CN110423269A - A kind of 2 type Cap protein of recombinant porcine circovirus and its application of Dominant Epitopes of connecting - Google Patents

A kind of 2 type Cap protein of recombinant porcine circovirus and its application of Dominant Epitopes of connecting Download PDF

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CN110423269A
CN110423269A CN201910610744.8A CN201910610744A CN110423269A CN 110423269 A CN110423269 A CN 110423269A CN 201910610744 A CN201910610744 A CN 201910610744A CN 110423269 A CN110423269 A CN 110423269A
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recombinant
recombinant baculovirus
protein
cap
tbcap
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赵明秋
吴珂珂
罗朝唯
易琳
陈金顶
丁红星
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South China Agricultural University
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Abstract

The invention belongs to field of biotechnology, and in particular to a kind of 2 type Cap protein of recombinant porcine circovirus and its application of Dominant Epitopes of connecting.The amino acid sequence of the 2 type Cap protein of recombinant porcine circovirus of the series connection Dominant Epitopes encodes the nucleotide sequence of above-mentioned albumen as shown in SEQ ID No.2 as shown in SEQ ID No.1.The present invention further constructs the double-promoter transfer vector containing above-mentioned nucleotide sequence, and converts Escherichia coli, obtains recombinant baculovirus plasmid；Then transfection insect cell obtains recombinant baculovirus, and recombinant protein expression quantity can be specifically bound up to 914 μ g/ml with positive serum, has good immunological response.Subunit vaccine provided by the invention can stimulate mouse to generate high-caliber humoral immune response after mouse is immunized；It can induce body after immune piglet and generate specific immune response, good immanoprotection action can be provided to attack malicious piglet.

Description

A kind of 2 type Cap protein of recombinant porcine circovirus and its application of Dominant Epitopes of connecting
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of 2 type Cap of recombinant porcine circovirus for Dominant Epitopes of connecting Albumen and its application.
Background technique
Porcine circovirus 2 type (Porcine circovirus type 2, PCV2) is to cause postweaning multisystemic failure The main pathogen of syndrome (PMWS).PCV2 mainly encroaches on the immune system of body, causes serious immunosupress, to be easy The secondary or mixed infection of other invasive organisms occurs, seriously endangers swinery health.From porcine circovirus 2 type epidemic disease in 2006 Since seedling comes out, the immunity inoculation of vaccine becomes the main means of prevention and treatment Porcine circovirus desease.The Cap egg of PCV2 ORF2 coding It is white to be the main structural proteins of virus and there is type specificity epitope, body can be induced to generate specific immune response, be Develop the ideal targeting object of PCV2 genetic engineering subunit vaccine.Current four kinds of commercialized vaccines worldwide make extensively With two of them is all using PCV2 Cap protein as immunogene, and experiment and work place study show PCV2 vaccine with being clarified above Have effects that reduce viremia virusemia, eliminate PCVAD, increase growth performance etc., but in the swinery of immunity inoculation still PCVAD can so occur, it means that the immunity inoculation of existing commercial vaccine can not prevent completely PCV2 to infect or propagate；This Outside, the virus titer obtained there are also totivirus culture is low, inactivation is not thorough that cause virulence to return strong, expensive and protective rate poor etc. Possibility.Therefore, developing new safely and effectively subunit vaccine is very necessary to control PCV2 infection.
In recent years, rhabdovirus expression vector can accommodate big target gene because it is with easy to operate, highly-safe, It is high-efficient to express foreign protein, there is posttranslational modification, expresses the immunogenicity, bioactivity and natural albumen of albumen The advantages that similar, the albumen that can be good at making up traditional prokaryotic expression exist with inclusion bodies, cause to produce Object purification difficult, and prokaryotic expression system post translational processing modification system is not perfect, the bioactivity of expression product is more low not Foot.To find a kind of method that can enhance Cap protein immunogenicity, the present invention is obtained with correct structure and is repaired using being easier The 2 type Cap protein of recombinant porcine circovirus of the rhabdovirus expression vector expression series connection Dominant Epitopes of active albumen is adornd, and it is new The subunit vaccine that type adjuvant is mixed with, to improve the immune effect of Cap protein subunit vaccine.
Summary of the invention
For overcome the deficiencies in the prior art and disadvantage, the primary purpose of the present invention is that providing a kind of series connection Dominant Epitopes 2 type Cap protein of recombinant porcine circovirus.
Another object of the present invention is to provide the 2 type Cap eggs of recombinant porcine circovirus for encoding above-mentioned series connection Dominant Epitopes White nucleotide sequence, the sequence can be improved expression quantity of the recombinant protein in rhabdovirus system and exempt from by manually modified Epidemic focus.
Third object of the present invention is to provide a kind of recombinant baculovirus transfer vector, which is double-promoter transfer Carrier, the nucleotide sequence of the 2 type Cap protein of recombinant porcine circovirus containing above-mentioned coding series connection Dominant Epitopes.
Fourth object of the present invention is to provide a kind of recombinant baculovirus, which being capable of secreting, expressing The 2 type Cap protein of recombinant porcine circovirus of series connection Dominant Epitopes.
Of the invention the 5th is designed to provide a kind of PCV2 subunit vaccine.
Of the invention the 6th is designed to provide the 2 type Cap protein of recombinant porcine circovirus of above-mentioned series connection Dominant Epitopes Application.
