CN111454336A

CN111454336A - Modified duck circovirus Cap protein and preparation method and application thereof

Info

Publication number: CN111454336A
Application number: CN202010079714.1A
Authority: CN
Inventors: 张勇; 王宏华; 王秀云; 曹阳; 王辉; 李佳礼; 焦绪娜; 辛瑞祥; 刘磊
Original assignee: Weifang Huaying Biotechnology Co Ltd
Current assignee: Weifang Huaying Biotechnology Co Ltd
Priority date: 2020-02-04
Filing date: 2020-02-04
Publication date: 2020-07-28
Anticipated expiration: 2040-02-04
Also published as: CN111454336B

Abstract

The invention aims to provide a modified duck circovirus Cap protein and a preparation method and application thereof, namely, a peptide segment rich in arginine at 21-36 sites at the N end of the duck circovirus Cap protein is deleted, a general T cell epitope is introduced into the deleted region to promote immunogenicity, the amino acid sequence of the modified duck circovirus Cap protein is SEQ ID NO 1, one nucleotide sequence is SEQ ID NO 2, the modified duck circovirus Cap protein gene is connected with an expression vector and then is transferred into escherichia coli B L21 (DE3) to realize high-efficiency soluble expression of the duck circovirus Cap protein, and the expressed protein is spontaneously assembled into virus-like particles and can be used for developing products such as duck circovirus genetic engineering subunit vaccines, yolk antibodies and the like.

Description

Modified duck circovirus Cap protein and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a modified duck circovirus Cap protein, and a preparation method and application thereof.

Background

Duck circovirus (DuCV), the first virus found in diseased Duck tissue in Hattermann, German scholarly, equal to 2003, was tentatively colonized by ICTV (the International Committee on Taxinomy of Virus) in the genus circovirus in 2007. At present, duck circovirus is reported in Germany, Hungary, America, Taiwan China and other areas, and China also detects the existence of the duck circovirus in Fujian areas in 2008. The duck circovirus can cause the symptoms of disorder of duck feather, growth retardation, weight loss and the like, can latently infect ducks and invade the immune system of the ducks to cause the reduction of the immune function of organisms, and often forms mixed infection with pathogens such as duck influenza virus, duck pox virus, duck reovirus, duck hepatitis virus, duck escherichia coli, riemerella anatipestifer and the like to cause the great improvement of the death rate of duck groups. Therefore, the duck circovirus as an important pathogenic microorganism causes great harm to the duck breeding industry in China, and the development of safe and effective preventive and therapeutic preparations such as vaccines, yolk antibodies and the like is urgently needed.

Limegalon et al (patent application No: CN201810793855) successfully realizes the in vitro separation of duck circovirus by using freshly prepared duck blood PBMC cells, and the virus titer reaches 8 × 10^7.0Copy/ml. However, the method can only be used for separating the virus, the production cost is too high for the antigen production of the duck circovirus vaccine, and the process is too complexZhang Heng et al (patent application No. CN201910707293) reports a method for separating and culturing duck circovirus by using a continuous cell line, namely chicken liver cancer cells (L MH cells), but the method does not show the virus titer of the duck circovirus and does not introduce large-scale culture.

Capsid protein (Cap protein) coded by the ORFC1 gene of duck circovirus is the only structural protein of the virus, has good immunogenicity, and is often used as target protein for developing genetic engineering vaccines. Cao wenlong et al (patent application No. CN201910530866) utilize a baculovirus expression system to realize the expression of the Cap protein of the duck circovirus, and the expressed protein can form virus-like particles, thereby showing good application prospects. However, the production cost of the biological agent prepared by the eukaryotic system expression protein is high for the industrial application of poultry biological products. The efficient expression of the duck circovirus Cap protein in escherichia coli is realized by deleting a region which affects the expression of the duck circovirus Cap protein and is rich in arginine at the N end (2014) of the duck circovirus Cap protein. However, the protein prepared by the method does not form virus-like particles with biological activity, and the expressed protein is an inactive inclusion body, so the application of the protein is greatly limited.

