CN111454336B - Modified duck circovirus Cap protein, and preparation method and application thereof - Google Patents
Modified duck circovirus Cap protein, and preparation method and application thereof Download PDFInfo
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Abstract
The invention aims to provide an improved duck circovirus Cap protein, a preparation method and application thereof, namely, deleting a peptide segment rich in arginine at the 21-36 positions of the N end of the duck circovirus Cap protein, introducing a universal T cell epitope into a deleted region to promote immunogenicity, and obtaining the amino acid sequence of the improved duck circovirus Cap protein as SEQ ID NO. 1, wherein one nucleotide sequence is SEQ ID NO. 2. The modified duck circovirus Cap protein gene is transferred into escherichia coli BL21 (DE 3) after being connected with an expression vector, so that the efficient soluble expression of the duck circovirus Cap protein is realized, and the expressed protein is spontaneously assembled into virus-like particles, and can be used for developing products such as subunit vaccines, yolk antibodies and the like of the duck circovirus genetic engineering.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a modified duck circovirus Cap protein, a preparation method and application thereof.
Background
Duck circovirus (DuCV), a tentative species listed in the genus circovirus by ICTV (The International Committee on Taxonomy of Viruses) in 2007, was first discovered in diseased Duck tissue by the German scholars Hattermann equal to 2003. At present, duck circovirus is reported in Germany, hungary, the United states and other areas, and the existence of the virus is detected in Fujian areas in 2008 in China. The duck circovirus can lead to symptoms such as messy duck feathers, slow growth, weight loss and the like, can potentially infect ducks, invade the immune system of the ducks, lead to the reduction of the immune function of organisms, often form mixed infection with pathogens such as duck influenza virus, duck poxvirus, duck reovirus, duck hepatitis virus, duck escherichia coli, riemerella anatipestifer and the like, and greatly promote the death rate of duck groups. Therefore, the duck circovirus is an important pathogenic microorganism, which causes great harm to the duck raising industry in China, and development of safe and effective preventive and therapeutic preparations such as vaccines, egg yolk antibodies and the like is urgently needed.
The industrialized development of biological agents such as vaccines, egg yolk antibodies and the like requires proper immunogens and the establishment of a rapid and economical production method thereof. Li Zhaolong et al (patent application number: CN 201810793855) successfully realized in vitro isolation of duck circovirus with freshly prepared duck blood PBMC cells, and virus titres reached 8X 10 7.0 Copy/ml. However, the method can only be used for separating viruses, and has high production cost and complicated process for producing the duck circovirus vaccine antigen. Zhang Hengdeng (patent application number: CN 201910707293) reports a method for separating and culturing duck circovirus by using a passage cell line, namely chicken liver cancer cells (LMH cells), but the invention does not describe the virus titer achieved by the duck circovirus, and is not introduced in large-scale culture. In addition to the two domestic invention patents, other duck circovirus in vitro culture success reports are not found at home and abroad. The development progress of biological agents of duck circovirus is severely limited by the immaturity of the separation and culture technology of the duck circovirus.
The capsid protein (Cap protein) encoded by the duck circovirus ORF C1 gene is the only structural protein of the virus, has good immunogenicity, and is often used as a target protein for developing genetic engineering vaccines. Cao Wenlong et al (patent application number: CN 201910530866) have realized the expression of duck circovirus Cap proteins by using a baculovirus expression system, and the expressed proteins can form virus-like particles, showing good application prospects. However, eukaryotic system expression proteins are used for preparing biological agents, and the production cost is high for industrial application of poultry biological products. The efficient expression of duck circovirus Cap proteins in E.coli is achieved by deleting the N-terminal arginine-rich region affecting the expression of duck circovirus Cap proteins, such as maize and the like (prokaryotic expression of duck circovirus Cap proteins, animal medicine progress, 2014). 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 that 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-bit peptide (RRFRRRRLRIARPRRR) rich in arginine at the N end of the duck circovirus Cap protein is replaced by a T cell epitope peptide (TAKSKKFPSYTATYQF) capable of promoting cell immunity, and the modified duck circovirus Cap protein is prepared through rare codon optimization, prokaryotic expression, nickel column purification and the like of escherichia coli. The protein can be used as antigen to further prepare biological agents such as duck circovirus genetic engineering subunit vaccine, yolk antibody and the like.
