CN109053896B

CN109053896B - Porcine circovirus bivalent gene engineering vaccine

Info

Publication number: CN109053896B
Application number: CN201810717823.4A
Authority: CN
Inventors: 李殿明; 蒲勤; 张晓丹; 田春辉; 齐春梅; 刘甜甜; 任百亮; 张导春; 吴启凡; 党将将
Original assignee: Qingdao Mingqin Biotechnology Co ltd
Current assignee: Qingdao Mingqin Biotechnology Co ltd
Priority date: 2018-07-03
Filing date: 2018-07-03
Publication date: 2021-08-17
Anticipated expiration: 2038-07-03
Also published as: CN109053896A

Abstract

The invention relates to a preparation method of a porcine circovirus bivalent genetic engineering vaccine. The vaccine comprises PCV1 structural protein Cap protein subunit, PCV3 structural protein Cap protein, porcine interleukin-5 and a purification label. The vaccine has stable preparation process and is suitable for large-scale production. Animal experiments show that the porcine circovirus bivalent gene engineering vaccine has good safety, can induce animal organisms to generate stronger immune response, and effectively prevents infection of porcine circovirus types 1 and 3.

Description

Porcine circovirus bivalent gene engineering vaccine

Technical Field

The invention belongs to the field of biotechnology genetic engineering, and relates to a fusion protein for preventing porcine circovirus type 1 and porcine circovirus type 3. Specifically, by utilizing a gene recombination technology, a porcine circovirus type 1 Cap protein subunit, a porcine circovirus type 3 Cap protein and a molecular adjuvant IL-5 are connected in series, cloned into a vector, transformed into host bacteria, fermented, purified, emulsified and the like to obtain a porcine circovirus bivalent genetic engineering vaccine and application of the vaccine in prevention of porcine circovirus type 1 and porcine circovirus type 3.

Background

Porcine Circovirus (PCV) belongs to the genus circovirus of the family circovirus, and is a circular single-stranded DNA virus. The PCV genome size is about 1.7kb, and the Cap protein is the only structural protein of PCV, and is immunologically related thereto (yankee et al, 2017). PCV is traditionally divided into two serotypes, namely PCV-1 and PCV-2, infection of the two serotypes can cause reproductive failure, so that the sow return rate is increased, mummy fetus is produced, abortion, stillbirth and weak farrowing are caused, and the like, although the reproductive failure caused by PCV-2 is more serious clinically, the PCV-1 has extremely high pollution rate in normal swinery and swine cells, and great challenge is brought to clinical prevention and treatment.

2016, American scholars, Palinski et al, reported that a novel porcine circovirus, designated porcine circovirus type 3 (PCV-3), was found in swine herds with PDNS. The PCV-3 genome comprises 2000 bases, has a similar genome structure with PCV-1 and PCV-2, and mainly encodes two genes, namely Cap and Rep. Since PCV-3 was discovered in the United states, the prevalence of the virus was also reported in China, Korea, Poland, Brazil, Italy, and other countries, and the wide range of detection found that the pathogen could be detected in 8 provinces and 1 prefecture city in China, and clinically showed typical local lesions of porcine dermatitis nephrotic syndrome, including necrotizing vasculitis, nephritis, granulomatous lymphadenitis, and interstitial pneumonia of bronchi.

Interleukin-5 (IL-5), which has been called B cell growth factor and T cell replacement factor, is a weakly acidic glycoprotein containing N-acetylgalactosamine residues produced by the second subtype of THC after PPD stimulation, has the effects of promoting the differentiation and growth of B cells, inducing the differentiation of eosinophils, and inducing the production of cytotoxic T Cells (CTL), and is a lymphokine with various biological functions.

At present, no domestic or international registered PCV-3 vaccine and PCV-1 and PCV-3 bivalent vaccine exist.

Disclosure of Invention

According to the invention, according to the effect of structural proteins in PCV virus infection, PCV-1 Cap protein subunits and PCV-3 Cap proteins are selected as vaccine framework structures, are connected through a flexible Linker and then are connected with interleukin-5 in series, are cloned into a pRSETB vector and then are transformed into escherichia coli, and the porcine circovirus bivalent genetic engineering vaccine with ideal immunogenicity is obtained through processes such as fermentation, purification, emulsification and the like. The vaccine prepared by the invention can effectively prevent infection of porcine circovirus type 1 and porcine circovirus type 3.