Disclosure of Invention

The invention aims to provide a modified duck circovirus Cap protein, namely, a 21-36 site peptide segment (RRFRRRR L RIARPRR) rich in arginine at the N end of the duck circovirus Cap protein is replaced by a T cell epitope peptide segment (TAKSKKFPSYTATYQF) capable of promoting cell immunity, and the modified duck circovirus Cap protein is prepared by escherichia coli rare codon optimization, prokaryotic expression, nickel column purification and the like.

The invention firstly provides a modified duck circovirus Cap protein, the amino acid sequence of the coded protein is SEQID NO. 1;

one nucleotide sequence of the protein is SEQ ID NO. 2;

the modified duck circovirus Cap protein gene is connected with a pET28a expression vector to construct recombinant plasmid, the recombinant plasmid is converted into escherichia coli B L21 (DE3) host bacteria, after induced expression, through SDS-PAGE analysis, the expression strain is broken to have 30kD recombinant target protein in supernatant, Western-blot identification shows that the recombinant plasmid can generate specific bands with duck circovirus positive serum, and electron microscope results show that the recombinant protein is spontaneously assembled into duck circovirus virus-like particles with the diameter of 20 nm.

The invention also provides a duck circovirus genetic engineering subunit vaccine, wherein the antigen is the duck circovirus virus-like particle, and the concentration of the antigen is more than 0.15 mg/ml.

The invention relates to a duck circovirus genetic engineering subunit vaccine, which comprises the following preparation steps:

1) carrying out induction fermentation on the duck circovirus recombinant gene engineering bacteria to prepare expression bacteria;

2) carrying out high-pressure homogeneous crushing and nickel column purification on the expression thallus to prepare purified target protein;

3) adding white oil adjuvant into the target protein to prepare the vaccine.

The invention also provides a duck circovirus egg yolk antibody, wherein the antigen is the duck circovirus virus-like particles, and the concentration of the duck circovirus egg yolk antibody is more than 0.15 mg/ml.

The preparation method of the duck circovirus egg yolk antibody comprises the following steps:

1) immunizing laying hens with the prepared duck circovirus gene engineering subunit vaccine for multiple times to prepare duck circovirus hyperimmune eggs;

2) the high-immunity egg is subjected to production processes of yolk separation, inactivation, extraction, filtration, subpackaging and the like to prepare a finished product of the yolk antibody, wherein the agar-agar titer of the antibody reaches 1: 16.

According to the invention, 21-36N-terminal arginine-rich peptide fragments of the duck circovirus Cap protein are deleted to promote recombinant expression and correct folding of the Cap protein, meanwhile, in order to promote immunogenicity of the duck circovirus Cap protein, a general T cell epitope capable of promoting immunity is introduced into a deletion region, and finally, biological software is adopted to carry out escherichia coli rare codon optimization, so that efficient soluble expression of the duck circovirus Cap protein is successfully realized.

Detailed Description

The applicant obtains a modified duck circovirus Cap protein amino acid sequence, obtains the gene of the modified duck circovirus Cap protein through optimization of rare codons of escherichia coli, then constructs a genetic engineering bacterium DP strain capable of expressing target protein after being connected with a pET28a prokaryotic expression vector, and prepares the modified duck circovirus Cap protein capable of forming virus-like particles through fermentation, induced expression, thallus fragmentation, protein purification and the like of the engineering bacterium. The protein can be used for preparing duck circovirus genetic engineering subunit vaccines and duck circovirus egg yolk antibodies.

The present invention is further described below with reference to specific embodiments, but it will be understood by those skilled in the art that modifications or substitutions in details and forms of the technical solution of the present invention may be made without departing from the technical solution of the present invention, and these modifications and substitutions fall within the scope of the present invention.