The invention firstly provides an improved duck circovirus Cap protein, the amino acid sequence of the encoded protein is SEQ ID NO. 1;
one nucleotide sequence of the protein is SEQ ID NO. 2;
the invention connects the modified duck circovirus Cap protein gene with pET28a expression vector to construct recombinant plasmid. The recombinant plasmid is transformed into escherichia coli BL21 (DE 3) host bacteria, after induced expression, SDS-PAGE analysis is carried out on the recombinant plasmid, 30kD recombinant target protein exists in the crushed supernatant of the expressed strain, western-blot identification shows that the recombinant plasmid can produce specific bands with duck circovirus positive serum, and an electron microscope result shows that the recombinant protein spontaneously assembles into duck circovirus virus-like particles with the diameter of 20nm.
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 induced fermentation on the duck circovirus recombinant genetic engineering bacteria to prepare expression thalli;
2) Carrying out high-pressure homogenization crushing and nickel column purification on the expressed thalli to prepare purified target protein;
3) Adding white oil adjuvant into target protein to obtain vaccine.
The invention also provides a duck circovirus egg yolk antibody, wherein the antigen is the duck circovirus virus-like particle, 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) The prepared duck circovirus genetic engineering subunit vaccine is immunized for a plurality of times with laying hen, so as to prepare duck circovirus hyperimmune egg;
2) The high-immunity egg is subjected to production processes such as yolk separation, inactivation, extraction, filtration, split charging and the like to prepare a yolk antibody finished product, and the agar-expanded titer of the antibody reaches 1:16.
According to the invention, the peptide segment rich in arginine at the 21-36 position of the N end of the Cap protein of the duck circovirus is deleted to promote the recombinant expression and correct folding of the Cap protein, meanwhile, in order to promote the immunogenicity of the Cap protein of the duck circovirus, a universal T cell epitope capable of promoting immunity is introduced into a deleted region, finally, the rare codon optimization of escherichia coli is carried out by adopting biological software, so that the efficient and soluble expression of the Cap protein of the duck circovirus is successfully realized, the expressed protein avoids a fussy renaturation process on one hand, and on the other hand, the expressed protein can be spontaneously assembled into virus-like particles, so that good application prospects are shown, and excellent effects are achieved when the subunit vaccine of the gene engineering of the duck circovirus and the yolk antibody of the duck circovirus are used for preventing or treating the virulence of the duck circovirus.
Detailed Description
The applicant obtains an amino acid sequence of a modified duck circovirus Cap protein, further obtains a gene of the modified duck circovirus Cap protein through rare codon optimization of escherichia coli, constructs a genetic engineering strain DP capable of expressing a 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 by fermenting, inducing expression, thallus crushing, protein purification and the like of engineering bacteria. The protein can be used for preparing duck circovirus genetic engineering subunit vaccine and duck circovirus egg yolk antibody.
The present invention will be further described with reference to the following specific embodiments, but it will be understood by those skilled in the art that modifications and substitutions can be made in the details and form of the technical solution of the present invention 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. DNAStar biological software analyzes Cap protein sequence of duck circovirus SDFC12 strain (GenBank accession number: KY 328304), and discovers that the 21-36 site peptide (RRFRRRRLRIARPRRR) at the N end of the protein is rich in arginine, which can seriously affect recombinant expression in an escherichia coli expression system. Meanwhile, the peptide (TAKSKKFPSYTATYQF) of the plague bacillus is a T cell epitope with good cell immunity function, and can obviously improve the cell immunity function of various immunogens. Therefore, the N-terminal 21-36 peptide segment of the duck circovirus Cap protein rich in arginine is replaced by the T cell epitope peptide segment (TAKSKKFPSYTATYQF) to obtain the modified duck circovirus Cap protein, and meanwhile, 6 pieces of histidine are added at the C terminal of the modified duck circovirus Cap protein in order to facilitate the subsequent modification of the nickel column purification of the protein, and the amino acid sequence of the final encoded protein is SEQ ID NO. 1.
2. And (3) performing 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 (3) respectively adding enzyme cutting sites of Nco I and Hind III at two ends of the newly obtained modified duck circovirus Cap protein nucleotide sequence, and then carrying out total gene synthesis.
4. The modified duck circovirus Cap protein gene synthesized by the whole gene is digested with Nco I and Hind III, and then is connected to the corresponding digestion site of the pET28a vector, so as to construct an expression vector.
