10 days.

The result of the challenge shows that the SD1 antiserum group ducklings survive 10/10; recombinant protein antiserum group duckling survives 5/10; while the green ducks of the non-immune group die. The DHV SD1 recombinant VP1 protein antiserum immune duckling can partially resist lethal attack of SD1 virulent virus, and the DHV SD1 recombinant VP1 protein has considerable immunogenicity, so that the duckling obtains immune protection.

TABLE 13 immune Effect of DHAV-3 SD1 VP1 protein

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

SEQUENCE LISTING

<110> Puleco bioengineering GmbH

<120> duck viral hepatitis bivalent yolk antibody and preparation method and application thereof

<130> 12NAP0195C-1

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 745

<212> DNA

<213> Duck hepatitis Virus

<400> 1

caatgatcag ggtgattcca atcagcttgg tgatgatgaa ccagtgtgtt ttctcaattt 60

tgagactgca aatgtgccaa tacaagggga gtcgcacacc ttggtgaaac atctttttgg 120

ccgtcaatgg ctggttcgta ctgttcaaca tactactgag gtacaagagt tggatttgcc 180

agtacctgac cagggtcatg catctctgtt gcgcttcttt gcctatttct ctggggaagt 240

gattctgacc attgttaata atggaacaac accctgcatg gttgcacact cttatacaat 300

ggacaatctc acttctgaat atgctgtcac tgccatgggg ggtattctta tcccagcaaa 360

ctctgccaag aatattaata tcccatttta ttctgttaca cctttacgcc ccacacgacc 420

catgccagca tctcaggggg gtggcttgac ttttggcagg ttgtatattt ggacacaatc 480

aggaagtgtt tctgttttta tgggcctcca taagccagct ttgtttttcc cactacctgc 540

accaacctac acaacacaca cgctgttgaa taagattgaa accatgaatc tgcataatca 600

atcagatcag ccagattgcc atctgtgtga gatttgtagg aaaatgaaaa aatggtctcg 660

caaccatcgc ccatttcgct tctgtttgag actcaaaaca cttgcctttg agctccattt 720

ggaaattgaa tcataccaat ttcga 745

<210> 2

<211> 240

<212> PRT

<213> Duck hepatitis Virus

<400> 2

Gly Asp Ser Asn Gln Leu Gly Asp Asp Glu Pro Val Cys Phe Leu Asn

1 5 10 15

Phe Glu Thr Ala Asn Val Pro Ile Gln Gly Glu Ser His Thr Leu Val

20 25 30

Lys His Leu Phe Gly Arg Gln Trp Leu Val Arg Thr Val Gln His Thr

35 40 45

Thr Glu Val Gln Glu Leu Asp Leu Pro Val Pro Asp Gln Gly His Ala

50 55 60

Ser Leu Leu Arg Phe Phe Ala Tyr Phe Ser Gly Glu Val Ile Leu Thr

65 70 75 80

Ile Val Asn Asn Gly Thr Thr Pro Cys Met Val Ala His Ser Tyr Thr

85 90 95

Met Asp Asn Leu Thr Ser Glu Tyr Ala Val Thr Ala Met Gly Gly Ile

100 105 110

Leu Ile Pro Ala Asn Ser Ala Lys Asn Ile Asn Ile Pro Phe Tyr Ser

115 120 125

Val Thr Pro Leu Arg Pro Thr Arg Pro Met Pro Ala Ser Gln Gly Gly

130 135 140

Gly Leu Thr Phe Gly Arg Leu Tyr Ile Trp Thr Gln Ser Gly Ser Val

145 150 155 160

Ser Val Phe Met Gly Leu His Lys Pro Ala Leu Phe Phe Pro Leu Pro

165 170 175

Ala Pro Thr Tyr Thr Thr His Thr Leu Leu Asn Lys Ile Glu Thr Met

180 185 190

Asn Leu His Asn Gln Ser Asp Gln Pro Asp Cys His Leu Cys Glu Ile

195 200 205

Cys Arg Lys Met Lys Lys Trp Ser Arg Asn His Arg Pro Phe Arg Phe

210 215 220

Cys Leu Arg Leu Lys Thr Leu Ala Phe Glu Leu His Leu Glu Ile Glu

225 230 235 240

Claims (10)

1. The duck viral hepatitis 3 virus strain is a duck viral hepatitis virus SD1strain, the SD1strain is a China center for type culture Collection, and the preservation number is CCTCC NO. V201225.