Preferably, the subunit of PCV-1 Cap protein of the invention is selected from the amino acid sequence of SEQ ID No.4 or a functional equivalent thereof.

Preferably, the PCV-3 Cap protein according to the present invention is selected from the amino acid sequence of SEQ ID number 6 or a functional equivalent thereof.

Preferably, the interleukin-5 of the present invention is selected from the amino acid sequence of SEQ ID number 8 or a functional equivalent thereof.

The fusion protein also comprises pharmaceutically acceptable salts and a vector required by expression.

The vaccine also comprises non-immunological active substances, namely, the connecting parts of the polypeptides, has no immunogenicity of antigen epitopes, also has no adjuvant activity, and mainly comprises a purification tag, a linker peptide, a chemical modification part and the like.

The porcine circovirus bivalent gene engineering vaccine provided by the invention has good safety and higher immunogenicity in clinic, stimulates an animal body to generate high-level specific antibodies, and effectively prevents infection of porcine circovirus type 1 and porcine circovirus type 3.

Drawings

The following drawings are included to illustrate specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.

FIG. 1 is a schematic diagram of expression vector pRSETB-PCV-IL-5 containing a gene encoding a fusion protein.

FIG. 2 shows the results of electrophoresis of the recombinant expression vector pRSETB-PCV-IL-5 digested with BamHI + HindIII, wherein lane 1 is a DNA Marker, lane 2 is an uncleaved control, lane 3 is a plasmid-digested map, and lane 4 is an empty plasmid.

FIG. 3 shows the SDS-PAGE identification of the fusion protein coding gene expression product, wherein the lane 1 is molecular Marker, which is 97.4KD, 66.2KD, 43KD, 31KD, 22.0KD and 14.4KD from top to bottom, the lane 2 is negative control, and the lane 3 is an induced purification sample.

FIG. 4 shows the Western Blot result of sample purification, wherein lane 1 is pre-stained Marker, and is 97.4KD, 66.2KD, 43KD, 31KD, 22.0KD and 14.4KD from top to bottom, lane 2 is negative control, and lane 3 is sample.

FIG. 5 shows the results of ELISA detection of serum-specific antibodies in immunized mice, where (-. diamond-solid. -) represents 20171107 groups; (- ■ -) is group 20171108; (. tangle-solidup-) is 20171109 groups; (× -) is blank control.

Detailed Description

The specific test methods described in the examples are only illustrative and are intended to illustrate the present invention in detail, but are not intended to limit the scope of the present invention, and the test methods described below, which are not specifically described, are performed according to the methods described in molecular cloning, a laboratory Manual (2002, third edition, scientific Press).

Example A source of fusion protein genes

The invention comprehensively analyzes the gene sequence, the antigen structure and the epidemiological research progress of the main epidemic strains of the porcine circovirus type 1 and type 3 at home and abroad, analyzes the hydrophilicity, the antigenicity, the plasticity, the surface accessibility and the secondary structure of the structural protein Cap protein by using related bioinformatics software according to the amino acid sequence of the Cap protein, predicts the possible B cell epitope and T cell epitope, and comprehensively reports the related information, thereby determining the PCV-1 and PCV-3 Cap protein subunits. The vaccine is connected with a flexible Linker to form a vaccine framework structure and then is connected with IL-5 in series, and the overall structure of the vaccine is as follows:

PCV1 Cap - PCV3 Cap - IL-5 - Tag

EXAMPLE two construction of E.coli expression vectors and expression strains

The nucleotide coding for the polypeptide designed in the first example is sent to Shanghai Invitrogen biotechnology company for synthesis, the two ends of the nucleotide fragment are respectively designed with BamHI (5 'end) and HindIII (3' end) restriction sites, the synthesized fragment is cloned to a pMD18T vector, and the sequence determination proves that the inserted gene fragment is consistent with the related sequence (see the sequence table). The recombinant plasmid is named as pMD18T-PCV-IL-5, the plasmid is cut by corresponding restriction enzyme, the Escherichia coli expression vector adopts pRSETB plasmid of Invitrogen company, and the same restriction enzyme is also used for treatment, and the cutting conditions are as follows: mu.L of plasmid 2. mu.L, 5 units of restriction enzyme activity (New England biolabs), 10 Xbuffer 1. mu.L, supplemented with deionized water, was added to the reaction system, and the reaction was incubated at 37.0 ℃ for 1.5 hours. After completion of the digestion, the reaction was stopped by adding 1. mu.L of 200mM EDTA. Electrophoresis was performed in a 1% agarose gel for 30 minutes. The pRSETB plasmid and the target fragment were excised under an ultraviolet lamp, and gel recovery was carried out according to the instructions of a gel recovery kit of QIAGEN. According to the carrier: the nucleotide fragment and an expression vector are mixed according to the proportion of 1: 2-3, a reaction system is 15 mu L, T4 DNA ligase is used for connection, overnight connection is carried out at 16 ℃, a recombinant plasmid named as pRSETB-PCV-IL-5 (shown in figure 1) is obtained, and transformation competent Escherichia coli BL21(DE 3) pLysS is obtained.

And (3) transformation: placing pRSETB-PCV-IL-5 on ice to melt, adding 1mL of connecting reaction solution, mixing uniformly again, carrying out ice water bath for 30 minutes at 42 ℃ for 30 seconds, then quickly placing back to the ice water bath for 90 seconds, adding 1mL of LB culture solution, carrying out standing culture at 37.0 ℃ for 1 hour, centrifuging at 4000g at low temperature for 10 seconds, discarding supernatant, suspending the bacteria by 200 mu L of LB culture medium, uniformly coating the bacteria solution on an LB agar culture plate containing 100 mu L/mL of ampicillin, and inversely placing the bacteria solution in an LB agar culture plate at 37 ℃ for culture for 12-16 hours until the clone is formed.

And (3) identification: picking single clone on the plate to LB culture medium, shaking culturing for 12 hours at 37 deg.C and 200rpm, extracting plasmid, using restriction enzyme BamH I and Hind III to double enzyme cutting, the clone which can cut out corresponding size fragment is about 1500bp, it can be primarily determined as positive clone (see figure 2), the positive clone is tested by DNA sequence to further verify its correctness (see sequence table).

Inducing expression: the positive clones were cultured overnight, transferred in the next morning at a ratio of 1:100, shake-cultured at 37 ℃ for 3 hours, induced with 0.5mM IPTG, and cultured for another 3 hours to prepare samples. Expression of the target protein was detected by conventional SDS-PAGE, and a specific band was observed as a correct clone at a molecular weight of about 55kD (see FIG. 3). After correct clones were taken, grown up and expression confirmed by SDS-PAGE, the expression accuracy was further confirmed using a conventional Western Blot (see FIG. 4). The engineering bacterium which is obtained by screening and efficiently secretes and expresses the fusion protein is named as pRSETB-PCV-IL-5/BL21(DE3, PLysS).

EXAMPLE three fermentation, purification and emulsification of engineering bacteria

Fermentation the production strain was inoculated into 2mL of LB liquid medium containing 100. mu.L/mL ampicillin, and shake-cultured at 37 ℃ and 180rpm for 12 hours to activate the strain. Inoculating the activated strain into a shake flask with the inoculation amount of 1:100, performing shake culture at 37 ℃ until OD600=3, and inoculating the strain into a fermentation tank according to the proportion of 10%. The fermentation medium is semisynthetic medium prepared with distilled water, and does not contain any antibiotic. Correcting the dissolved oxygen and pH value electrodes, starting the tank body for stirring at the rotation speed of 300rpm, sterilizing the tank body on line, and calibrating the pH and dissolved Oxygen (OD) zero point when the temperature of the culture solution in the tank is reduced to 37 ℃. The fermentation temperature is 37.0 +/-0.1 ℃, the dissolved oxygen is controlled to be about 20 percent, the pH is controlled to be 7.0, the culture thalli OD600= 1.0-1.2 after inoculation is fed with 500mL of fed-batch materials, IPTG (final concentration is 0.5 mM) is added 1 hour after the fed-batch materials are used for induction and expression, the fermentation is finished after 6 hours of continuous induction, and samples are taken for SDS-PAGE to detect the expression condition.