The purpose of the invention is achieved by the following technical solution:
A kind of 2 type Cap protein of recombinant porcine circovirus for Dominant Epitopes of connecting, amino acid sequence such as SEQ ID No.1 It is shown；
Encode the nucleotide sequence such as SEQ ID of the 2 type Cap protein of recombinant porcine circovirus of above-mentioned series connection Dominant Epitopes Shown in No.2；
A kind of recombinant baculovirus transfer vector, 2 type of recombinant porcine circovirus comprising above-mentioned coding series connection Dominant Epitopes The nucleotide sequence of Cap protein；
The carrier that sets out of the recombinant baculovirus transfer vector is preferably pFastBacTMDual carrier；
The recombinant baculovirus transfer vector is baculovirus transfer vector pFBD-TBCap-2, i.e., respectively in P10, PH The nucleotide sequence of the 2 type Cap protein of recombinant porcine circovirus of above-mentioned coding series connection Dominant Epitopes is cloned under promoter；
A kind of recombinant baculovirus converts DH10Bac Escherichia coli by above-mentioned recombinant baculovirus transfer vector, by turning Seat recombination, obtains recombinant baculovirus plasmid；Finally utilize cationic-liposome cellfectin II by recombinant baculovirus matter After grain transfection insect cell, collects supernatant and obtain；
The insect cell is preferably sf9 cell or High-five cell；
Above-mentioned recombinant baculovirus is inoculated with insect cell by a kind of PCV2 subunit vaccine, after expanding culture, collects purpose Destination protein and immunologic adjuvant are mixed to get by albumen；
The preparation method of the PCV2 subunit vaccine, preferably comprises following steps:
Above-mentioned recombinant baculovirus is inoculated with to the High-Five insect cell for the culture that suspends, collects culture after infection, from The heart removes culture supernatant；Sonicated cells, centrifugation removal cell fragment, collect ultrasonic supernatant；By purpose egg in ultrasonic supernatant It is white to emulsify or be mixed to prepare with immunologic adjuvant；
The infective dose of the recombinant baculovirus is preferably MOI=1；
The infection time is preferably 96h；
The immunologic adjuvant is preferably ISA 201VG adjuvant and MontanideTMAt least one of 01 adjuvant of GEL；
The immunologic adjuvant is more preferably MontanideTM01 adjuvant of GEL；
The concentration of destination protein is preferably 200~500 μ g/ml in the PCV2 subunit vaccine；
The 2 type Cap protein of recombinant porcine circovirus of the series connection Dominant Epitopes is preparing the application in PCV2 vaccine；
The present invention has the following advantages and effects with respect to the prior art:
(1) for the present invention according to the preferences of baculoviral codon, engineer, which has synthesized, contains multiple dominant antigen epitopes 2 type Cap protein of recombinant porcine circovirus nucleotide sequence, for sequence as shown in SEQ ID NO.2, which improves mesh Gene in baculoviral expression and immunogenicity.
(2) present invention constructs the core of the 2 type Cap protein of recombinant porcine circovirus containing above-mentioned coding series connection Dominant Epitopes The double-promoter recombinant baculovirus transfer vector of nucleotide sequence, the recombinant transfer vector and single promoter recombinant baculovirus turn Transfer body is compared, and expressing quantity is higher.
(3) recombinant baculovirus provided by the invention can secreting, expressing connect multiple dominant antigen epitopes Recombinant Swine circle 2 type Cap protein of circovirus virus, can specifically bind with positive serum, have good reactionogenicity, and through being overexpressed item The recombinant protein TBCap expression quantity of piece optimization is up to 914 μ g/ml, applied to can largely reduce life in actual production Produce cost.
(4) the 2 type Cap of recombinant porcine circovirus of the expressed dominant antigen epitope that must connect of recombinant baculovirus of the invention The subunit vaccine of albumen preparation after mouse is immunized, can stimulate mouse to generate high-caliber humoral immune response；Immune piglet Afterwards, it can induce body and generate specific immune response, good immanoprotection action can be provided to attack malicious piglet.The present invention provides Subunit vaccine can be used as treatment PCV2 infection effective, safe PCV2 subunit vaccine it is candidate.
Detailed description of the invention
Fig. 1 is restructuring rod granule PCR appraisal principle figure.
Fig. 2 is the plasmid figure spectrogram of recombinant baculovirus transfer vector pFBD-Cap-1 and pFBD-Cap-2.
Fig. 3 is the plasmid figure spectrogram of recombinant baculovirus transfer vector pFBD-TBCap-1 and pFBD-TBCap-2.
Fig. 4 is normal Sf9 and the microscope figure of the Sf9 cell of CPE occurs after transfecting；Wherein, A: normal Sf9 cell, B: Occurs the Sf9 cell of CPE after transfection.
Fig. 5 is the cell sample for infecting recombinant baculovirus Ac-Cap-1, Ac-Cap-2, Ac-TBCap-1 and Ac-TBCap-2 The Western-blot qualification result figure of product；Wherein, A:P1 is for recombinant baculovirus albumen, and B:P2 is for recombinant baculovirus egg White, C:P3 is for recombinant baculovirus albumen.
Fig. 6 is the Western-blot result figure that recombinant baculovirus harvest time influences recombinant protein expression；Wherein, It is 72h that A, which is harvest time, in A, 1~4:Ac-Cap-1, Ac-Cap-2, Ac-TBCap-1, Ac-TBCap-2 (MOI=1), 5~ 8:Ac-Cap-1, Ac-Cap-2, Ac-TBCap-1, Ac-TBCap-1 (MOI=2), 9~12:Ac-Cap-1Ac-Cap-2, Ac- TBCap-1, Ac-TBCap-2 (MOI=4)；It is 96h that B, which is harvest time, in B, 11~14:Ac-Cap-1, Ac-Cap-2, Ac- TBCap-1, Ac-TBCap-2 (MOI=1), 15~18:Ac-Cap-1, Ac-Cap-2, Ac-TBCap-1, Ac-TBCap-1 (MOI =2), 19~22:Ac-Cap-1, Ac-Cap-2, Ac-TBCap-1, Ac-TBCap-2 (MOI=4).