Example 1

Preparation of modified duck circovirus Cap protein and obtaining of engineering bacteria

1. DNASAR biological software analyzes the Cap protein sequence of the duck circovirus SDFC12 strain (GenBank accession number: KY328304), finds that the peptide segment (RRFRRRR L RIARPR) at the N end is rich in arginine and can seriously affect the recombinant expression of the arginine in an escherichia coli expression system, meanwhile, the peptide segment (TAKSKKFPSYTATYQF) of plague bacillus is a T cell epitope with good cellular immune function and can obviously improve the cellular immune function of various immunogens, therefore, the invention replaces the peptide segment (TAKSKKFPSYTATYQF) at the N end 21-36 of the duck circovirus Cap protein rich in arginine with the T cell epitope peptide segment to obtain a modified duck circovirus Cap protein, and simultaneously, 6 histidines is added at the C end of the duck circovirus Cap protein for the convenience of nickel column purification of subsequent modified protein, and the amino acid sequence of the final coding protein is SEQ ID NO 1.

2. And (2) performing escherichia coli rare codon optimization on the modified duck circovirus Cap protein by using online biological software DNAworks to obtain a nucleotide sequence SEQ ID NO 2.

3. And adding Nco I and HindIII enzyme restriction sites to both ends of the newly obtained modified duck circovirus Cap protein nucleotide sequence respectively, and then carrying out whole gene synthesis.

4. The modified duck circovirus Cap protein gene synthesized by the whole gene is subjected to double enzyme digestion by Nco I and Hind III and then is connected with the corresponding enzyme digestion site of a pET28a vector to construct an expression vector.

5. With CaCl₂The method comprises the steps of transforming an expression vector into Escherichia coli B L21 (DE3), coating the Escherichia coli B L (DE3) on an agar plate containing 50 mu g/ml kanamycin, carrying out overnight culture at 37 ℃, selecting 10 single colonies to extract plasmids, carrying out double enzyme digestion to verify positive colonies by Nco I and Hind III, further sequencing and identifying, adding 0.2-0.5 mM IPTG into the positive clones after sequencing verification in a L B culture medium for fermentation culture till 0.6-0.8, inducing for 4-9 hours, carrying out ultrasonic disruption after centrifugally collecting the thalli, carrying out centrifugation to obtain supernatant, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to detect protein expression, and simultaneously setting uninduced thalli as a control, wherein the protein band is more than that of the control bacteria at the position of 30kD after the result is induced, the protein band is consistent with the theoretical molecular weight of recombinant protein, and.

6. And (3) breaking the induced expression strain in the step (5) to carry out supernatant electrophoresis, and carrying out Western-blot identification by using duck circovirus positive serum, wherein the result shows a positive reaction.

7. And (3) dropwise adding the ultrasonication supernatant of the induced expression strain in the step (5) onto a copper net with a carbon film, naturally air-drying, dropwise adding a 2% sodium phosphotungstate solution for negative dyeing, and carrying out electron microscope observation after the negative dyeing. The modified duck circovirus Cap protein can be spontaneously assembled into virus-like particles, and the diameter is about 20 nm.

The results prove that the obtained positive clone is duck circovirus Cap protein engineering bacteria and is named as DP strain.

Example 2

Preparation and inspection of duck circovirus gene engineering subunit vaccine

1 bacterial species

1.1 the strain used for manufacturing is a DP strain of a duck circovirus gene engineering subunit vaccine production strain.

1.2 Strain standards for production

1.2.1 morphological and biochemical Properties

The culture was performed overnight on L B agar plates containing kanamycin, round, well-edged, protruding, milky and glossy smooth colonies appeared on the plates, gram-negative Brevibacterium was observed under the microscope after gram staining, and the results of biochemical tests were glucose fermentation +, indole test +, methyl red test +, VP-, and citric acid utilization test-.

1.2.2 culture characteristics growth in kanamycin-containing medium was possible.

1.2.3 authentication test

1.2.3.1 PCR detection Using L B liquid culture of this strain as a template, the following PCR primers should be used for PCR amplification to amplify a fragment of about 574 bp.

P1：5′-TTGGTTTCTTCGGTTCTC-3′

P2：5′-ATGTCGTATTGTGCGTTA-3′

The amplification conditions were as follows: denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30S, denaturation at 50 ℃ for 30S, denaturation at 72 ℃ for 1min, 30 cycles, and elongation at 72 ℃ for 10 min.