The collected cells were purified, suspended in an inclusion body wash I (1% Triton X-100, 20Mm Tris-cl pH 8.0), and then sonicated at 2000W for 1 hour. The inclusion bodies were collected by centrifugation at 12000rpm at 4 ℃ and washed by secondary ultrasound in suspension with inclusion body wash II (1% DOC,4M urea, 20mM Tris-cl pH 8.0) and collected by secondary low temperature centrifugation. The inclusion body pellet was mixed well with 8M urea, 0.3% beta-ME, 20mM Tris-cl (pH = 8.0), stirred at room temperature for 4 hours, centrifuged at 8000rpm for 30 minutes, and the pellet was discarded. The denatured protein 1:100 was diluted, and the renaturation solution was renatured with Tris (pH = 8.0) buffer, 0.3M arginine was added, and stirring was carried out at 4 ℃ for 24 hours. The renaturation solution was eluted with 20mM phosphate buffer pH =8.0, 0.5M sodium chloride, 20mM imidazole, equilibrated on an affinity column, with 20mM phosphate buffer pH =8.0, 0.5M sodium chloride, 0.5M imidazole. And then balancing the semi-finished product on a hydrophobic chromatographic column by using 1.5M ammonium sulfate, 100mM EDTA and 10mM disodium hydrogen phosphate with pH =8.5, then balancing, eluting by using 10mM disodium hydrogen phosphate with pH =8.5 to obtain the semi-finished product stock solution of the porcine circovirus bivalent gene engineering vaccine, and carrying out SDS-PAGE and Western Blot to test whether the purified product is the target protein.

Emulsification the purified semi-finished stock solution was diluted to 200. mu.g/mL with sterile PBS. Sterilizing imported white oil mineral oil adjuvant DUOPRIME (pharmaceutical grade) at 121 deg.C for 15 min. The water-in-oil single-phase vaccine is prepared according to the proportion that an oil phase and a water phase are =50:50, the oil phase is firstly added into an emulsification cylinder, a stirrer is started to slowly stir at the speed of 80-100 r/min, the water phase is slowly added, stirring is carried out for 2 minutes after the addition is finished, and then the water-in-oil single-phase vaccine is prepared by high-speed circulation emulsification at 5500r/min for 9 minutes. And (4) performing sterile inspection, viscosity measurement and stability measurement according to the appendix of the Chinese veterinary pharmacopoeia of the current edition, and storing at 2-8 ℃ for later use.

Example four porcine circovirus bivalent genetic engineering vaccine safety experiments

Material

Vaccine: porcine circovirus bivalent gene engineering vaccines,

lot numbers

20171107, 20171108, 20171109, were provided by the company research and development center.

Test animals: BALB/c mice at 8 weeks of age were purchased from Jinanpunyue laboratory animal breeders, Inc. The 28-day-old healthy ternary hybrid weaned piglet is provided by Guangdong Yongshun pharmacy.

Method

Safety of vaccines against white mice

40 8-week-old BALB/c mice were randomly divided into 3 batch groups and 1 control group, 10 mice/group. 3 different batches of porcine circovirus bivalent gene engineering vaccines are respectively injected subcutaneously in batches of 0.5 mL/vaccine. The control group was injected subcutaneously with 0.5 mL/mouse of saline-white oil emulsion. The observation was continued for 14 days, and the health status of the mice was recorded.

Safety of vaccines to piglets

The 20 healthy ternary hybrid weaned piglets with the age of 28 days are randomly divided into 3 batch groups, 1 control group and 5 piglets per group. The batch groups are injected with 3 different batches of porcine circovirus bivalent gene engineering vaccines by muscle after the ears respectively, and each batch is 2 mL. The control group was injected with 2 mL/body of saline-white oil emulsion. The piglets were kept under observation for 14 days and their health was recorded.

Test results

Safety test of vaccine against mice

The results are shown in table 1, after immunization, the mice of the three test groups have no anaphylactic reaction or toxic symptom, good mental state, normal body temperature, food intake, drinking water and the like, no clinical side reaction such as obvious local inflammation and the like, no death, and consistency with the control group, and the porcine circovirus bivalent gene engineering vaccine is safe for the mice.