Fig. 7 is the Western-blot result figure that different culture medium and different insects cell influence recombinant protein expression； Wherein, recombinant protein expression quantity after A:SF900 and SIM SF culture medium culture sf9 cell；B: different insects cell recombinant protein Expression quantity, 1: the sf9 cell sample of expression recombinant C ap-2 albumen, 2: the High-five cell of expression recombinant C ap-2 albumen is super Sample before sound, 3: the sf9 cell sample of expression recombination TBCap-2 albumen, 4: the High-five of expression recombination TBCap-2 albumen Cell sample.
Fig. 8 is recombinate shape virus infection dosage, harvest time on the Western-blot knot of recombinant protein expression influence Fruit figure；Wherein, 1:48h, MOI=1；2:48h, MOI=2；3:48h, MOI=4；4:72h, MOI=1；5:72h, MOI=2；6: 72h, MOI=4；7:96h, MOI=1；8:96h, MOI=2；9:96h, MOI=4.
Fig. 9 is the soluble analysis result figure of recombinant Cap protein and recombination TBCap albumen；Wherein, A: recombinant Cap protein, In A, 1: the High-five cell being uninfected by；2: infecting the High-five cell sample of Ac-Cap-2；3: infection Ac-Cap-2 High-five ultrasound on final proof；B:TBCap albumen, 1 in B: the High-five cell being uninfected by；2: infection Ac-TBCap-2 High-five cell sample；3: infecting the High-five ultrasound Supernatant samples of Ac-TBCap-2.
Figure 10 is recombinant Cap protein and recombination TBCap protein secretion Expression temporal result analysis chart；Wherein, A: recombinant C ap Albumen, in A, 1~5: being respectively 24,48,72,84,96h harvest cell training after recombinate shape virus infection High-five cell Support Supernatant samples；B:TBCap albumen, in B, 1~5: be respectively 24 after recombinate shape virus infection High-five cell, 48, 72,84,96h harvest cells and supernatant sample.
Figure 11 is ELISA antibody test result analysis chart after mouse immune.
Figure 12 is the measurement result analysis chart of piglet body temperature after attacking poison；Wherein, CC: malicious control group is attacked, NC: blank group.
Figure 13 is ELISA antibody test (S/P) result analysis chart after piglet immunological.
Figure 14 is Real-time PCR canonical plotting.
Figure 15 is each group piglet serum viral load result analysis chart after attacking poison.
Figure 16 is each group Lymph Nodes of Piglets virus load result analysis chart after attacking poison.
Figure 17 is to attack poison group tissue microscopic lesion result analysis chart；Wherein, A: poison group lungs HE dyeing (40 ×) is attacked, B: is attacked Poison group lungs HE dyeing (100 ×) C: attacks poison group lymph node microscopic lesion HE dyeing (100 ×), D: it is micro- to attack poison group lymph node Lesion HE dyes (400 ×).
Figure 18 is immunohistochemistry testing result figure.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited In this.
Unless stated otherwise, the present invention uses reagent, method and apparatus for the art conventional reagent, method and are set It is standby.
Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
In embodiment, expression vector pFastBacTMDual), competent escherichia coli cell DH10Bac, DH5a, wild Type virus AcNPV is saved by College of Veterinary Medicine, South China Agricultural University's microbiology and immunology teaching and research room, commercially available；Pig annulus Viral 2 type baculovirus vector inactivated vaccines (Ingelvac CircoFLEX) are Boehringer Ingelheim Products；Pig PCV2 Positive serum is given by guangdong agricultural science institute veterinary institute.
The building of 1 recombinant baculovirus transfer vector of embodiment
(1) the gene order design and synthesis of recombinant protein
With LG plants of complete genome sequences (HM038034.1) of PCV2 for being included in GenBank for template, in PCV2 cap albumen Upper introducing Cap/Rep linear epitope, the specific method is as follows: carrying out sequence to epitope using DNAStar biosoftware Analysis filters out on the peptide fragment i.e. Rep albumen that antigenicity is good and specificity is high in 81-100,201-220 and Cap protein 61-85,113-131,169-180,192-202 property epitopes, and determine the series sequence between dominant antigen epitope, and Introduce bee venom signal peptide sequence (honeybee melittin signal peptide, HBM) to realize secretion type expression, in The base sequence of 6 histidines of design coding, final design go out the nucleotide of recombinant protein PCV2-TBCap before terminator codon Sequence after carrying out the codon optimization for insect cell to its nucleotide sequence, is sent to the raw limited public affairs of work bioengineering in Shanghai Department's synthesis.The nucleotide sequence of PCV2-Cap albumen and recombinant protein PCV2-TBCap are as follows:
The nucleotide sequence of PCV2-Cap albumen:
Wherein, grey protrusion is bee venom signal peptide, and dashed part is Cap gene order, grey protrusion+italicized item For 6 × His；
The amino acid sequence of PCV2-Cap albumen:
MKFLVNVALVFMVVYISYIYATYPRRRFRRRRHRPRSHLGLILRRRPWLVHPRHRYRWRRKNGIFNTR LSCTFGYTVKATTVRTPSWAVDMMRFNINDFVPPGGGTNEISIPFEYYRIRKVKVEFWPCSPITQGDRGVGSTAVI LDDNFVTRATALTYGPYVNYSSRHTIPQPFSYHSRYFTPKPVLDSTIDYFQPNNKRNQLWLRLQTSANVDHVGLGI AFENSTYDQDYNIRVTMYVQFREFNLKDPPLKPHHHHHH.
