CN108159409A - A kind of 3 type Cap protein vaccine of pig circular ring virus and its preparation method and application - Google Patents

A kind of 3 type Cap protein vaccine of pig circular ring virus and its preparation method and application Download PDF

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CN108159409A
CN108159409A CN201711420604.1A CN201711420604A CN108159409A CN 108159409 A CN108159409 A CN 108159409A CN 201711420604 A CN201711420604 A CN 201711420604A CN 108159409 A CN108159409 A CN 108159409A
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circular ring
cap
ring virus
pig circular
protein
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朱燕锋
卢业云
杨德明
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Nanjing Dayao Network Technology Co Ltd
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Abstract

A kind of 3 type Cap protein vaccine of pig circular ring virus and its preparation method and application, belongs to veterinary biological product technical field.3 type Cap protein antigen of pig circular ring virus of the present invention, comprising soluble protein, which obtains for 3 type Cap protein gene of Bacillus coli expression pig circular ring virus.Wherein 1 ~ 30aa of Cap protein removal N-terminal（Nuclear localization sequence）, while increase antigen Synergistic structure fragment gene, by the codon optimization of Escherichia coli, can not only in Escherichia coli great expression, and soluble protein can be formed.Using the 3 type Cap protein subunit vaccine of pig circular ring virus of the preparation of the present invention, have the characteristics that antigen purity height, immunogenicity are strong, while prepare that vaccination process is simple, and production cost is relatively low.

Description

A kind of 3 type Cap protein vaccine of pig circular ring virus and its preparation method and application
Technical field
The present invention relates to veterinary biologics technical fields, are related to a kind of 3 type Cap protein vaccine of pig circular ring virus and its system Preparation Method and application.
Background technology
Pig circular ring virus (porcine circovirus, PCV) be circovirus section Circovirus important member it One.The virus includes two genotype (genotypes)：1 type and 2 types, i.e. PCVl and PCV2.PCVl is from pig renal epithelial cell (PK-15) the non-pathogenic virus particle separated in, and PCV2 is then cause of disease very harmful to world's pig breeding industry at present One of body has brought tremendous economic losses to global aquaculture.
In October, 2016, the R.Palinski of Kansas state universities of the U.S. etc. and University of California San Francisco divide School T.G.Phan etc. almost reports a new PCV genotype, also known as PCV3 in the same time.The virus is from there is illness It is isolated in sow or piglet, while PCV2 is detected as feminine gender.Genomic sequence analysis finds that PCV3 genomes include 2000 Base has the genome structure similar to PCV1 and PCV2, two genes of main code cap and rep.
With the relevant diseases of PCV (PCV2 associated diseases, PCVAD) including postweaning multisystemic failure Syndrome (postweaning multisystemic wasting syndrome, PMWS), pigskin inflammation nephrotic syndrome (porcine dermatitis and nephropathy syndrome, PDNS), respiratory disease (respiratory Disease), breeding difficulty (reproductive failure) and intestines problem (enteric disease) etc..
3 type of pig circular ring virus is detected in the pig farm case in China, and from miscarriage sow and the small of dermatitis nephrosis occurs Find a large amount of viral load on pig, and with porcine circovirus 2 type infection morbidity simultaneously, cause larger harm.Therefore, exist Clinically in practical application, it is desirable to be able to prevent the new generation vaccine of 3 type of pig circular ring virus, will using the method for genetic engineering The Cap protein gene expression of 3 type of pig circular ring virus comes out, and it is following important need to prepare safe and efficient vaccine.
Invention content
The technical issues of solution：The present invention provides a kind of 3 type Cap protein vaccine of pig circular ring virus and preparation method thereof and should With.