Table 1 safety test results of vaccines against mice

Group of

Number of animals

Body temperature

Appetite stimulation

Spirit of the invention

Health condition

Inflammatory reaction

Number of deaths

20171107

10

Is normal

Good effect

Is free of

0

20171108

10

Is normal

Good effect

Is free of

0

20171109

10

Is normal

Good effect

Is free of

0

Control group

10

Is normal

Good effect

Is free of

0

Safety testing of vaccines on piglets

The results are shown in table 2, the body temperature and appetite of the piglets of all the immunization groups are normal, the mental state is good, no clinical abnormal phenomenon occurs, no allergic or inflammatory reaction is found at the immune part, no death occurs, and the results are consistent with those of the control group, so that the porcine circovirus bivalent gene engineering vaccine is safe for weaned piglets.

TABLE 2 safety test results of vaccines on piglets

Group of

Number of animals

Body temperature

Appetite stimulation

Spirit of the invention

Health condition

Inflammatory reaction

Number of deaths

20171107

5

Is normal

Good effect

Is free of

0

20171108

5

Is normal

Good effect

Is free of

0

20171109

5

Is normal

Good effect

Is free of

0

Control group

5

Is normal

Good effect

Is free of

0

Example five detection of antibody levels following immunization with porcine circovirus bivalent genetic engineering vaccine

Vaccine: porcine circovirus bivalent gene engineering vaccines,

lot numbers

Test animals: BALB/c mice at 8 weeks of age were purchased from Jinanpunyue laboratory animal breeders, Inc.

Method

20 BALB/c mice 8 weeks old were randomly divided into 4 groups, 3 vaccine immunized groups and 1 placebo group, 5 mice/group. Test mice were immunized according to the grouping, and injected subcutaneously with 0.2 ml/mouse. Blood was collected by tail breaking 7, 14, 21, 28, 35, 42, 49, 56 days before and after immunization, respectively, and serum was isolated for antibody detection.

Diluting the purified recombinant protein antigen to 1 mu g/mL by using 50mmol/L CBS (pH9.6), adding the diluted recombinant protein antigen into an enzyme label plate, keeping the diluted recombinant protein antigen at 100 mu L/hole, standing at 4 ℃ and coating overnight; removing liquid, washing the plate with PBST (containing 0.05% Tween-20, pH7.4) for three times, adding blocking solution (containing 5% horse serum PBST), sealing at 37 deg.C for 1 hr at 100 μ L/well; washing the plate for three times, diluting the sample to be detected by 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200 and 1:6400 times by using a serum diluent (PBST containing 5% horse serum), adding an enzyme label plate into the diluted sample, adding 100 mu L/hole of the diluted sample, setting a negative control at the same time, and incubating the diluted sample for 1 hour at 37 ℃; washing the plate for three times, adding HRP (horse radish peroxidase) labeled goat anti-mouse IgG diluted by 1:5000, 100 mu L/hole, and incubating for 1 hour at 37 ℃; washing the plate for four times, adding 100 mu L/hole of TMB substrate, and developing for 15 minutes in a dark place; 2M H was added₂SO₄The reaction was stopped and the absorbance was measured at a wavelength of 450nm (BIORAD 680 enzyme-linked microplate reader).

And (3) test results:

as shown in fig. 5, specific antibodies were detected in the serum of mice in the three vaccine immunization groups at 14 days after immunization, then the antibody level was continuously increased and reached a peak at 35 days after immunization, and then stably decreased until the end of the experiment, no significant difference was observed among the three vaccine groups, and no antibody was detected in the blank control group.

EXAMPLE six immunoprotection assays

Vaccine: porcine circovirus bivalent gene engineering vaccines,

lot numbers

Test animals: the 28-day-old healthy ternary hybrid weaned piglet is provided by Guangdong Yongshun pharmacy.