1.2.3.2 Western-blot detection, the thalli induced to express are resuspended by PBS and then are homogenized and crushed under high pressure, and specific bands are required to appear when the supernatant and duck circovirus resistant positive serum are subjected to Western-blot test.

1.2.4 purely with appropriate medium check, should be pure.

1.2.5 the freeze-dried strain is preserved at-20 ℃ and the preservation period is 2 years.

2 vaccine preparation and semi-finished product inspection,

2.1 seed preparation for production

2.1.1 first-order seed propagation and identification lyophilized strains were inoculated respectively in L B liquid medium containing kanamycin, shake-cultured at 37 ℃ for 8-10 hours, and then streaked on L B solid medium containing kanamycin, and cultured at 37 ℃ for 16-18 hours, as first-order seeds.

2.1.2 propagation of Secondary seeds 10 typical colonies meeting the criteria of 1.2.1 were selected from the primary seeds and mixed in a small amount of L B culture medium, inoculated in L B culture medium containing kanamycin, cultured at 37 ℃ for 8-10 hours, and subjected to a pure test.

2.2 the culture medium for preparing the vaccine is an improved L B culture medium, and each 1000ml of the culture medium contains 10g of tryptone, 5g of yeast extract powder, 10g of sodium chloride, 5g of glucose and MgSO₄·7H₂O5g。

2.3 preparation of antigen solution for preparing vaccine

2.3.1 aerobic culture in a culture tank for bacterial liquid culture, charging a proper amount of culture medium (about 70%) and antifoaming agent according to the volume of the culture tank, sterilizing, inoculating secondary seed bacterial liquid according to 1-10% of the culture medium, aerobic culture at 37 deg.C, and waiting for OD of the bacterial liquid₆₀₀When the value reaches 7.0, 2-10 g/L of α -lactose is added for induction, then the culture is continued for 6-8 hours, the pH is adjusted by using 20% NaOH, the dissolved oxygen is controlled by the association of the rotating speed, and when the dissolved oxygen rapidly rises, the feeding is carried out.

2.3.2 after the completion of the disruption culture, the cells were collected by centrifugation. The collected bacteria are cleaned and made into 1-10% suspension by PBS, and bacteria are crushed by a high-pressure homogenizer. The crushed bacterial liquid is centrifuged for 15 minutes at 8000r/min, and the supernatant is collected.

2.3.3 purifying the conventional nickel column by using a nickel column chromatography, putting the collected recombinant protein eluent into a dialysis bag, taking PBS (phosphate buffer solution) as dialysis external liquid, and dialyzing and desalting to obtain the recombinant protein liquid.

2.3.4 inactivation A10% formaldehyde solution was added to the purified supernatant in proportion to a final concentration of 0.2% and was inactivated at 37 ℃ for 12 hours. Taking a small amount of samples to carry out semi-finished product inspection.

2.4 inspection of semi-finished products

2.4.1 protein content detection the protein concentration of the supernatant is detected by BCA method, diluted to 0.5mg/ml final concentration, and sterile filtered for later use.

2.4.2 sterility test the sterility test is carried out according to the current Chinese veterinary pharmacopoeia, and the sterility growth is required.

2.4.3 detection of endotoxin the endotoxin detection was carried out by limulus reagent method, and the vaccine was prepared in the case where the endotoxin content was not higher than 1000 EU/ml.

2.5 vaccine preparation

2.5.1 preparing oil phase 94 parts of high-quality white oil for injection and 2 parts of aluminum stearate. And (2) uniformly mixing in an oil phase tank, heating to melt the mixture to be semitransparent, adding 806 parts of span, keeping the temperature for 30 minutes when the temperature reaches 125-130 ℃, and cooling to room temperature for later use.

2.5.2 preparing water phase by adding 96 parts of qualified semi-finished product into 804 parts of sterilized Tween-80, and stirring until Tween-80 is completely dissolved.