Technical solution：A kind of preparation method of 3 type Cap protein vaccine of pig circular ring virus, includes the following steps：（1）Synthesis Target gene：According to the Cap protein amino acid sequence of 3 type ORF2 of pig circular ring virus, 1 ~ 30aa of N-terminal is removed, while C-terminal adds in Linker adds in antigen synergy segment, and adds in restriction enzyme site at whole gene segment both ends, such as SEQ ID NO:Ammonia shown in 2 Base acid sequence, then optimized with e. coli codon, reversely it is compiled as nucleotide sequence such as SEQ ID NO:Shown in 3, it is named as rPCV3-Cap；（2）The structure of recombinant expression plasmid：By step（1）The rPCV3-Cap and carrier PGEX-4T-1 of acquisition carry out double Then digestion is recycled, connection overnight, converts；After obtaining positive colony, plasmid is extracted, carries out PCR identifications and double digestion identification, It identifies that correct plasmid saves backup, is named as PGEX-4T-1-rPCV3-Cap；（3）The screening of recombinant bacterial strain：By step（2） The PGEX-4T-1-rPCV3-Cap plasmids conversion E. coli expression strains BL21 of acquisition（DE3）, after obtaining positive colony, into Row PCR identifies that identification correctly carries out SDS-PAGE analyses again, it is BL21 to identify correct recombinant bacterial strain（DE3）-PGEX-4T- 1-rPCV3-Cap；（4）The expression of recombinant protein：By step（3）The expression bacterial strain BL21 of acquisition（DE3）-PGEX-4T-1- RPCV3-Cap is cultivated, and is expressed using IPTG inducible proteins；（5）Label is removed in purifying, the digestion of recombinant protein：It is collected by centrifugation Step（4）The recombinant bacterium of acquisition carries out ultrasonic disruption, is centrifuged after broken, abandon deposit, and supernatant is passed through 0.45 μm of filter Device filters, then by supernatant gst fusion protein affinity chromatography column purification, finally with fibrin ferment digestion 20 hours again, removal GST-TAG finally obtains the fusion protein PCV3-Cap of purifying；（6）The preparation of vaccine：The antigen protein PCV3- that will be prepared Cap is sterile filtered, and carries out concentration mensuration, has diluted antigen protein PCV3-Cap according to concentration dose setting, has then been mixed with adjuvant It closes, emulsification, obtains 3 type subunit vaccine of pig circular ring virus.
Step（1）In, the double enzyme site that both ends add in is BamH I and Xho I.
Step（4）In culture medium main component be：Glucose, tryptone, yeast extract powder, casein peptone, dimension life Plain B1, NaCl, MgSO4。
Step（6）The adjuvant used matches Bick GEL01 adjuvants for France.
Step（6）Volume ratio of the adjuvant in preparation system is 25%.
The 3 type subunit vaccine of pig circular ring virus that above-mentioned preparation method obtains..
Application of the above-mentioned vaccine in pmws, pigskin inflammation nephrotic syndrome drug is prepared.
Advantageous effect：1st, the present invention provides a kind of 3 type Cap protein vaccine of pig circular ring virus and its preparation method and application, Using the method for removal Cap protein N-terminal nuclear localization sequence, while add linker in C-terminal and enhance the ammonia of immunogenicity Base acid sequence increases the solubility of 3 type Cap protein of pig circular ring virus, in cell conditioned medium, obtain expression quantity it is higher can Dissolubility albumen, while also enhance immunogenicity.
2nd, using PGEX-4T-1 carriers as expression vector, the fusion protein of formation is carried with fibrin ferment digestion removal GST The part of body, the purifying protein finally obtained reach higher immunogenicity close to 3 type natural structure of pig circular ring virus.
3rd, adopting said method prepares 3 type subunit vaccine of pig circular ring virus, is not related to strong poison, can without pathogenic Higher immunogenicity is generated after Clinical practice, while safety is guaranteed.
4th, 3 type Cap protein antigen of pig circular ring virus is prepared and its is applied, and has preparation method simply, conveniently, low cost, epidemic disease The characteristics of seedling potency is high.
Description of the drawings
Fig. 1 is the expression plasmid PGEX-4T-1-rPCV3-Cap double digestion schematic diagrames built, and M1 swimming lanes represent DNA maker（100-2000bp）, M2 swimming lanes expression DNA maker（1000-10000bp）, 1,2 swimming lanes are the purpose base of PCV3-Cap Because of 600bp, simultaneously containing 5000 bp or so PGEX-4T-1 carrier segments.
Fig. 2 PGEX-4T-1-rPCV3-Cap express albumen schematic diagram, and M swimming lanes represent protein maker（15-170 KDa）, the thalline before the expression induction of 1 swimming lane, the thalline after the expression induction of 2 swimming lanes（There is apparent expression band in 49 kDa）, 3 swimming lanes Represent the bacterial sediment after ultrasonication（Do not occur significantly expressing band）, the thalline supernatant after the expression ultrasonication of 4 swimming lanes（49 There is apparent expression band in kDa）.
The western blotting pictures of the Cap-GST fusion proteins and GST monoclonal antibodies of Fig. 3 purifying, M swimming lanes represent egg White matter maker（20-80 KDa）, 1 swimming lane represent purifying Cap-GST fusion proteins reacted with GST monoclonal antibodies（49 kDa occur bright Aobvious reaction zone）, 2 swimming lanes represent GST protein controls reacted with GST monoclonal antibodies（There is significant reaction band in 26 kDa）.
Rear antibody level variation diagram is immunized for 3 type subunit vaccine of pig circular ring virus in Fig. 4.
Specific embodiment
The present invention is further described with reference to specific embodiment.These embodiments are merely illustrative of, and are not construed as To any restrictions of invention overall range.Those skilled in the art are without departing from the spirit and scope of the invention to skill of the present invention Any modification and replacement that the details and form of art scheme carry out, each fall in protection scope of the present invention.
Illustrate the present invention for preparing and its apply for 3 type Cap protein antigen of pig circular ring virus in the embodiment of the present invention.