Method

40 piglets of 28 days old were randomly divided into 8 groups, 5/group. Test piglets were immunized according to the grouping, wherein 1 and 5 groups of vaccine were injected intramuscularly behind the ear in 20171107 batches, 1 ml/pig; 2 and 6 groups of vaccine 20171108 batches were injected intramuscularly behind the ear, 1 ml/mouse; groups 3 and 7 had 20171109 batches of vaccine injected intramuscularly behind the ear, 1 ml/mouse; groups 4 and 8 are controls. And on 35 days after immunization, 1-4 groups are subjected to virus attack by adopting PCV1 BJ-1, and 5-8 groups are subjected to virus attack by adopting PCV3-China/GD 2016. The morbidity and mortality of the test piglets were observed and recorded. After the challenge, the mental state, clinical symptoms and death condition of the swinery are continuously observed for 14 days, the morbidity is counted, and the protection rate is calculated.

Protection rate = [ (incidence of challenge control group-incidence of vaccine immunization group)/incidence of challenge control group ] × 100%.

And (3) test results:

the results are shown in table 3, the PCV1 strain is adopted to challenge, one 20171107 vaccine group is attacked, no death occurs, and the protection rate is 80%; 20171108 group and 20171109 group have no morbidity and death, and the protection rate is 100%; five control groups all developed disease, and one control group died. PCV3 strain is adopted to challenge, no morbidity and no mortality exist in both 20171107 group and 20171108 group, and the protection rate is 100%; 20171109 one group is attacked without death, and the protection rate is 80%; five control groups all developed disease, and three died.

TABLE 3 protective efficacy of porcine circovirus bivalent genetically engineered vaccines on piglets

Sequence listing

Ming-Du Biotechnology Ltd of 110 Qingdao

Bivalent gene engineering vaccine of porcine circovirus (120)

〈160〉10

〈210〉1

〈211〉1473

〈212〉DNA

Artificial synthesis of < 213 >

〈220〉

Divalent gene engineering vaccine protein DNA code sequence of < 221 > porcine circovirus

〈222〉（1）…（1473）

〈400〉

ctgccgctgc cgtttcagta ttaccgcatt cgtaaagcaa aatatgaatt ctacccgcgc 60

gatccgatca ccagcaatga acgtggcgtc ggttctacgg tggttattct ggatgcaaac 120

tttgtgaccc cgtccacgaa tctggcttat gacccgtaca ttaactatag ctctcgccat 180

accatccgtc agccgtttac gtaccacagc cgctatttca ccccgaaacc ggaactggat 240

aaaacgattg actggttcca tccgaacaat aaacgtaatc aactgtggct gcatctgaac 300

acccacacga atgtggaaca caccggtctg ggtggttctg gtttacttag agaacggact 360

tgtaacgaat ccaaacttct ttggtgccgt agaagtctgt cattccagtt ttttccggga 420

cataaatgct ccaaagcagt gctccccatt gaacggtggg gtcatatgtg ttgagccatg 480

gggtgggtct ggagaaaaag aagaggcttt gtcctgggtg agcgctggta gttcccgcca 540

gaattggttt gggggtgaag taacggctgt gttttttttt agaagtcata actttacgag 600

tggaactttc cgcataaggg tcgtcttgga gccaagtgtt tgtggtccag gcgccgtcta 660

gatctatggc tgtgtgcccg aacatagttt ttgtttgctg agctggagaa attacagggc 720

tgagtgtaac tttcatcttt agtatcttat aatattcaaa gctaatcgca gtttcccatt 780

cgtttaggcg ggtaatgaag tggttggcgt gccacggctt gttattctga ggggttccaa 840

cggaaatgac gttcatggtg gagtatttct ttgtgtagta tgtgccagct gtgggcctcc 900

taatgaatag ttttcttctg acatagcgcc ttctgtggcg tcgtcgtctc cttgggcggg 960

gtcttcttct gaatatagct ctgtgtctca tttggttctg gtatgagaat gcttctgcat 1020

ttgagtctgc taggtcttgg agctgcctac gttagtgcca ttgctgtaga aaataccatg 1080

aatagactgg tggcagagac cttgacactg ctctccattc atcgaactct gctgataggc 1140

gatgggaact tgatgatttc aactcctgta catacaaatc accaactatg cattgaagaa 1200

gtctttcagg gaatagacac gttgaagaat caaactgcac gaggggatgc cgtggaaaaa 1260

ctattccaaa acttgtcttt aataaaagaa tatatagacc gccaaaaaaa aaattgtgga 1320

ggggaaagat ggagagtaac gcaattcctg gactacttgc aagtttttct tggtgtgata 1380

aataccgagt ggacaatgga aagttaacta gaacaaaaac tcatctcaga agaggatctg 1440

aatagcgccg tcgaccatca tcatcatcat cat 1473

〈210〉2

〈211〉487

Protein of < 212 >

Artificial design of < 213 >

〈220〉

Divalent gene engineering vaccine amino acid sequence of < 221 > porcine circovirus