The nucleotide sequence of PCV2-TBCap:
Wherein, grey protrusion is bee venom signal peptide, and thickened portion is t cell epitope, grey protrusion+wave part For B cell epitope, underscore part is Cap gene order, and grey protrusion+italicized item is 6 × His；
The amino acid sequence of PCV2-TBCap is as follows:
MKFLVNVALVFMVVYISYIYACHIEKAKGTDQQNKEYCSKEGGKWWDGYHGEEVVVIDDFYGWGGTVR TPSWAVDMMRFNINDFVPPGGGGGQGDRGVGSTAVILDDNFVTGGSTIDYFQPNNKRGGNVDHVGLGIAFGGTYPR RRFRRRRHRPRSHLGLILRRRPWLVHPRHRYRWRRKNGIFNTRLSCTFGYTVKATTVRTPSWAVDMMRFNINDFVP PGGGTNEISIPFEYYRIRKVKVEFWPCSPITQGDRGVGSTAVILDDNFVTRATALTYGPYVNYSSRHTIPQPFSYH SRYFTPKPVLDSTIDYFQPNNKRNQLWLRLQTSANVDHVGLGIAFENSTYDQDYNIRVTMYVQFREFNLKDPPLKP HHHHHH.
(2) building of recombinant baculovirus transfer vector
1. the nucleotide sequence application Primer5.0 according to above-mentioned PCV2-Cap albumen and recombinant protein PCV2-TBCap is soft Part separately designs primer (being shown in Table 1), and wherein Cap-p10-F/R and Cap-pH-F/R is used to expand the Cap without more epitopes Gene order, TBCap-p10-F/R and TBCap-pH-F/R introduce BamH I for expanding recombination TBCap, the end of upstream primer 5 ' Or Xho I restriction enzyme site, the end of downstream primer 5 ' introduce Sac I or Nhe I restriction enzyme site.With the PCV2-Cap albumen of synthesis and again The nucleotides sequence of histone PCV2-TBCap is classified as template, respectively with above-mentioned primer amplification recombinant C ap and recombination TBCap segment. PCR system is as follows: 5 × PrimeSTAR Buffer, 10.0 μ L, PrimeSTAR HS DNA Polymerase 0.5 μ L, DNA 1.0 μ L of template, 1.0 μ L of upstream primer, 1.0 μ L, dNTP Mixture of downstream primer 4.0 μ L, 32.5 μ L of distilled water；PCR reaction Condition are as follows: 98 DEG C of 3min；98 DEG C of 15s, 55 DEG C of 15s, 72 DEG C of 5min, totally 30 recycle；72℃10min.The mesh that amplification is obtained Segment carry out agarose nucleic acid electrophoresis, and to purpose band carry out glue recovery purifying.
2. the 1. target fragment and pFastBac with Xho I and Nhe I restriction enzyme site that step is recycledTMDual is carried Body carries out double digestion with Xho I and Nhe I restriction endonuclease respectively, and digestion system is as follows: 10 × FastDigest 2.0 μ L, FastDigest Xho I of Buffer, 1.0 μ L, FastDigest Nhe I, 1.0 μ L, PCR recovery product/carrier 10.0 μ L/2 μ L, distilled water add to 20 μ L of total volume；37 DEG C of water-bath double digestion 30min, product distinguish glue recovery purifying.By double enzymes Target fragment and pFastBac after cuttingTMDual carrier is attached, and connection product is converted DH5 α competent cell, with The screening of LB (AMP) solid medium.Picking positive colony carries out bacterium colony PCR identification, and upstream and downstream identifies that primer is pFBD2-F/R, And positive colony is expanded, illustrate that step extracts plasmid by Omga plasmid extraction kit, and digestion is identified.Finally weighed respectively Group baculovirus transfer vector pFBD-Cap-1, pFBD-TBCap-1.
3. the 1. target fragment and recombinant baculovirus transfer with BamH I and Sac I restriction enzyme site that step is recycled Carrier pFBD-Cap-1, pFBD-TBCap-1 carry out double digestion with BamH I and Sac I restriction endonuclease respectively, and glue returns After receipts connect, convert, identification (identify primer be pFBD1-F/R and pFBD2-F/R, specific method referring to step 2., appraisal principle See Fig. 1), recombinant baculovirus transfer vector pFBD-Cap-2, pFBD-TBCap-2 are obtained respectively, are saved in -20 DEG C.
Table 1 is used to expand the primer sequence and detection primer of different genes
Include pFBD-2F/R primer binding site in transfer vector pFastBac Dual, is located under p10 promoter more grams The two sides in grand site, electrophoresis result are shown: using pFBD-2F/R primer as amplimer, recombinant baculovirus transfer vector pFBD- Cap-1 and pFBD-Cap-2 pcr amplification product length and recombinant baculovirus transfer vector pFBD-TBCap-1 and pFBD- TBCap-2 pcr amplification product length and expection are in the same size.
Include pFBD1-F/R primer binding site in transfer vector pFastBac Dual, is located at polyhedron promoter (PH Promoter) under multiple cloning sites two sides, electrophoresis result shows: using pFBD-1F/R primer as amplimer, recombinant baculovirus Transfer vector pFBD-Cap-2 and pFBD-TBCap-2 pcr amplification product length is consistent with expection.