5. By CaCl 2 Method the expression vector was transformed into E.coli BL21 (DE 3), plated on agar plates containing 50. Mu.g/ml kanamycin and incubated overnight at 37 ℃.10 single colonies were selected to extract plasmids, and colonies positive for double restriction verification of Nco I and Hind III were further sequenced and identified. And fermenting and culturing the positive clone subjected to sequencing verification in an LB culture medium to 0.6-0.8, adding 0.2-0.5 mM IPTG, inducing for 4-9 hours, centrifugally collecting thalli, ultrasonically crushing, centrifugally taking supernatant, detecting protein expression by SDS-PAGE electrophoresis, and setting up uninduced thalli as a control. The result shows that the positive clone has one more protein band at 30kD than the control bacterium, and the expression quantity is over 20% in accordance with the theoretical molecular weight of recombinant protein.
6. Crushing the induced expression strain in step 5, performing electrophoresis on the supernatant, and performing Western-blot identification by using duck circovirus positive serum, wherein the result shows a positive reaction.
7. And (3) dripping ultrasonic disruption supernatant of the induced expression strain in step (5) onto a copper mesh with a carbon film, naturally air-drying, dripping 2% sodium phosphotungstate solution for negative dyeing, and performing electron microscope observation after negative dyeing. The modified duck circovirus Cap protein can be spontaneously assembled into virus-like particles, and the diameter size is about 20nm.
The result proves 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 genetic engineering subunit vaccine
1. Bacterial and toxic species
1.1 The strain for manufacturing is a duck circovirus genetic engineering subunit vaccine production strain DP.
1.2 Production strain standard
1.2.1 Morphological and biochemical characteristics
Culturing on LB agar plate containing kanamycin overnight, and displaying round, neat edge, protruding, milky and glossy smooth colony on the culture plate, and displaying gram-negative Brevibacterium under the mirror after gram staining; the biochemical test results are glucose fermentation+, indole test+, methyl red test+, VP-, and citric acid utilization test-.
1.2.2 The culture characteristics can be grown in a medium containing kanamycin.
1.2.3 Authentication verification
1.2.3.1 PCR detection the LB liquid culture of the bacterium is used as a template, and PCR amplification is carried out by using the following PCR primers, so that a fragment of about 574bp can be amplified.
P1：5′- TTGGTTTCTTCGGTTCTC -3′
P2：5 ′- ATGTCGTATTGTGCGTTA-3′
The conditions for amplification were as follows: denaturation at 94℃for 5min, denaturation at 94℃for 30S, denaturation at 50℃for 30S, denaturation at 72℃for 1min for 30 cycles, and elongation at 72℃for 10min.
1.2.3.2 Western-blot detection after the induced expression thalli are resuspended by PBS, the thalli are crushed by high-pressure homogenization, and the supernatant and the positive serum of the anti-duck circovirus are subjected to a Western-blot test, so that a specific band is formed.
1.2.4 Examination purely with a suitable medium should be purely.
1.2.5 Preserving the freeze-dried strain at-20deg.C for 2 years.
Vaccine manufacture and semi-finished product inspection,
2.1 Seed preparation for production
2.1.1 And (3) carrying out primary seed propagation and identification, respectively inoculating freeze-dried strains into an LB liquid medium containing kanamycin, carrying out shaking culture at 37 ℃ for 8-10 hours, and then carrying out streak inoculation on an LB solid medium containing kanamycin for 16-18 hours at 37 ℃ to serve as primary seeds.
2.1.2 The second seed breeding is to select 10 typical colonies meeting 1.2.1 standard from the first seed, mix them in a small amount of LB culture solution, inoculate them in LB culture solution containing kanamycin, culture at 37 deg.C for 8-10 hours, and carry out pure test.
2.2 The culture medium for seedling preparation is an improved LB culture medium, and each 1000ml of culture mediumThe nutrient medium contains tryptone 10g, yeast extract 5g, sodium chloride 10g, glucose 5g and MgSO 4 ∙ 7H 2 O 5g。
2.3 Preparation of antigen liquid for preparing seedling
2.3.1 Aeration culture in culture tank with proper amount of culture medium (about 70%) and defoaming agent, sterilizing, inoculating secondary seed bacterial liquid in 1-10% of culture medium, aeration culturing at 37deg.C, and standing until OD of bacterial liquid is reached 600 When the value reaches 7.0, adding 2-10 g/L of alpha-lactose to induce, and further culturing for 6-8 hours. The pH was adjusted using 20% NaOH and dissolved oxygen was controlled by rotational speed correlation. When dissolved oxygen rises rapidly, feeding material is fed.