〈222〉（1）…（487）

〈220〉

Subunit amino acid sequence of PCV1 Cap protein

〈222〉（1）…（111）

〈220〉

< 221 > linker peptide

〈222〉（112）…（114）

〈220〉

(221) PCV3 Cap protein amino acid sequence

〈222〉（115）…（328）

〈220〉

< 221 > linker peptide

〈222〉（329）…（331）

〈220〉

Interleukin 5 amino acid sequence of < 221 > pig

〈222〉（332）…（465）

〈220〉

221 Tag amino acid sequence

〈222〉（466）…（487）

〈400〉

LPLPFQYYRI RKAKYEFYPR DPITSNERGV GSTVVILDAN FVTPSTNLAY DPYINYSSRH 60

TIRQPFTYHS RYFTPKPELD KTIDWFHPNN KRNQLWLHLN THTNVEHTGL GGSGMRHRAI 120

FRRRPRPRRR RRHRRRYVRR KLFIRRPTAG TYYTKKYSTM NVISVGTPQN NKPWHANHFI 180

TRLNEWETAI SFEYYKILKM KVTLSPVISP AQQTKTMFGH TAIDLDGAWT TNTWLQDDPY 240

AESSTRKVMT SKKKHSRYFT PKPILAGTTS AHPGQSLFFF SRPTPWLNTY DPTVQWGALL 300

WSIYVPEKTG MTDFYGTKEV WIRYKSVLGS GMRMLLHLSL LGLGAAYVSA IAVENTMNRL 360

VAETLTLLSI HRTLLIGDGN LMISTPVHTN HQLCIEEVFQ GIDTLKNQTA RGDAVEKLFQ 420

NLSLIKEYID RQKKNCGGER WRVTQFLDYL QVFLGVINTE WTMESLEQKL ISEEDLNSAV 480

DHHHHHH 487

〈210〉3

〈211〉333

〈212〉DNA

Artificial synthesis of < 213 >

〈220〉

DNA coding sequence of < 221 > PCV1 Cap protein subunit

〈222〉（1）…（333）

〈400〉

ctgccgctgc cgtttcagta ttaccgcatt cgtaaagcaa aatatgaatt ctacccgcgc 60

gatccgatca ccagcaatga acgtggcgtc ggttctacgg tggttattct ggatgcaaac 120

tttgtgaccc cgtccacgaa tctggcttat gacccgtaca ttaactatag ctctcgccat 180

accatccgtc agccgtttac gtaccacagc cgctatttca ccccgaaacc ggaactggat 240

aaaacgattg actggttcca tccgaacaat aaacgtaatc aactgtggct gcatctgaac 300

acccacacga atgtggaaca caccggtctg ggt 333

〈210〉4

〈211〉111

Polypeptide of < 212 >

Artificial synthesis of < 213 >

〈220〉

Subunit amino acid sequence of PCV1 Cap protein

〈222〉（1）…（111）

〈400〉

LPLPFQYYRI RKAKYEFYPR DPITSNERGV GSTVVILDAN FVTPSTNLAY DPYINYSSRH 60

TIRQPFTYHS RYFTPKPELD KTIDWFHPNN KRNQLWLHLN THTNVEHTGL G 111

〈210〉5

〈211〉651

〈212〉DNA

Artificial synthesis of < 213 >

〈220〉

DNA coding sequence of < 221 > PCV3 Cap protein

〈222〉（1）…（651）

〈400〉

ttacttagag aacggacttg taacgaatcc aaacttcttt ggtgccgtag aagtctgtca 60

ttccagtttt ttccgggaca taaatgctcc aaagcagtgc tccccattga acggtggggt 120

catatgtgtt gagccatggg gtgggtctgg agaaaaagaa gaggctttgt cctgggtgag 180

cgctggtagt tcccgccaga attggtttgg gggtgaagta acggctgtgt ttttttttag 240

aagtcataac tttacgagtg gaactttccg cataagggtc gtcttggagc caagtgtttg 300