It is prepared by 1 pig circular ring virus of embodiment, 3 type Cap protein antigen
1 materials and methods
1.1 bacterial strains and carrier PGEX-4T-1 carriers, primer, sequent synthesis, bacillus coli DH 5 alpha competence and Escherichia coli BL21（DE3）Competence gives birth to work purchased from Shanghai.
The BALB/c female mices of 1.2 experimental animal weight about 25g, purchased from Nanjing Medical University's zoopery The heart tests and derives from academy of agricultural sciences of Jiangsu Province veterinary institute with pig.
The 1.3 small extraction reagent kits of main agents DNA plasmid, DNA plastic recovery kits, T4 ligases, the anti-histidine mark of mouse Sign (His) IgG, GST affinity chromatography fillers, BCA protein quantification kits, the mountain sheep anti-mouse igg of HRP labels, horseradish peroxidating Object enzyme（HRP）, I restriction enzyme of BamH I and Xhol etc. gives birth to work bioengineering purchased from Shanghai（Shanghai）Limited company.
1.4 pig circular ring virus, 3 type Cap protein expression vector establishment
1.4.1 3 type Cap protein gene chemical synthesis of pig circular ring virus
With 3 strain CN/Hubei-618/2016 of Porcine circovirus（GenBank: KY354039.1）Cap Based on the amino acid sequence of albumen, such as SEQ ID NO:1, pass through sequence analysis, 1 ~ 30aa of removal N-terminal（Nuclear localization sequence）, C-terminal adds in linker and adds in antigen synergy segment simultaneously, finally obtains the amino acid sequence such as SEQ ID NO of demand:2 sequence Row, then nucleotide sequence is reversely compiled into, and add in restriction enzyme site at whole fragment both ends by e. coli codon optimization, Make it suitable in expression in escherichia coli, commission Shanghai Sangon Biotech Company synthesizes, and last gene order is such as：SEQ ID NO:3, life Entitled rPCV3-Cap is connected to pMD-18T, converts in bacillus coli DH 5 alpha strain.
1.4.2 the double digestion of target fragment and carrier recycles
With plasmid extraction kit plasmid of the extraction containing rPCV3-Cap genes and PGEX-4T-1 empty carriers, it is carried out at the same time BamH I and Xhol, I double digestions, 50 μ L reaction systems are I 1 μ L of restriction enzyme BamH；I 1 μ L of restriction enzyme Xhol；4 μ of plasmid L；10x K buffer 5μL；ddH2O 39μL；It is placed in 37 DEG C to react 3 hours, the rPCV3-Cap for having 600bp is found after running glue The PGEX-4T-1 segments of the 4000bp of genetic fragment and carrier recycle practical box with glue and carry out gel extraction.
1.4.2 the connection conversion of target fragment and carrier
It by rPCV3-Cap segments and PGEX-4T-1 segments, is attached, is configured according to following 20 μ L reaction systems：rPCV3- 8 μ L of Cap segments；2 μ L of PGEX-4T-1 segments；2 μ L of T4 ligases；10 x T4 buffer 2μL；ddH26 μ L of O, as 16 DEG C Reaction 12 hours.Connection product is converted in bacillus coli DH 5 alpha competence later.Response procedures are as follows：It, will under aseptic condition 10 μ L connection products are added in bacillus coli DH 5 alpha competence, ice bath 30min;Taking-up is placed in 42 DEG C, heat shock 90s；It is placed in ice again Bathe 10min；Add in 700 μ L LB fluid nutrient mediums, 37 DEG C of constant-temperature table recovery culture 45min；6000r/min centrifuges 5min, Remove supernatant 600ul；Even spread ammonia benzyl tablet after thalline is resuspended in residue, is placed in 37 DEG C and cultivates 12 hours.Picking single bacterium colony, connects It plants in the LB fluid nutrient mediums containing ampicillin, cultivates 6 hours,（T7 promoter upstream and downstream sequences）It carries out PCR identifications and carries Plasmid BamH I and I double digestions of Xhol is taken to identify.
1.4.3 BamH I and the identification of I double digestions of Xhol are carried out, 50 μ L reaction systems are III 1 μ L of restriction enzyme Hind； I 1 μ L of restriction enzyme Xhol；4 μ L of plasmid；10x K buffer 5μL；ddH2O 39μL；It is placed in 37 DEG C to react 3 hours, production Object finds there is the carrier segments of 600bp target fragments and 4000bp or so through gel electrophoresis.
1.4.4 PCR identifications are carried out, 50 μ L amplification systems are 20.5 μ L of ddH2O；2xPCRmix 25μL；Primer 1 （50mmol/L）2μL；Primer 2（50mmol/L）2μL；Plasmid template（100mmol/L）0.5μL；Reaction condition is 94 DEG C 5min；94 DEG C of 45s, 60 DEG C of 45s, 72 DEG C of 90s are recycled 30 times；72 DEG C of 10min, product amplify a 600bp left sides through gel electrophoresis Right purpose band.