2.3.2 After the completion of the bacterial culture, the cells were collected by centrifugation. The collected thalli are washed and then made into 1-10% suspension by PBS, and bacteria are crushed by a high-pressure refiner. The crushed bacterial liquid is centrifuged for 15 minutes at 8000r/min, and the supernatant is collected.
2.3.3 Purifying by nickel column chromatography, and collecting recombinant protein eluent, placing into dialysis bag, and dialyzing with PBS solution as dialysis external solution to desalt to obtain recombinant protein solution.
2.3.4 Inactivation the purified supernatant was proportionally added to a 10% formaldehyde solution having a final concentration of 0.2% and inactivated at 37 ℃ for 12 hours. A small amount of sample was taken for semi-finished product inspection.
2.4 Inspection of semifinished products
2.4.1 Protein content detection supernatant protein concentration was detected by BCA method, diluted to final concentration of 0.5mg/ml, and sterile filtered for use.
2.4.2 The sterility test is carried out according to the current Chinese animal pharmacopoeia, and the sterility test should be carried out.
2.4.3 Endotoxin content detection endotoxin detection was carried out according to the limulus reagent method, and the samples with endotoxin content of not more than 1000EU/ml could be used for preparing seedlings.
2.5 Vaccine preparation
2.5.1 94 parts of high-quality white oil for injection and 2 parts of aluminum stearate are prepared from the oil phase. Mixing uniformly in an oil phase tank, heating to melt to be semitransparent, adding about 80 parts of sauce again, maintaining for 30 minutes when the temperature reaches 125-130 ℃, and cooling to room temperature for standby.
2.5.2 Preparing water phase, namely preparing sterilized Tween-80 by 4 parts, adding 96 parts of qualified semi-finished products, and fully stirring until the Tween-80 is completely dissolved.
2.5.3 2 parts of oil phase is emulsified and taken, the oil phase is placed in a high-speed shearing machine, a motor is started to stir at a low speed, 1 part of water phase is slowly added, the emulsion is carried out for 40 minutes at 3600r/min, and 1% of merthiolate solution is added before the stirring is stopped, so that the final concentration reaches 0.01%. After emulsification, 10ml of the sample was added to a centrifuge tube and centrifuged at 3000r/min for 15 minutes, and no water phase was precipitated at the bottom of the tube.
2.5.4 Packaging, quantifying and packaging, and sealing the bottle mouth.
Inspection of finished products
3.1 Physical properties
The appearance is milky emulsion.
The dosage form is water-in-oil type. A cleaning straw is taken, a small amount of vaccine is sucked and dripped into cold water, and the vaccine is not diffused except the 1 st drip.
10ml of the stable sucking vaccine is added into a centrifuge tube, and the mixture is centrifuged for 15 minutes at 3000r/min, and no water phase is separated out from the bottom of the tube.
The viscosity is carried out according to the current Chinese animal pharmacopoeia, and meets the regulations.
The filling quantity inspection is carried out according to the current Chinese animal pharmacopoeia, and meets the regulations.
3.2 The sterility test is carried out according to the current Chinese animal pharmacopoeia, and the sterility growth is carried out.
3.3 Safety inspection 20 healthy susceptible ducks of 7 days old are equally divided into two groups of 10 ducks each. The first group of leg muscles were injected with the vaccine prepared according to the present invention, 0.4 ml/one, and the second group of leg muscles were injected with the same volume of physiological saline. The duckling group is observed 21 days after immunization, and as a result, the ducklings of both groups are all healthy and alive without any adverse reaction.
3.4 The residual amount of the formaldehyde and mercury preservative is measured according to the current Chinese animal pharmacopoeia, and the residual amount meets the regulations.
Example 3
Efficacy test of duck circovirus genetic engineering subunit vaccine
1. Preparation of control vaccine
Referring to the preparation procedure of tissue inactivated vaccine of China animal pharmacopoeia of 2015 edition, the duck circovirus WF strain is proportionally added into 10% formaldehyde solution, the final concentration of the formaldehyde solution is 0.2%, and the duck circovirus WF strain is inactivated for 12 hours at 37 ℃. After the inactivation is completed, the duck circovirus tissue inactivated vaccine is prepared according to the method of the preparation part of the vaccine and the inspection part of the 3 finished products in the example 2.