2.5.3 emulsifying, namely placing 2 parts of oil phase in a high-speed shearing machine, starting a motor to stir at a low speed, slowly adding 1 part of water phase, emulsifying for 40 minutes at 3600r/min, and adding 1% thimerosal solution before stopping stirring until the final concentration reaches 0.01%. After emulsification, 10ml of the sample is added into a centrifuge tube and centrifuged at 3000r/min for 15 minutes, and an anhydrous phase is separated out at the bottom of the tube.

2.5.4 subpackaging and quantitatively subpackaging, and sealing the bottle mouth.

3 inspection of finished products

3.1 physical Properties

The appearance was a milky white emulsion.

The dosage form is water-in-oil type. A clean pipette is taken, a small amount of vaccine is sucked and dropped into cold water, and the vaccine is not diffused except for the 1 st drop.

Adding 10ml of the stable suction vaccine into a centrifuge tube, centrifuging for 15 minutes at 3000r/min, and separating out an anhydrous phase at the bottom of the tube.

The viscosity is carried out according to the current Chinese animal pharmacopoeia and conforms to the regulations.

The inspection of the loading amount is carried out according to the current Chinese animal pharmacopoeia, which conforms to the regulations.

3.2 sterile test according to the existing Chinese animal pharmacopoeia, and sterile growth.

3.3 safety inspection 20 healthy and susceptible ducks of 7 days old were equally divided into two groups of 10 ducks each. The first group of legs were injected intramuscularly with the vaccine prepared according to the invention at a dose of 0.4ml per leg, and the second group of legs were injected intramuscularly with the same volume of physiological saline. The duck group is observed 21 days after immunization, and as a result, both groups of ducklings are healthy and alive without any adverse reaction.

3.4 the residual quantity of the formaldehyde and mercury preservatives is determined according to the current Chinese animal pharmacopoeia, and the determination conforms to the regulations.

Example 3

Efficacy test of duck circovirus gene engineering subunit vaccine

1 preparation of control vaccine

Referring to the preparation procedure of tissue inactivated vaccine of 2015 edition of Chinese veterinary pharmacopoeia, duck circovirus WF strain is proportionally added into 10% formaldehyde solution, the final concentration of the formaldehyde solution is 0.2%, and inactivation is carried out for 12 hours at 37 ℃. After complete inactivation, the duck circovirus tissue inactivated vaccine is prepared by the method of the 2.5 vaccine preparation part and the 3 finished product inspection part in the example 2.

2 animal efficacy test

The 7-day-old healthy susceptible ducks are divided into two groups of 20 ducks on average. The first group is Cap protein vaccine immunization group, and 0.2 ml/duck circovirus gene engineering subunit vaccine prepared by the invention is used for leg muscle immunization. The second group is a tissue inactivated vaccine group, and the prepared duck circovirus tissue inactivated vaccine is used for leg muscle immunization, wherein each duck circovirus tissue inactivated vaccine is 0.2 ml. The third group was a saline control group, which was injected with the same volume of sterile saline. After 21 days of immunization, 0.2ml of cyclovirulent WF (containing 10) strain of muscle meat ducks of each group^4.0ID₅₀) The immune protection rate (percentage of the number of diseased to the total number) was counted 14 days after challenge. Wherein the pathogenesis standard is as follows: 1. feather malnutrition, shaft bleeding, burrs and gradual emaciation; 2. and death. One of the two is considered to be the onset of disease.

As a result: the immune protection rate of the duck group after the immunization of the Cap protein vaccine immunization group vaccine is 100%, the immune protection rate of the duck group after the immunization of the contrast vaccine group is 55%, and the disease incidence rate of the duck group of the normal saline contrast group is 90%. Proved that the duck circovirus genetic engineering subunit vaccine has good clinical protection effect on duck groups, is superior to duck circovirus tissue inactivated vaccine, and can be clinically popularized and applied.