Finally, correct bacterium solution and plasmid are identified through PCR and double digestion, serves the raw work sequencing in sea correctly, recombinant plasmid structure Completion is built, is named as PGEX-4T-1-rPCV3-Cap.
1.5 pig circular ring virus, 3 type Cap protein is expressed
By recombinant expression carrier PGEX-4T-1-rPCV3-Cap plasmids conversion e. coli bl21 (DE3), picking positive transformant Culture reaches 0.5 to OD600, adds in final concentration of 1mmol/L IPTG, is induced 5 hours respectively at 37 DEG C；28 DEG C of inductions 5 are small When；16 DEG C induce 10 hours, and 4 DEG C of 8000 r/min centrifugations 10min collects thalline.It adds in 6mL physiological saline and thalline, ultrasound is resuspended Broken bacteria suspension is limpid to solution, and 12000r/min centrifugation 10min, collected supernatant precipitated liquid is analyzed using SDS-PAGE The expressive site of testing goal albumen.
Then, by expression albumen transfer nitrocellulose filter, and it is successively incubated mouse anti-GST antibody at room temperature（Primary antibody, 1: 2000）With sheep anti-mouse igg-HRP antibody（Secondary antibody, 1:2000）Each 2 hours；Nitrocellulose filter is then placed in DAB dyeing liquors Middle immersion 3-15min carries out western blotting detections.
1.6 pig circular ring virus, 3 type Cap protein purifies
1.6.1 the Bacillus coli cells cracking supernatant containing 3 type Cap protein of pig circular ring virus and 50% glutathione-sepharose tree Fat homogenate mixes, and resets and add 2mL resins on every 100mL, jog 8-12 hours at room temperature;
1.6.2 cell cracking supernatant centrifuges 5min in 4 DEG C with resin compound with 500g (2100r/min), carefully removes supernatant And a little progress SDS-PAGE that keeps sample;
1.6.3 the PBS of 10 times of normal volumes is added in the precipitation left, overturns centrifuge tube mixing for several times, wash away not with resin knot The albumen of conjunction；
1.6.4 5min is centrifuged with 500g (2100r/min) 4 DEG C, carefully removes supernatant and keep sample and a little carry out SDS-PAGE;
1.6.5 step 1.6.3 and 1.6.4 are repeated twice；
1.6.6 3 type Cap protein of pig circular ring virus can be cut with gst fusion protein with blood clotting enzymes factor, and every milliliter of resin adds in The fibrin ferment of 50 unit 1mL PBS overturns centrifuge tube mixing for several times, shakes 4~6h at room temperature.
1.6.7 5min is centrifuged with 500g (2100r/min) 4 DEG C, finally obtains supernatant and carefully move in new pipe, as pig 3 type Cap protein of circovirus；
1.6.8 10%SDS-PAGE analyzes each step washing, the protein of elution samples forms.
2 results and analysis
2.1 pig circular ring virus, 3 type Cap protein expression vector establishment
The PGEX-4T-1-rPCV3-Cap recombinant plasmids built identify that product is through gel electricity through BamH I and I double digestions of Xhol Swimming finds there is the carrier segments of 600bp target fragments and 4000bp or so.（See Fig. 1）
2.2 pig circular ring virus, 3 type Cap protein is expressed
12000 rpm of culture solution after induction is taken to centrifuge 5 min, removes supernatant, PBS liquid is added in and precipitation is resuspended, be eventually adding SDS-PAGE sample-loading buffers heat 10 min of sample at 100 DEG C, are then centrifuged for taking supernatant electrophoresis.Before electrophoresis during 10 min, 100 V voltage stabilizing electrophoresis, 200 V voltage stabilizings electrophoresis to bromophenol blue band is migrated to from gel bottom after bromophenol blue indicator enters separation gel 1 cm of portion takes out gel and is dyed with Coomassie brilliant blue dyeing liquor, then continues in destainer, decolourize to clear background.It is it was found that broken There is the purpose band of 49KD in full bacterium in broken cell conditioned medium, and cell precipitation and empty control plasmid do not occur, is consistent with expection It closes.（See Fig. 2）
2.3 pig circular ring virus, 3 type Cap protein purifies
Using GST affinity chromatographys in method, purify 3 type Cap protein of pig circular ring virus, grope Bacillus coli cells cracking supernatant with The conjugation condition and concentration of 50% Glutathione-agarose resin.Finally grope the concentration with blood coagulation cleavage gst fusion protein And the parameters such as reaction time, temperature, finally obtain the 3 type Cap protein of pig circular ring virus of purifying.