The acquisition of 2 recombinant baculovirus of embodiment and recombinant protein expression condition optimization
(1) acquisition of recombinant baculovirus plasmid
To identify successful recombinant baculovirus transfer vector pFBD-Cap-1, pFBD-Cap-2, pFBD-TBCap-1, PFBD-TBCap-2 is converted respectively to DH10Bac competence and is gently blown and beaten mixing with liquid-transfering gun, stands 25min on ice；2. will Competent cell takes out from ice bath, is immediately placed in heat shock 45s in 42 DEG C of water-baths, and rear take out is incubated for 2min in ice bath；3. to 900 μ L SOC culture mediums are added in EP pipe, in 37 DEG C of Bacteria Culture shaking table 200rpm shaken cultivation swivel base 4h；4. being cultivated with SOC Base carries out gradient dilution (10 to transformed bacteria-1, 10-2, 10-3), the 100 μ L of transformed bacteria for drawing above-mentioned dilution gradient respectively is coated on (Gen, Kan, Tet, IPTG, X-Gel press Bac-to-Bac Baculoviruse Expression System using concentration to LB Operational manual) on solid medium；5. plate is placed in 37 DEG C of bacteriological incubators after cultivating 48h and observes colony colour, Middle white colony is target bacterium colony；6. in order to be further purified, after picking white colony again LB (Gen, Kan, Tet, IPTG, X-Gel) it crosses on solid medium, 48h is cultivated in 37 DEG C of bacteriological incubators；7. picking white colony, is inoculated in In 5mL LB (Gen, Kan, Tet) fluid nutrient medium, 37 DEG C of 200rpm cultivate 24~36h；Lifting manipulation extracts restructuring rod granule, finally Recombinant baculovirus plasmid rBac-Cap-1, rBac-TBCap-1, rBac-Cap-2, rBac-TBCap-2 are obtained, rod granule is detected Concentration, 4 DEG C save backup.
(2) identification of recombinant baculovirus plasmid
Since the baculovirus plasmid molecular weight after recombination is larger, conventional digestion identification cannot be used to examine foreign gene Whether segment is inserted into success.Since foreign gene insertion point is located at the Tn7 swivel base site left and right arms of recombinant baculovirus plasmid Between, thus using M13-F and M13-R universal primer (M13F:CCCAGTCACGACGACGTTGTAAAACG, M13R: AGCGGATAACAATTTCACACAGG PCR identification) is carried out to restructuring rod granule, can be amplified as the result is shown and expected size one The specific band of cause shows that target gene fragment is successfully recombined into Baculovirus Gene group in Escherichia coli.
(3) acquisition of recombinant baculovirus
Using cationic-liposome cellfectin II mediated transfection, by recombinant baculovirus plasmid rBac-Cap-1, RBac-TBCap-1, rBac-Cap-2, rBac-TBCap-2 transfect sf9 cell monolayer (purchased from Wuhan University's Chinese Typical Representative respectively Object collection), 3~5h is incubated in 27 DEG C of incubators；After the completion of transfection, the supernatant in cell hole is removed, and supplement 2mL Sf900III culture medium, stationary culture 72h or more in 27 DEG C of incubators；
After transfection, Transfected cells are observed once every for 24 hours, when transfection cell starts typical cytopathy occurred Feature, such as cell stops increasing, cell edges are coarse, appearance intracellular a large amount of vesica feature (Fig. 4) when, show successfully to save Rescue recombinant baculovirus.Continue culture cell can be observed to start largely take off wall and crack broken (about 4~6d after transfection), receipts Cell culture is obtained, room temperature 1000rpm is centrifuged 5min, and supernatant is recombinant baculovirus (P1 generation), in the virus liquid of harvest Final concentration of 2% FBS is added, -80 DEG C save backup.The recombinant baculovirus difference obtained after restructuring rod granule is transfected respectively It is named as Ac-Cap-1, Ac-TBCap-1, Ac-Cap-2, Ac-TBCap-2.Since P1 is for the virus titer of recombinant baculovirus It is lower, it needs to pass on it and expanded, to increase virus titer, referring to Bac-to-Bac Baculoviruse Expression System operational manual, obtains second generation recombinant baculovirus (P2 generation) respectively and third generation recombination is rod-shaped Viral (P3 generation).
(4) recombinant baculovirus titer determination
1. the building of standard plasmid
Using 5 software of Primer, using baculoviral (wild-type virus AcNPV) full-length genome as template, design a pair is drawn Object Ac-F/R is used for real-time fluorescence quantitative PCR, and the another pair of primers AcMK107-F/R that designs is used for amplification fluorescent quantitative PCR standard Plasmid fragments, and connect carrier T.Primer sequence is shown in Table 2.
2 quantitative fluorescent PCR of table and standard plasmid construct primer sequence
Baculoviral (wild type) total DNA, using baculoviral total DNA as template, AcMK107- are extracted with portable plasmid method F/R is primer, expands aim sequence, PCR system are as follows: DNA profiling 1 μ L, AcMK107-F 1 μ L, AcMK107-R 1 μ L, 2 × 10 μ L of Taq PCR StarMix, 7 μ L of distilled water；PCR reaction condition are as follows: 94 DEG C of 3min；94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C 30s, totally 30 recycle；72℃10min.The target fragment of amplification glue recovery purifying target fragment after agarose gel electrophoresis, It is connected in pMD18-T carrier, linked system is as follows: 1 μ L of pMD18-T Vector, 115 μ of μ L, Solution of target fragment L, 3 μ L of distilled water；16 DEG C of incubation 30min after linked system are prepared, connection product are converted into DH5 α competent cell, and with LB (AMP) solid medium screens.Picking positive colony is inoculated in 5mL LB (AMP) fluid nutrient medium, 37 DEG C of constant-temperature tables 200rpm shaken cultivation 16h illustrates that step extracts plasmid by Omga plasmid extraction kit, and with BamH I/Xba I to plasmid Carry out double digestion identification.Mentioned plasmid concentration is measured with micro ultraviolet specrophotometer, and calculates plasmid copy as follows Number, plasmid are named as pMD-Ac, save backup in -20 DEG C.