Animal efficacy experiment
60 healthy susceptible ducks of 7 days old are equally divided into two groups of 20 ducks each. The first group is Cap protein vaccine immune group, leg muscle immunity is carried out by using the duck circovirus genetic engineering subunit vaccine prepared by the invention, and the volume is 0.2 ml/one. The second group is tissue inactivated vaccine group, leg muscle immunity is carried out by using the prepared duck circovirus tissue inactivated vaccine, and the volume is 0.2 ml/animal. The third group was a saline control group, and the same volume of sterile saline was injected. After 21 days of immunization, each group of leg muscle duck circular ring virulent WF strain is 0.2ml (containing 10 4.0 ID 50 ) The immune protection rate (percentage of the number of incidences to the total number) was counted 14 days after challenge. Wherein the incidence criteria are: 1. feather malnutrition, feather shaft bleeding, burrs and gradual emaciation; 2. death. One of the two is judged as the onset of the disease.
Results: the immune protection rate of the duck group after the Cap protein vaccine immunization group is immunized is 100%, the immune protection rate of the duck group after the control vaccine group is immunized is 55%, and the morbidity of the duck group in the normal saline control group is 90%. The duck circovirus genetic engineering subunit vaccine has good clinical protection effect on duck groups, is superior to the duck circovirus tissue inactivated vaccine, and can be clinically popularized and applied.
Example 4
Preparation and inspection of duck circovirus egg yolk antibody
1. Immunization of laying hens
The duck circovirus genetic engineering subunit vaccine prepared in example 2 is immunized into laying hens, 0.2ml of vaccine prepared by subcutaneous injection of each chicken neck is immunized for the first time, the second immunization is carried out after 14 days, 0.4ml of vaccine prepared by subcutaneous injection of each chicken neck is immunized for the third time after 14 days after the second immunization, 0.4ml of vaccine prepared by subcutaneous injection of each chicken neck is immunized for the third time, the boosting is carried out after 14 days, 0.4ml of vaccine prepared by subcutaneous injection of each chicken neck is immunized, 14 days after the boosting, yolk is collected and the egg yolk is collected to determine the agarob titer of the duck circovirus, and the agarob titer is more than or equal to 1:64.
Yolk antibody production
2.1 Eggshell sterilization the collected hyperimmune eggs were immersed in a 0.1% aqueous solution of benzalkonium bromide at 42 c for 15 minutes. The eggshells are seriously polluted, are selected in advance, are washed by sterilizing water alone, and are soaked and sterilized for 1 time.
2.2 The egg yolk is separated by manually or mechanically beating eggs. Egg white (white), blastoderm and laces were removed thoroughly, and yolk was collected.
2.3 Inactivating the yolk into uniform paste by fully stirring, adding sterilizing injection water with the same volume of pH value of 6.5, uniformly stirring, heating at 60-65 ℃ for inactivating for 30 minutes, and cooling to room temperature.
2.4 Extracting, adding acidified water (hydrochloric acid is used for adjusting the pH value to 4.2) with the volume of 6 times of the original yolk into a stainless steel tank, cooling to 4 ℃, adding the inactivated yolk liquid I, stirring while adding, standing at 4 ℃ for 4-8 hours, centrifuging at low temperature after acidification is completed, separating the supernatant, and transferring into another reaction tank.
2.5 And (3) adding n-octanoic acid with the final concentration of 0.2% into the supernatant, uniformly stirring, and standing at room temperature for 5-10 hours.
2.6 Filtration is carried out by a suitable filtration method (e.g., filtration using a cartridge filter) to clarify the mixture.
2.7 And (3) adding formaldehyde solution with the final concentration of 0.05% into the filtrate, fully and uniformly stirring, standing at room temperature for 24 hours, and stirring for 4-6 times.
2.8 Filtering and sterilizing, adding proper amount of sodium hydroxide to regulate pH value to about 6.8-7.2, and filtering and sterilizing with 0.22 micron microporous filter element.
2.9 The sterility test is carried out according to the annex of the current Chinese animal pharmacopoeia, and the sterility test should be carried out.
2.10 The titer of the agarobotics is not lower than 1:16.
2.11 And subpackaging, namely quantitatively subpackaging the qualified semi-finished product aseptically, and capping and sealing.
Inspection of finished products
3.1 The physical property of the product is clear liquid. After 48 hours of standing, there was a little sediment at the bottom of the flask. The pH value is 6.8-7.2.