By purified albumen, SDS-PAGE electrophoresis is carried out, after cutting, is attached on nitrocellulose filter, uses graphite electrode Nitrocellulose filter is transferred, and is successively incubated mouse anti-GST antibody at room temperature（Primary antibody, 1:2000）Resist with sheep anti-mouse igg-HRP Body（Secondary antibody, 1:2000）Each 2 hours；Then nitrocellulose filter is placed in DAB dyeing liquors and impregnates 3-15min colour developings, is occurred The reaction of purpose band size, analysis result（See Fig. 3）.
It is prepared by 2 pig circular ring virus of embodiment, 3 type Cap protein subunit vaccine
The 3 type Cap protein original liquid concentration of purifying pig circular ring virus prepared with BCA protein quantifications kit measurement by embodiment 1, Protein concentration is adjusted final concentration of to group 1：0.2mg/mL, group 2：0.1mg/mL, group 3：0.05 mg/mL, group 4：0.01 mg/ ML is matched again with France match gram adjuvant GEL01, and adjuvant addition volume of the total volume 25% is carried out after being uniformly mixed stirring, pressed After current edition Chinese veterinary pharmacopoeia appended claims steriling test, viscosimetric analysis, Stability Determination qualification, be positioned over 2-8 DEG C it is spare.
3 pig circular ring virus of embodiment, 3 type Cap protein subunit vaccine application
The 3 type Cap protein subunit vaccine of pig circular ring virus prepared with embodiment 2 is grouped immune son according to gradient concentration Pig program is shown in Table 1.Blood sampling is primary every 2 weeks（2nd, 4,6,8 week）, assess antibody level changing rule, the pig annulus of various concentration Viral 3 type Cap protein subunit vaccines, generating antibody level after being immunized, there were significant differences compared to control group, uses pig circular ring virus 2 Malicious 3 type Cap proteins coating elisa plate, establishes indirect ELISA method and measures antibody level, see Fig. 4.
1 piglet immunological pig circular ring virus of table, 3 type Cap protein subunit vaccine program
Grouping Size of animal Side reaction The age of immune Immune programme
Group 1 5 Nothing 21 days 2mL/ times
Group 2 5 Nothing 21 days 2mL/ times
Group 3 5 Nothing 21 days 2mL/ times
Group 4 5 Nothing 21 days 2mL/ times
Blank group 5 Nothing 21 days 2mL/ times
It is as follows to be related to gene order：
Porcine circovirus 3 strain CN/Hubei-618/2016（GenBank: KY354039.1）Cap protein Amino acid sequence
SEQ ID NO:1
MRHRAIFRRRPRPRRRRRHRRRYARRRLFIRRPTAGTYYTKKYSMNVISVGTPQNNKPWHANHFITRLNEWET AISFEYYKILKMKVTLSPVISPAQQTKTMFGHTAIDLDGAWTTNTWLQDDPYAESSTRKVMTSKKKHSRYFTPKPIL AGTTSAHPGQSLFFFSRPTPWLNTYDPTVQWGALLWSIYVPEKTGMTDFYGTKEVWIRYKSVL
It removes nuclear localization sequence and rear end adds in linker and synergy amino acid sequence
SEQ ID NO:2
AGTYYTKKYSTMNVISVGTPQNNKPWHANHFITRLNEWETAISFEYYKILKMKVTLSPVISPAQQTKTMFGHT AIDLDGAWTTNTWLQDDPYAESSTRKVMTSKKKHSRYFTPKPILAGTTSAHPGQSLFFFSRPTPWLNTYDPTVQWGA LLWSIYVPEKTGMTDFYGTKEVWIRYKSVLGGGSMAGCKNFFWKTFTSC
Cap protein gene sequences after e. coli codon optimization
SEQ ID NO:3
Restriction enzyme site：BamHI/XhoI (before XhoI plus terminator)
GGATCCGCGGGCACCTACTACACTAAAAAATATAGCACCATGAACGTGATTAGCGTAGGCACCCCACAGAATA ATAAACCGTGGCACGCTAACCACTTTATAACCCGTCTGAATGAATGGGAAACCGCGATCAGCTTCGAATACTACAAA ATCCTGAAAATGAAAGTTACCCTGTCTCCGGTTATCAGCCCGGCGCAGCAGACCAAAACCATGTTCGGCCACACCGC GATCGATCTGGATGGCGCGTGGACCACCAACACCTGGCTGCAGGATGATCCGTACGCGGAAAGCAGCACCCGTAAAG TTATGACCAGCAAAAAGAAACACAGCCGTTACTTCACCCCGAAACCGATCCTGGCCGGTACCACCAGCGCTCATCCG GGCCAGAGCCTGTTCTTCTTCAGCCGTCCGACCCCGTGGCTGAACACCTACGATCCGACCGTTCAGTGGGGCGCACT GCTGTGGAGCATCTACGTTCCGGAAAAAACCGGCATGACCGATTTCTACGGCACCAAAGAAGTTTGGATTCGTTACA AAAGCGTTCTGGGCGGCGGCAGCATGGCGGGTTGCAAAAACTTTTTCTGGAAAACCTTCACCAGCTGCTAACTCGAG