tggtccaggc gccgtctaga tctatggctg tgtgcccgaa catagttttt gtttgctgag 360

ctggagaaat tacagggctg agtgtaactt tcatctttag tatcttataa tattcaaagc 420

taatcgcagt ttcccattcg tttaggcggg taatgaagtg gttggcgtgc cacggcttgt 480

tattctgagg ggttccaacg gaaatgacgt tcatggtgga gtatttcttt gtgtagtatg 540

tgccagctgt gggcctccta atgaatagtt ttcttctgac atagcgcctt ctgtggcgtc 600

gtcgtctcct tgggcggggt cttcttctga atatagctct gtgtctcattt 651

〈210〉6

〈211〉214

Polypeptide of < 212 >

Artificial synthesis of < 213 >

〈220〉

(221) PCV3 Cap protein amino acid sequence

〈222〉（1）…（214）

〈400〉

MRHRAIFRRR PRPRRRRRHR RRYVRRKLFI RRPTAGTYYT KKYSTMNVIS VGTPQNNKPW 60

HANHFITRLN EWETAISFEY YKILKMKVTL SPVISPAQQT KTMFGHTAID LDGAWTTNTW 120

LQDDPYAESS TRKVMTSKKK HSRYFTPKPI LAGTTSAHPG QSLFFFSRPT PWLNTYDPTV 180

QWGALLWSIY VPEKTGMTDF YGTKEVWIRY KSVL 214

〈210〉7

〈211〉405

〈212〉DNA

Artificial synthesis of < 213 >

〈220〉

221 pig interleukin 5 DNA coding sequence

〈222〉（1）…（405）

〈400〉

atgagaatgc ttctgcattt gagtctgcta ggtcttggag ctgcctacgt tagtgccatt 60

gctgtagaaa ataccatgaa tagactggtg gcagagacct tgacactgct ctccattcat 120

cgaactctgc tgataggcga tgggaacttg atgatttcaa ctcctgtaca tacaaatcac 180

caactatgca ttgaagaagt ctttcaggga atagacacgt tgaagaatca aactgcacga 240

ggggatgccg tggaaaaact attccaaaac ttgtctttaa taaaagaata tatagaccgc 300

caaaaaaaaa attgtggagg ggaaagatgg agagtaacgc aattcctgga ctacttgcaa 360

gtttttcttg gtgtgataaa taccgagtgg acaatggaaa gttaa 405

〈210〉8

〈211〉134

Protein of < 212 >

213 pig (spine)

〈220〉

Interleukin 5 amino acid sequence of < 221 > pig

〈222〉（1）…（134）

〈400〉

MRMLLHLSLL GLGAAYVSAI AVENTMNRLV AETLTLLSIH RTLLIGDGNL MISTPVHTNH 60

QLCIEEVFQG IDTLKNQTAR GDAVEKLFQN LSLIKEYIDR QKKNCGGERW RVTQFLDYLQ 120

VFLGVINTEW TMES 134

〈210〉9

〈211〉66

〈212〉DNA

Artificial synthesis of < 213 >

〈220〉

221 Tag DNA coding sequence

〈222〉（1）…（66）

〈400〉

ctagaacaaa aactcatctc agaagaggat ctgaatagcg ccgtcgacca tcatcatcat 60

catcat 66

〈210〉10

〈211〉22

Polypeptide of < 212 >

Artificial design of < 213 >

〈220〉

221 Tag amino acid sequence

〈222〉（1）…（22）

〈400〉

LEQKLISEED LNSAVDHHHH HH 22

Claims

1. A fusion protein has an amino acid sequence shown as SEQ ID No. 2.

2. A vaccine for preventing porcine circovirus type 1 and porcine circovirus type 3 comprising the protein of claim 1 and a pharmaceutically acceptable carrier.