3.2 The filling quantity inspection is carried out according to the annex of the current Chinese animal pharmacopoeia, and the filling quantity inspection meets the regulations.
3.3 The sterility test is carried out according to the annex of the current Chinese animal pharmacopoeia, and the sterility test should be carried out.
3.4 The mycoplasma test is carried out according to the annex of the current Chinese animal pharmacopoeia, and no mycoplasma grows.
3.5 The exogenous virus test is carried out according to the annex of the current Chinese animal pharmacopoeia, and no exogenous virus pollution is needed.
3.6 Safety experiments 20 healthy susceptible ducks of 7 days old are equally divided into two groups of 10 ducks each. The first group of leg muscles are injected with the yolk antibody prepared by the invention, 1.0 ml/one, and the second group of leg muscles are injected with the same volume of physiological saline. The ducks should be healthy and alive completely and have no adverse reaction after 21 days of immunization.
Example 5
Efficacy test of duck circovirus egg yolk antibody
1. Preparation of control egg yolk antibody
The control vaccine prepared in example 3 was used to immunize a layer chicken and to prepare and test a yolk antibody according to the experimental procedure of example 4, i.e., the control yolk antibody, and the titer of the agave antibody should be not less than 1:16.
Animal efficacy experiment
60 healthy susceptible ducks of 28 days old are equally divided into two groups of 20 ducks each. The first group is Cap protein treatment group, leg intramuscular injection of duck circular ring virulent WF strain 0.2ml (containing 10) 4.0 ID 50 ) After 24 hours, the duck circovirus egg yolk antibody prepared by the invention is injected into muscle, and the volume of the egg yolk antibody is 0.5 ml/egg yolk antibody. The second group is control egg yolk antibody treatment group, and the leg muscle injection duck circular ring virulent WF strain is 0.2ml (containing 10) 4.0 ID 50 ) After 24h, the control egg yolk antibody prepared by the invention is injected into the muscle, and the volume of the egg yolk antibody is 0.5 ml/egg yolk antibody. The third group was a normal saline control group, leg muscle duck circular virulent WF strain 0.2ml (containing 10 4.0 ID 50 ) After 24h, sterile physiological saline was injected intramuscularly at 0.5 ml/min. The treatment effect is counted after 14 days of toxin expelling. Wherein the incidence criteria are: 1. feather malnutrition, feather shaft bleeding, burrs and gradual emaciation; 2. death. One of the two is judged as the onset of the disease.
Results: the duck group is 100% protected after Cap protein treatment, the duck group is 60% protected after control egg yolk antibody treatment, and 80% disease occurs in the duck group in normal saline control group. 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 by duck circovirus tissue inactivated vaccine, and can be clinically popularized and applied.
Sequence listing
<110> Weifang Huaying Biotechnology Co., ltd
<120> an improved duck circovirus Cap protein, and preparation method and application thereof
<130> P200051-HYS
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 263
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Met Arg Gly Arg Thr Tyr Arg Arg Ala Tyr Arg Gly Arg Arg Lys Arg
1 5 10 15
Arg Gly Leu Arg Thr Ala Lys Ser Lys Lys Phe Pro Ser Tyr Thr Ala
20 25 30
Thr Tyr Gln Phe Phe Ser Val Val Thr Tyr Lys Val Thr Arg His Thr
35 40 45
Val Phe Gly Phe Phe Gly Ser Gln Thr Gly Pro Thr Ala Ala Gly Lys
50 55 60
Trp Gln Ser Leu Ser Leu Glu Asp Gly Ala Gln Tyr Thr Asp Pro Pro
65 70 75 80
Ala Arg Gly Asn Asn Ile Cys Gly Leu Asn Met Arg Trp Ala Met Phe
85 90 95