3 type of pig circular ring virus and gst fusion protein express amino acid sequence and predicted molecular weight
SEQ ID NO:4
Protein Length=425 MW=49044.0 Predicted pI=8.92
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIR YIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTH PDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSA GTYYTKKYSTMNVISVGTPQNNKPWHANHFITRLNEWETAISFEYYKILKMKVTLSPVISPAQQTKTMFGHTAIDLD GAWTTNTWLQDDPYAESSTRKVMTSKKKHSRYFTPKPILAGTTSAHPGQSLFFFSRPTPWLNTYDPTVQWGALLWSI YVPEKTGMTDFYGTKEVWIRYKSVLGGGSMAGCKNFFWKTFTSC。
Sequence table
<110>Nanjing great Yao network technology Co., Ltd
<120>A kind of 3 type Cap protein vaccine of pig circular ring virus and its preparation method and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 213
<212> PRT
<213>3 type Cap protein of pig circular ring virus (3 strain CN/Hubei-618/2016 of Porcine circovirus)
<400> 1
Met Arg His Arg Ala Ile Phe Arg Arg Arg Pro Arg Pro Arg Arg Arg
1 5 10 15
Arg Arg His Arg Arg Arg Tyr Ala Arg Arg Arg Leu Phe Ile Arg Arg
20 25 30
Pro Thr Ala Gly Thr Tyr Tyr Thr Lys Lys Tyr Ser Met Asn Val Ile
35 40 45
Ser Val Gly Thr Pro Gln Asn Asn Lys Pro Trp His Ala Asn His Phe
50 55 60
Ile Thr Arg Leu Asn Glu Trp Glu Thr Ala Ile Ser Phe Glu Tyr Tyr
65 70 75 80
Lys Ile Leu Lys Met Lys Val Thr Leu Ser Pro Val Ile Ser Pro Ala
85 90 95
Gln Gln Thr Lys Thr Met Phe Gly His Thr Ala Ile Asp Leu Asp Gly
100 105 110
Ala Trp Thr Thr Asn Thr Trp Leu Gln Asp Asp Pro Tyr Ala Glu Ser
115 120 125
Ser Thr Arg Lys Val Met Thr Ser Lys Lys Lys His Ser Arg Tyr Phe
130 135 140
Thr Pro Lys Pro Ile Leu Ala Gly Thr Thr Ser Ala His Pro Gly Gln
145 150 155 160
Ser Leu Phe Phe Phe Ser Arg Pro Thr Pro Trp Leu Asn Thr Tyr Asp
165 170 175
Pro Thr Val Gln Trp Gly Ala Leu Leu Trp Ser Ile Tyr Val Pro Glu
180 185 190
Lys Thr Gly Met Thr Asp Phe Tyr Gly Thr Lys Glu Val Trp Ile Arg
195 200 205
Tyr Lys Ser Val Leu
210
<210> 2
<211> 199
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 2
Ala Gly Thr Tyr Tyr Thr Lys Lys Tyr Ser Thr Met Asn Val Ile Ser
1 5 10 15
Val Gly Thr Pro Gln Asn Asn Lys Pro Trp His Ala Asn His Phe Ile
20 25 30
Thr Arg Leu Asn Glu Trp Glu Thr Ala Ile Ser Phe Glu Tyr Tyr Lys
35 40 45
Ile Leu Lys Met Lys Val Thr Leu Ser Pro Val Ile Ser Pro Ala Gln
50 55 60
Gln Thr Lys Thr Met Phe Gly His Thr Ala Ile Asp Leu Asp Gly Ala
65 70 75 80
Trp Thr Thr Asn Thr Trp Leu Gln Asp Asp Pro Tyr Ala Glu Ser Ser
85 90 95
Thr Arg Lys Val Met Thr Ser Lys Lys Lys His Ser Arg Tyr Phe Thr
100 105 110
Pro Lys Pro Ile Leu Ala Gly Thr Thr Ser Ala His Pro Gly Gln Ser
115 120 125
Leu Phe Phe Phe Ser Arg Pro Thr Pro Trp Leu Asn Thr Tyr Asp Pro
130 135 140
Thr Val Gln Trp Gly Ala Leu Leu Trp Ser Ile Tyr Val Pro Glu Lys
145 150 155 160
Thr Gly Met Thr Asp Phe Tyr Gly Thr Lys Glu Val Trp Ile Arg Tyr
165 170 175
Lys Ser Val Leu Gly Gly Gly Ser Met Ala Gly Cys Lys Asn Phe Phe
180 185 190
Trp Lys Thr Phe Thr Ser Cys
195
<210> 3
<211> 612
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ggatccgcgg gcacctacta cactaaaaaa tatagcacca tgaacgtgat tagcgtaggc 60
accccacaga ataataaacc gtggcacgct aaccacttta taacccgtct gaatgaatgg 120
gaaaccgcga tcagcttcga atactacaaa atcctgaaaa tgaaagttac cctgtctccg 180
gttatcagcc cggcgcagca gaccaaaacc atgttcggcc acaccgcgat cgatctggat 240
ggcgcgtgga ccaccaacac ctggctgcag gatgatccgt acgcggaaag cagcacccgt 300
aaagttatga ccagcaaaaa gaaacacagc cgttacttca ccccgaaacc gatcctggcc 360
ggtaccacca gcgctcatcc gggccagagc ctgttcttct tcagccgtcc gaccccgtgg 420
ctgaacacct acgatccgac cgttcagtgg ggcgcactgc tgtggagcat ctacgttccg 480