TABLE 1 EXAMPLE 3 challenge protection assay following immunization with Duck circovirus genetically engineered subunit vaccine

Example 4

Preparation and inspection of duck circovirus egg yolk antibody

1 immunization of layer chickens

Immunizing laying hens with the duck circovirus gene engineering subunit vaccine prepared in example 2, immunizing 0.2ml of vaccine prepared by subcutaneous injection of each chicken neck for the first time, immunizing for the second time after 14 days, immunizing for the third time after 0.4ml of vaccine prepared by subcutaneous injection of each chicken neck, immunizing for the boosting time after 14 days, and collecting and determining the egg yolk circovirus agar titer, wherein the egg yolk circovirus agar titer is not less than 1:64 after 14 days after boosting immunization, and the egg yolk circovirus titer is determined by subcutaneous injection of 0.4ml of vaccine prepared by subcutaneous injection of each chicken neck.

2 yolk antibody production

2.1 Eggshell Sterilization the collected hyperimmune eggs were immersed in 0.1% aqueous benzalkonium bromide at 42 ℃ for 15 minutes. The eggshell is selected in advance when the pollution is serious, and is soaked and disinfected for 1 time after being washed by the disinfection water independently.

2.2 egg breaking and yolk separation by manual or mechanical egg breaking. Egg white, blastoderm and frenulum were removed sufficiently and egg yolk was collected.

2.3 the inactivation I is fully stirred to make the yolk into uniform paste, sterile water for injection with the same volume and the pH value of 6.5 is added, the mixture is uniformly stirred, heated and inactivated at the temperature of 60-65 ℃ for 30 minutes, and cooled to the room temperature.

2.4 extraction in a stainless steel tank adding 6 times volume of acidified water (water for injection with pH adjusted to 4.2 by hydrochloric acid) of original yolk, cooling to 4 deg.C, adding inactivated I yolk solution while stirring, standing at 4 deg.C for 4-8 hr, acidifying, centrifuging at low temperature to separate supernatant, and transferring into another reaction tank.

2.5 adding n-octanoic acid with the final concentration of 0.2% into the inactivated II supernatant, uniformly stirring, and standing for 5-10 hours at room temperature.

2.6 filtration the mixture is clarified by filtration using a suitable filtration method, for example filtration using a cartridge filter.

2.7 adding a formaldehyde solution with the final concentration of 0.05% into the filtrate, fully and uniformly stirring, standing for 24 hours at room temperature, and stirring for 4-6 times.

2.8, filtering, sterilizing, adding a proper amount of sodium hydroxide to adjust the pH value to be about 6.8-7.2, and filtering and sterilizing by using a 0.22 mu m microporous filter element.

2.9 sterility test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the growth is carried out aseptically.

The titer of the 2.10 agar-agar antibody is determined to be not less than 1: 16.

2.11 subpackaging, aseptically and quantitatively subpackaging the qualified semi-finished products, and covering and sealing.

3 inspection of finished products

3.1 physical Properties this product is a clear liquid. After standing for 48 hours, there was a little precipitate at the bottom of the bottle. The pH value is 6.8-7.2.

3.2 the filling quantity is checked according to the appendix of the current Chinese animal pharmacopoeia, and the filling quantity is in accordance with the regulations.

3.3 sterility test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the growth is carried out aseptically.

3.4 Mycoplasma examination was performed according to the appendix of the current "Chinese veterinary pharmacopoeia" and no mycoplasma should grow.

3.5 the test of exogenous virus is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and exogenous virus pollution is avoided.

3.6 safety experiments 20 healthy susceptible ducks of 7 days old were equally divided into two groups of 10 ducks each. The first group of legs are injected with 1.0ml of the yolk antibody prepared by the invention per egg, and the second group of legs are injected with the same volume of physiological saline. The duck group is observed 21 days after immunization, and the duck group should be healthy and alive without any adverse reaction.

Example 5

Potency assay for duck circovirus egg yolk antibodies

1 preparation of control egg yolk antibody

The control vaccine prepared in example 3 is used for immunizing laying hens according to the experimental steps in example 4 and preparing and testing yolk antibodies, namely the control yolk antibodies, wherein the titer of the agar-agar antibody is not less than 1: 16.