Gly Asp Thr Asn Ser Tyr Met Thr Gly Ser Thr Pro Phe Tyr His Tyr
100 105 110
Pro Tyr Asp Tyr Tyr Met Ile Lys Gly Val Ala Ile Thr Leu Arg Pro
115 120 125
Ala Tyr Asn Ile Tyr Gln Lys Ser Lys Thr Gln Gly Ser Thr Val Ile
130 135 140
Asp Lys Asp Gly Gln Ile Val Lys Thr Ser Thr Thr Gly Trp Ser Ile
145 150 155 160
Asp Pro Tyr Gly Ser Thr Ser Ser Arg Arg Thr Trp Asp Pro Ser Arg
165 170 175
Val His Arg Arg Tyr Phe Ile Pro Lys Pro Ile Ile Gln Gly Ala Gly
180 185 190
Glu Gly Thr Lys His Ser Thr Phe Phe Leu Gly Gly Lys Asn Phe Thr
195 200 205
Trp Ile Asn Cys Thr Gln Asp Gln Val Val His Tyr Gly Met Gly Met
210 215 220
Ser Leu Arg Lys Pro Asp Asn Thr Thr Gly Val Asn Ala Gln Tyr Asp
225 230 235 240
Ile Glu Ala Gln Phe Thr Phe Tyr Ile Lys Phe Gly Gln Phe Thr Gly
245 250 255
Phe His His His His His His
260
<210> 2
<211> 789
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
atgcgtggcc gtacctaccg tcgcgcgtac cgtggtcgtc gtaaacgtcg tggtctgcgt 60
actgcgaaaa gcaagaagtt cccgtcctac accgcgacct accagttttt ctctgttgtt 120
acctacaaag ttacccgtca caccgttttt ggtttcttcg gttctcagac gggtccaact 180
gctgcgggca aatggcagtc tctgtctctg gaagacggcg ctcagtacac cgacccgcca 240
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
tacttcatcc cgaaaccgat catccagggt gcgggtgaag gtaccaaaca ctctaccttc 600
ttcctgggtg gtaaaaactt cacctggatc aattgcaccc aagaccaggt tgttcactac 660
ggtatgggta tgtctctccg taaaccagac aatactaccg gtgttaacgc acaatacgac 720
atcgaagcgc aattcacctt ctacatcaaa ttcggtcagt ttaccggctt ccatcatcat 780
catcatcac 789
Claims (5)
1. The modified duck circovirus Cap protein is characterized in that the amino acid sequence of the Cap protein is SEQ ID NO. 1, and the modified duck circovirus Cap protein is prepared by replacing a 21-36-site peptide RRFRRRRLRIARPRRR rich in arginine at the N end of the duck circovirus Cap protein with a T cell epitope TAKSKKFPSYTATYQF.
2. The engineered duck circovirus Cap protein of claim 1, wherein one nucleotide sequence of said Cap protein is SEQ ID No. 2.
3. A method for preparing the modified duck circovirus Cap protein according to any one of claims 1-2, wherein the nucleotide sequence of claim 2 SEQ ID NO. 2 is prepared by total gene synthesis, vector construction, escherichia coli fermentation and nickel column purification methods, and the prepared target protein spontaneously assembles into virus-like particles.
4. A duck circovirus subunit vaccine, characterized in that the antigen of the vaccine is the engineered duck circovirus Cap protein of any one of claims 1-2.
5. A duck circovirus egg yolk antibody, characterized in that the immunized laying hen antigen of the egg yolk antibody is the modified duck circovirus Cap protein of any one of claims 1-2.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103007273A (en) * | 2012-11-16 | 2013-04-03 | 中国农业科学院兰州兽医研究所 | Foot-and-mouth disease genetic engineering mixed epitope vaccine and preparation method thereof |
| CN110237243A (en) * | 2019-06-19 | 2019-09-17 | 苏州世诺生物技术有限公司 | Duck circovirus genetic engineering subunit vaccine and its preparation method and application |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103007273A (en) * | 2012-11-16 | 2013-04-03 | 中国农业科学院兰州兽医研究所 | Foot-and-mouth disease genetic engineering mixed epitope vaccine and preparation method thereof |
| CN110237243A (en) * | 2019-06-19 | 2019-09-17 | 苏州世诺生物技术有限公司 | Duck circovirus genetic engineering subunit vaccine and its preparation method and application |
Non-Patent Citations (3)
| Title |
|---|
| 嵌合口蹄疫病毒中和表位的猪圆环病毒衣壳蛋白表达与免疫原性研究;李艳丽;《中国优秀博硕士学位论文全文数据库(硕士) 农业科技辑》;20140215(第2期);第35页第3.2.2节 * |
| 鸭圆环病毒Cap蛋白的原核表达;粟艳琼等;《动物医学进展》;20141231;第35卷(第3期);第19页第1.2.1节，第20页第2.4节，第22-23页第3节，图7 * |
| 鸭圆环病毒基因组序列分析及其C1截短基因的原核表达;施少华等;《中国兽医学报》;20091031;第29卷(第10期);第1269-1273页 * |
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