gaaaaaaccg gcatgaccga tttctacggc accaaagaag tttggattcg ttacaaaagc 540
gttctgggcg gcggcagcat ggcgggttgc aaaaactttt tctggaaaac cttcaccagc 600
tgctaactcg ag 612
<210> 4
<211> 425
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 4
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg
210 215 220
Gly Ser Ala Gly Thr Tyr Tyr Thr Lys Lys Tyr Ser Thr Met Asn Val
225 230 235 240
Ile Ser Val Gly Thr Pro Gln Asn Asn Lys Pro Trp His Ala Asn His
245 250 255
Phe Ile Thr Arg Leu Asn Glu Trp Glu Thr Ala Ile Ser Phe Glu Tyr
260 265 270
Tyr Lys Ile Leu Lys Met Lys Val Thr Leu Ser Pro Val Ile Ser Pro
275 280 285
Ala Gln Gln Thr Lys Thr Met Phe Gly His Thr Ala Ile Asp Leu Asp
290 295 300
Gly Ala Trp Thr Thr Asn Thr Trp Leu Gln Asp Asp Pro Tyr Ala Glu
305 310 315 320
Ser Ser Thr Arg Lys Val Met Thr Ser Lys Lys Lys His Ser Arg Tyr
325 330 335
Phe Thr Pro Lys Pro Ile Leu Ala Gly Thr Thr Ser Ala His Pro Gly
340 345 350
Gln Ser Leu Phe Phe Phe Ser Arg Pro Thr Pro Trp Leu Asn Thr Tyr
355 360 365
Asp Pro Thr Val Gln Trp Gly Ala Leu Leu Trp Ser Ile Tyr Val Pro
370 375 380
Glu Lys Thr Gly Met Thr Asp Phe Tyr Gly Thr Lys Glu Val Trp Ile
385 390 395 400
Arg Tyr Lys Ser Val Leu Gly Gly Gly Ser Met Ala Gly Cys Lys Asn
405 410 415
Phe Phe Trp Lys Thr Phe Thr Ser Cys
420 425

Claims (7)

1. a kind of preparation method of 3 type Cap protein vaccine of pig circular ring virus, it is characterised in that include the following steps：
（1）Synthesize target gene：According to the Cap protein amino acid sequence of 3 type ORF2 of pig circular ring virus, 1 ~ 30aa of N-terminal is removed, together When C-terminal add in linker add in antigen synergy segment, and whole gene segment both ends add in restriction enzyme site, such as SEQ ID NO: Amino acid sequence shown in 2, then optimized with e. coli codon, reversely it is compiled as nucleotide sequence such as SEQ ID NO:3 institutes Show, be named as rPCV3-Cap；
（2）The structure of recombinant expression plasmid：By step（1）The rPCV3-Cap and carrier PGEX-4T-1 of acquisition carry out double digestion, Then it recycles, connection overnight, converts；After obtaining positive colony, plasmid is extracted, carries out PCR identifications and double digestion identification, identification is just True plasmid saves backup, and is named as PGEX-4T-1-rPCV3-Cap；
（3）The screening of recombinant bacterial strain：By step（2）The PGEX-4T-1-rPCV3-Cap plasmids conversion Bacillus coli expression of acquisition Bacterial strain BL21（DE3）, after obtaining positive colony, PCR identifications are carried out, identification correctly carries out SDS-PAGE analyses again, identification is correct Recombinant bacterial strain be BL21（DE3）-PGEX-4T-1-rPCV3-Cap；
（4）The expression of recombinant protein：By step（3）The expression bacterial strain BL21 of acquisition（DE3）- PGEX-4T-1-rPCV3-Cap into Row culture, is expressed using IPTG inducible proteins；
（5）Label is removed in purifying, the digestion of recombinant protein：Step is collected by centrifugation（4）The recombinant bacterium of acquisition carries out ultrasonic disruption, It is centrifuged after broken, abandons deposit, supernatant is filtered, then supernatant gst fusion protein is affine by 0.45 μm of filter Column chromatography finally with fibrin ferment digestion 20 hours again, removes GST-TAG, finally obtains the fusion protein PCV3- of purifying Cap；
（6）The preparation of vaccine：The antigen protein PCV3-Cap prepared is sterile filtered, concentration mensuration is carried out, according to concentration agent Amount setting has diluted antigen protein PCV3-Cap, then mixes, emulsifies with adjuvant, obtains 3 type subunit vaccine of pig circular ring virus.