2 animal efficacy test

The 60 healthy and susceptible ducks of 28 days old are evenly divided into two groups, and each group comprises 20 ducks. The first group is a Cap protein treatment group, and 0.2ml (containing 10 parts) of duck circular ring virulent WF strain is injected into leg muscles^4.0ID₅₀) And after 24 hours, 0.5ml of duck circovirus egg yolk antibody is intramuscularly injected. The second group is control yolk antibody treatment group, and 0.2ml (containing 10 parts of virulent WF strain of duck circular ring is injected into leg muscle^4.0ID₅₀) After 24h, the control yolk antibody prepared by the invention is injected into the muscle at 0.5 ml/mouse. The third group is normal saline control group, and leg muscle duck circular ring virulent WF strain 0.2ml (containing 10)^4.0ID₅₀) After 24h, sterile normal saline, 0.5 ml/tube, was injected intramuscularly. The treatment effect is counted 14 days after the toxic materials are attacked. Wherein the pathogenesis standard is as follows: 1. feather malnutrition, shaft bleeding, burrs and gradual emaciation; 2. and death. One of the two is considered to be the onset of disease.

As a result: the duck group after the treatment of the Cap protein treatment group obtains 100% protection, the duck group after the treatment of the contrast yolk antibody treatment group obtains 60% protection, and the duck group of the normal saline contrast group has 80% morbidity. The duck circovirus egg yolk antibody prepared by the invention has good clinical protection effect on duck flocks, is superior to a control egg yolk antibody prepared from duck circovirus tissue inactivated vaccine, and can be clinically popularized and applied.

TABLE 2 EXAMPLE 5 Experimental results of Duck circovirus egg yolk antibody therapeutic effect

Sequence listing

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<120> modified duck circovirus Cap protein, preparation method and application

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Thr Tyr Gln Phe Phe Ser Val Val Thr Tyr Lys Val Thr Arg His Thr

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gcgcgtggca acaatatttg cggtctgaac atgcgttggg cgatgttcgg tgacaccaac 300

tcttacatga ccggttccac cccgttctac cattatccgt acgactacta catgatcaaa 360

ggcgttgcga tcaccctgcg tccggcgtac aacatctacc agaaatctaa aacccagggt 420

tctacggtta tcgacaaaga cggtcagatc gttaaaacct ctaccaccgg ttggtctatc 480

gatccttacg gctctacttc cagccgtcgt acctgggacc cgagccgtgt tcatcgtcgt 540

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ttcctgggtg gtaaaaactt cacctggatc aattgcaccc aagaccaggt tgttcactac 660

ggtatgggta tgtctctccg taaaccagac aatactaccg gtgttaacgc acaatacgac 720

atcgaagcgc aattcacctt ctacatcaaa ttcggtcagt ttaccggctt ccatcatcat 780

catcatcac 789

Claims

1. The modified duck circovirus Cap protein is characterized in that the amino acid sequence of the encoding protein is SEQID NO. 1.

2. The modified duck circovirus Cap protein of claim 1, wherein one nucleotide sequence of the encoded protein is SEQ ID NO 2.

3. The modified duck circovirus Cap protein of claim 1 or 2, prepared by replacing the arginine-rich 21-36 peptide fragment (RRFRRRR L RIARPRR) at the N-terminus of the duck circovirus Cap protein with a T-cell epitope (TAKSKKFPSYTATYQF).

4. The preparation method of the modified duck circovirus Cap protein as defined in any one of claims 2 to 3, wherein the nucleotide sequence as defined in claim 2 is prepared by whole gene synthesis, vector construction, Escherichia coli fermentation and nickel column purification, and the prepared target protein is spontaneously assembled into virus-like particles.

5. A duck circovirus subunit vaccine, wherein the antigen of the vaccine is the modified duck circovirus Cap protein of any one of claims 1 to 3.

6. A duck circovirus egg yolk antibody, wherein an immune layer antigen of the egg yolk antibody is the modified duck circovirus Cap protein of claims 1-3.

7. The use of the duck circovirus subunit vaccine and the duck circovirus egg yolk antibody according to claims 5 to 6 for preventing and treating diseases caused by duck circovirus.