2. A DNA sequence of a duck viral hepatitis type 3 virus strain VP1, wherein the DNA sequence is shown as SEQ ID No. 1.

3. A duck virus hepatitis 3 virus VP1 antigen protein, wherein the amino acid sequence of the VP1 antigen protein is shown in SEQ ID No.2, and the antigen protein has three amino acid substitution sites of 49-T, 196-N and 207-E and three common sites of 197-Q, 198-S and 199-D.

4. A duck viral hepatitis divalent yolk antibody, which comprises a duck viral hepatitis DHAV-1 type DRL-62 strain and a yolk antibody prepared from the duck viral hepatitis DHAV-3 type SD1strain of claim 1, wherein the DHAV-1 type DRL-62 strain is deposited by American type culture Collection ATCC with the deposit number being VR-1313.

5. A preparation method of a duck virus hepatitis bivalent yolk antibody comprises the following steps:

(1) respectively inoculating 9-day-old SPF chicken embryos and 10-day-old susceptible duck embryos with the duck virus hepatitis DHAV-1 strain DRL-62 and the DHAV-3 strain SD1, which are described in claim 4, then harvesting allantoic fluid, inactivating the harvested virus fluid with formaldehyde, mixing the inactivated virus fluid with the allantoic fluid in proportion, and preparing inactivated vaccines for immunization;

(2) immunizing laying hens by using the prepared inactivated vaccine for immunization, sampling and measuring the neutralizing titer of anti-DHAV-1 and anti-DHAV-3 antigen antibodies in the hyperimmune egg yolk of the laying hens after immunization to be more than or equal to 1: 8192, and then collecting hyperimmune eggs of the laying hens;

(3) sterilizing the high-immunity egg shells, collecting egg yolks, adding distilled water with the same volume as the egg yolks, uniformly stirring and inactivating the egg yolks by low-temperature pasteurization; purifying by an acidification distilled water method and an octanoic acid method; and (4) performing microfiltration and ultrafiltration.

6. The preparation method according to claim 5, wherein the low temperature pasteurization conditions are heat inactivation at 62.5 ℃ for 30 min.

7. The method of claim 5, wherein the acidified distilled water purification step is: adding sterilized distilled water with the volume 6 times that of the yolk into an interlayer reaction tank, wherein the pH value of the distilled water is 4.2, cooling to 4 ℃, adding the inactivated yolk liquid, stirring, standing at 4 ℃ for 4 hours, and centrifugally separating supernatant;

the method for purifying the caprylic acid comprises the following steps: adding caprylic acid according to 0.2% of the total volume of the supernatant, stirring, standing at room temperature for 4h, filtering to be clear, adding a saturated formaldehyde solution according to 0.1% of the total volume of the filtrate, stirring, and standing at room temperature for 24 h.

8. The preparation method according to claim 5, wherein the microfiltration is performed by filtration sterilization using a 0.22 μm microporous filter cartridge, and the ultrafiltration is performed by virus removal by ultrafiltration using an ultrafiltration membrane having a molecular weight cutoff of 1000 kDa.

9. The use of the divalent yolk antibody against duck viral hepatitis according to claim 4 in the preparation of a medicament for preventing and treating duck viral hepatitis.

10. A vaccine composition for preventing and treating duck viral hepatitis, which comprises an antigen prepared from the DHAV-3 type SD1strain of duck viral hepatitis of claim 1 and a pharmaceutically acceptable carrier.

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