2. absolute fluorescence quantitative PCR
It is 10 by standard items plasmid doubling dilution according to the copy number of the standard items plasmid pMD-Ac of measurement8、107、106、 105、104Copies/ μ L, using the series of concentrations plasmid as template, each concentration does 3 repetitions, as standard curve；To check weighing Group baculovirus DNA does 3 repetitions, quantitative fluorescent PCR reaction system are as follows: 2 × T5Fast qPCR Mix, 10 μ L, Ac-F0.8 μ L, Ac-R 0.8 μ L, standard plasmid/recombinant baculovirus DNA 2.0 μ L, 6.4 μ L of distilled water；Reaction condition are as follows: 95 DEG C of 1min； 95 DEG C of 10s, 60 DEG C of 15s, totally 35 recycle.60 DEG C of acquisition fluorescence signals；
Standard curve is drawn according to fluorescent quantitative PCR result, calculates recombinant virus dna copy number, and count as follows Calculate virus titer:
Recombinant baculovirus Ac-Cap-1, Ac-TBCap-1, Ac-Cap-2, Ac-TBCap-2 are expanded through continuous two generation, are obtained Obtain each 50ml of P3 generation virus.The standard plasmid pMD-Ac 1. constructed using step measures recombination bar by absolute fluorescence quantitative PCR The titre of shape virus, recombinant baculovirus Ac-Cap-1, Ac-Cap-2, Ac-TBCap-1 and Ac-TBCap-2 is copied as the result is shown Number is respectively 6.4 × 107copies/μL、1.5×108copies/μL、5.7×107Copies/ μ L and 1.1 × 108copies/ μ L, calculating virus titer is respectively 1.2 × 109PFU/mL、2.8×109PFU/mL、1.1×109PFU/mL and 2.1 × 109PFU/mL。
(5) Western-blot detects recombinant protein expression
1. preparing PAGE gel according to PAGE gel rapid configuration kit specification.Respectively by P1, P2, P3 For the metainfective sf9 cell sample of recombinant virus Ac-Cap-1, Ac-Cap-2, Ac-TBCap-1 and Ac-TBCap-2 through SDS- After PAGE gel electrophoresis, Western blot identification is carried out after transferring pvdf membrane, detects Cap the and TBCap albumen pair of expression The reactivity of PCV2 positive serum.
As a result as shown in figure 5, the cell sample of infection Ac-Cap-1 and Ac-Cap-2 recombinant virus has specifically at 28kDa Property band；Ac-TBCap-1 and Ac-TBCap-2 have specific band at 44kDa.The above band meets expected size, and Wild baculoviral has no specific band, it was demonstrated that recombinant virus constructs successfully and can correct express express target protein.
2. by P3 for recombinant baculovirus Ac-Cap-1, Ac-Cap-2, Ac-TBCap-1 and Ac-TBCap-2 with identical MOI Value infection logarithmic phase growth sf9 cell, specific method with step 1..
The expressing quantity of identical harvest time compares discovery, and rBac-Cap-2, rBac-TBCap-2 of double startup are obtained Recombinant baculovirus than single promoter rBac Cap-1, rBac-TBCap-1 obtain recombinate shape virus infection cell Sample expression quantity is high, so the recombinant baculovirus Ac- that subsequent experimental is obtained using double-promoter rod granule without special instruction Cap-2,Ac-TBCap-2.(Fig. 6).
(6) recombinant protein expression condition optimization
The semi-quantitative analysis of recombinant protein: using the His label protein of known concentration as standard items albumen, Western is carried out Blot semi-quantitative analysis measures recombinant protein concentration.The cell sample of preparation is loaded according to predetermined order, while series is dense The standard His label protein of degree is loaded in order, rear to carry out PAGE gel electrophoresis, successively carries out Western blot points Analysis.Exposure image carries out gray value analysis to each purpose band with ImageJ software, is drawn based on standard protein band Gray value-protein concentration linear function calculates recombinant protein concentration.
1. with two kinds of different culture medium culture sf9 cells of SF900 and SIM SF to logarithmic growth phase, recombinant baculovirus Ac-Cap-2, Ac-TBCap-2 distinguish infection cell with the infective dose of MOI=1, and harvest cell sample, system with same time Standby protein sample carries out Western-blot quantitative detection by the semi-quantitative analysis method of above-mentioned recombinant protein, dense with standard items Degree be ordinate, corresponding gray value be abscissa draw standard protein curve, calculate recombinant C ap-2 protein concentration is respectively 333 μ g/mL (SF900) and 115 μ g/mL (SIM SF), recombination TBcap-2 protein concentration be respectively 424 μ g/mL (SF900) and 66μg/mL(SIM SF).(Fig. 7 A).Know that the selection of culture medium has a certain impact to recombinant protein expression quantity, it is thin using sf9 When born of the same parents are used for express express target protein, if do not considered culture medium cost, SF900 culture medium can be preferentially selected.