2. a kind of preparation method of 3 type Cap protein vaccine of pig circular ring virus according to claim 1, it is characterised in that：Step （1）In, the double enzyme site that both ends add in is BamH I and Xho I.
3. a kind of preparation method of 3 type Cap protein vaccine of pig circular ring virus according to claim 1, it is characterised in that：Step （4）In culture medium main component be：Glucose, tryptone, yeast extract powder, casein peptone, vitamin B1, NaCl, MgSO4。
4. a kind of preparation method of 3 type Cap protein vaccine of pig circular ring virus according to claim 1, it is characterised in that：Step （6）The adjuvant used matches Bick GEL01 adjuvants for France.
5. a kind of preparation method of 3 type Cap protein vaccine of pig circular ring virus according to claim 1, it is characterised in that：Step （6）Volume ratio of the adjuvant in preparation system is 25%.
6. the 3 type subunit vaccine of pig circular ring virus that any preparation methods of claim 1-5 obtain.
7. the vaccine described in claim 6 is preparing pmws, pigskin inflammation nephrotic syndrome drug In application.
CN201711420604.1A 2017-12-25 2017-12-25 A kind of 3 type Cap protein vaccine of pig circular ring virus and its preparation method and application Pending CN108159409A (en)

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CN108823231A (en) * 2018-07-09 2018-11-16 荣俊 A kind of 3 type genetic engineering subunit vaccine of pig circular ring virus and preparation method thereof
CN109001461A (en) * 2018-06-26 2018-12-14 安阳工学院 The immune quick testing reagent box of PCV-3 immunity evaluation and wild virus infection diagnosis
CN109021115A (en) * 2018-08-06 2018-12-18 青岛明勤生物科技有限公司 A kind of pig circular ring virus trivalent subunit vaccine
CN109207522A (en) * 2018-08-12 2019-01-15 扬州大学 It expresses 3 type of pig circular ring virus and truncates Cap protein recombinant baculovirus and its construction method and primer
CN109550045A (en) * 2018-12-26 2019-04-02 哈药集团生物疫苗有限公司 3 type of pig circular ring virus, pig parvoviral and swine flu triple inactivated vaccine and preparation method thereof
CN109762052A (en) * 2019-01-18 2019-05-17 南京农业大学 3 type Cap recombinant protein of pig circular ring virus and its encoding gene and application
CN109852622A (en) * 2019-01-18 2019-06-07 南京农业大学 A kind of solubility PCV3Cap albumen and its encoding gene and application
CN110257428A (en) * 2019-07-01 2019-09-20 武汉科前生物股份有限公司 A kind of recombined adhenovirus that expressing 3 type ORF2 gene of pig circular ring virus and preparation method and application
CN110283235A (en) * 2019-07-18 2019-09-27 华南农业大学 PCV3 Cap protein B cell linear epitope polypeptide and its screening and application
CN110684084A (en) * 2019-10-25 2020-01-14 天津瑞普生物技术股份有限公司 Preparation method and application of porcine circovirus type 3 (PCV-3) Cap protein virus-like particles
CN111187781A (en) * 2019-09-12 2020-05-22 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） Optimized porcine circovirus type 3 capsid protein gene and application thereof in preparation of virus-like particles
CN111253477A (en) * 2020-03-10 2020-06-09 天康生物(上海)有限公司 Porcine circovirus type 3Cap protein, nucleic acid, virus-like particle, vaccine, preparation method and application
WO2020206452A1 (en) * 2019-04-04 2020-10-08 Boehringer Ingelheim Animal Health USA Inc. Porcine circovirus type 3 (pcv3) vaccines, and production and uses thereof
CN112501186A (en) * 2020-11-26 2021-03-16 浙江鼎持生物制品有限公司 Porcine circovirus 2 d-type CAP protein and application thereof in preparation of subunit vaccine
CN113416236A (en) * 2021-05-21 2021-09-21 武汉科前生物股份有限公司 Porcine circovirus type 3 virus-like particle and preparation method and application thereof
CN114409741A (en) * 2021-12-02 2022-04-29 吉林大学 PCV2, PCV3 and PCV4 triplet subunit vaccine and preparation method and application thereof
CN114807059A (en) * 2022-04-28 2022-07-29 山东信得科技股份有限公司 Novel PCV3 virus strain and PCV3 genetic engineering subunit vaccine

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Application publication date: 20180615