2. recombinant baculovirus Ac-Cap-2, Ac-TBCap-2 infect logarithmic growth phase with the infective dose of MOI=1 respectively Sf9 (consider cost, herein sf9 using SIM SF cultivate) and High-five cell (use and the same price of SIM SF Culture medium), protein sample is prepared in 96h harvest cell, and carry out by the semi-quantitative analysis method of above-mentioned recombinant protein Western-blot quantitative detection, using standard concentration as ordinate, corresponding gray value is that abscissa draws standard protein song Line, calculate recombinant C ap-2 protein concentration is respectively 335 μ g/mL (sf9 cell) and 709 μ g/mL (High-five cell), weight Group TBcap-2 protein concentration is respectively 278 μ g/mL (sf9 cell) and 914 μ g/mL (High-five cell) (Fig. 7 B).Known to It is significantly larger than sf9 expression cell amount using High-five cell expression protein content, therefore subsequent experimental uses High-five Cell is to express recombinant protein.
3. by P3 for recombinant baculovirus Ac-Cap-1, Ac-Cap-2, Ac-TBCap-1 and Ac-TBCap-2 respectively with 1,2 With the High-five cell of 4 MOI value inoculation logarithmic phase growth, 27 DEG C of constant-temperature table 140rpm shaken cultivations, respectively at inoculation 48h, 72h, 96h after virus take 100 μ l cell cultures to prepare protein sample, carry out Western blot detection, determine Recombinant protein optimal expression condition.
As a result as shown in figure 8, in cell pyrolysis liquid, it can be detected the table of Cap and TBCap recombinant protein after 48h It reaches, recombinant protein expression quantity is increase with time in cell pyrolysis liquid；With the increase of infective dose, the cell of various time points is trained The expression protein concentration of object is supported without more apparent difference.In view of the cost problem in actual production process, this experiment is not attempted to make With higher infective dose.Suggest using the infective dose of MOI=1 in actual production, and harvests Cap and TBCap in 96h and recombinate egg White, expression quantity respectively can be of about 709 μ g/mL and 914 μ g/mL.
(7) solubility of recombinant protein and secreting, expressing analysis
According to it is above-mentioned probe into it is optimal connect toxic dose, by P3 for recombinant baculovirus Ac-Cap-2 and Ac-TBCap-2 distinguish Being inoculated in concentration is 2 × 106The volume of cells/mL is in the suspension High Five cell of 10mL, carefully in different time harvest Born of the same parents' culture supernatant, and prepare protein sample；According to optimal harvesting time, cell suspending culture solution is poured into the centrifuge tube of 50mL In, room temperature 1000rpm is centrifuged 10min, and cell precipitation is resuspended, is placed in and carries out ultrasonic disruption on ice by the PBS that 5mL is added, Remaining ultrasonic disruption sample is placed in 4 DEG C of centrifuges by the ultrasonic disruption sample for taking 80 μ L, 11000rpm centrifugation 15min harvests supernatant, takes out the supernatant solution of 80 μ L.Upper final proof after the ultrasonic disruption sample of 80 μ L and centrifugation 5 × SDS sample-loading buffer of 20 μ L is added in product, boils 10min for 100 DEG C in constant-temperature metal bath, sample is put in -20 DEG C of guarantors It deposits.By the protein sample after denaturation by Western blot with the recombinant protein of the His source of mouse Identification of the antibodies expression marked It is soluble.
As a result as shown in figure 9, being inoculated with P3 for the High Five cell of recombinant baculovirus Ac-Cap-2 through ultrasonic disruption Recombinant protein c ap, size about 28kDa (Fig. 9 A) can be detected in cell fragment and supernatant afterwards；It is rod-shaped to be inoculated with P3 generation recombination The High Five cell of viral Ac-TBCap-2 can detect recombination egg in the cell fragment and supernatant after ultrasonic disruption White TBCap, size about 44kDa.The result shows that: recombinant protein c ap and TBCap is after ultrasonic disruption centrifugal treating, part egg Bai Jun is dissolved in PBS；It is good anti-to show that recombinant protein c ap and TBCap still has after ultrasonication centrifugal treating simultaneously Answer originality.
With the P3 of 1 MOI for recombinate shape virus infection High-five cell, appropriate time takes a small amount of cell after infection Culture supernatant, through Western-blot testing goal protein expression situation, 48h begins with recombination after cell infection as the result is shown The expression of TBCap protein secretion reaches maximum in 72h, and nothing obviously increases later；96h just begins with recombinant C ap egg after cell infection White secreting, expressing, this secreting, expressing difference may be related (Figure 10) with recombinant protein architectural characteristic.
The immune efficacy experiment of the subunit vaccine of 3 recombinant protein of embodiment preparation
(1) preparation of PCV2 subunit vaccine
3rd generation recombinant baculovirus liquid Ac-Cap-2 and Ac-TBCap-2 is inoculated in concentration according to the best poison amount that connects respectively It is 2 × 106In the High Five suspension cell of cells/mL, 27 DEG C of constant-temperature table 120rpm shaking cultured cells are placed in, point Cell precipitation is not collected by centrifugation according to best harvest time, suitable PBS is added, cell is resuspended, is placed on ice, carries out ultrasound Wave smudge cells, then 11000rpm is centrifuged 15min under the conditions of 4 DEG C.Collect supernatant, the immunizing antigen liquid as expressed.
The concentration of recombinant protein is adjusted to every milliliter of 1000 μ g, by recombinant protein c ap, TBCap of this concentration respectively and ISA201VG adjuvant is emulsified according to the ratio of 1:1, is prepared into the PCV2 subunit that every 200 μ l contains 100 μ g destination proteins Vaccine is for being immunized mouse experiment.The concentration of recombinant protein is adjusted to every milliliter of 625 μ g, by the recombinant protein c ap of this concentration, TBCap is respectively and MontanideTM01 adjuvant of GEL is uniformly mixed according to the ratio of 4:1, is prepared into every 200 μ l and is contained The PCV2 subunit vaccine of 100 μ g destination proteins is for being immunized mouse experiment.
The concentration of recombinant protein is adjusted to every milliliter of 250 μ g recombinant proteins, by recombinant protein c ap, TBCap of this concentration Respectively and MontanideTM01 adjuvant of GEL is uniformly mixed according to the ratio of 4:1, is prepared into every milliliter and is contained 200 μ g mesh Albumen PCV2 subunit vaccine for immune swine test.
(2) mouse immune is tested
1. mice group and immune
The SPF grade BALB/c mouse of 30 4 week old is randomly divided into 6 groups, it is every group 5, corresponding using subcutaneous multi-point injection Vaccine, each group immunization time, dosage and injection reagent are shown in Table 1.
1 experimental animal of table is grouped (mouse)
Group Inject reagent Head exempts from dosage Two exempt from dosage (after head exempts from 2 weeks)
1 Cap+MontanideTM01 adjuvant of Gel 200 μ l/ are only 200 μ l/ are only
2 TBCap+MontanideTM01 adjuvant of Gel 200 μ l/ are only 200 μ l/ are only
3 Cap+ISA 201VG adjuvant 200 μ l/ are only 200 μ l/ are only
4 TBCap+ISA 201VG adjuvant 200 μ l/ are only 200 μ l/ are only
5 Porcine circovirus type 2 subunit vaccine is commercialized One part One part
6 Blank control 200 μ l PBS/ are only 200 μ l PBS/ are only
Before immune, head exempt from after the 7th, 14 day, two exempt from after eye socket blood sampling, every batch of blood sample point were carried out to every group of mouse in the 7th, 14 day It is saved from serum in -80 DEG C, is used for ELISA antibody test.Immune group mouse survival situation is observed simultaneously, inoculation position is no different Often wait to carry out subunit vaccine security inspection.
2. ELISA antibody test
Serum to be checked is detected with 2 type ELISA antibody assay kit of pig annulus (Wuhan Ke Qian Biological Co., Ltd.) Antibody level.
Mouse, and evaluation comparison immune protective effect is immunized in the result is shown in Figure 11.With indirect ELISA measurement PCV2 specificity Antibody, OD value when as a result diluting 50 times with serum indicate.The result shows that the vaccine immunity group being mixed with different adjuvants, 14 days ELISA antibody reaches higher level after the first exemption, after the first exemption with Cap the and TBCap group of 01 adjuvant of GEL mixing 14 days ELISA antibody has reached higher level, remains at higher level 21 days, 28 days；And with the preparation of 201 adjuvant emulsions Cap and TBCap group 28 days ELISA antibody reaches highest level after the first exemption, can deduce aqueous adjuvants GEL 01 mix epidemic disease The humoral immune response of Miao Zuneng induced higher levels within the immune rear short period, oil adjuvant 201 emulsify vaccine group and are increasing Also it is capable of the humoral immune response of induced high levels after being immunized by force, therefore follow-up immunization piglets preferentially use MontanideTM 01 aqueous adjuvants of GEL prepare subunit vaccine.Furthermore, the results showed that, the subunit vaccine of two kinds of recombinant proteins preparation is immunized small Mouse can be stimulated to generate specific antibody after mouse, wherein the TBCap albumen of recombinant baculovirus expression is in identical adjuvant situation Under compared with Cap protein have better immunogenicity.
It lives 3. immune group mouse is all strong in subunit vaccine security inspection one week, inoculation position is no different paradoxical reaction.
(3) piglet immunological Protection
1. piglet grouping and Immunization
It is negative pig by 20 PCV2 and PRRSV antigens, antibody test, is randomly divided into 4 groups, every group 5, using warp Intramuscular injection path injects corresponding vaccine, and each group immunization time, dosage and injection reagent are shown in Table 2.
2 experimental animal of table is grouped (pig)
Every morning measures the body temperature of all piglets after attacking poison, and observes the clinical manifestation of piglet.7 after exempting from one, 14, 21, it takes a blood sample within 28 days, separation serum is used for ELISA antibody test.It attacks after poison to cut open all pigs on the 28th day and kill, take a blood sample, separate serum It is detected for viremia virusemia, observes the organ diseases situation such as every pig lungs and lymph node, take lymph node, lungs in 4% poly It is fixed in formaldehyde, for histotomy production and immunohistochemical experiment.Specific method and result are as follows:
2. attacking piglet clinical manifestation and Temperature changing after poison
Before attacking poison, the clinical lesion of any experimental group is not observed.After first immunisation 4 weeks, to two immune groups and It attacks malicious control group to carry out attacking poison, measures body temperature daily, continuous 4 weeks, statistical result was shown in Figure 12.In same time point, each group Body temperature does not have apparent difference, without the phenomenon that generating heat (being greater than 40 DEG C) yet.But from the point of view of clinical manifestation, attacking malicious group has Both ends pig occur it is obvious thin, tremble, coat thick disorderly drowsiness and undergrowth the phenomenon that.Two immune groups and blank control group are then Show it is normal, without apparent clinical symptoms.
3. ELISA antibody test
The same mouse experiment of specific method, the result is shown in Figure 13, according to mouse immuning test as a result, determining with GEL01 adjuvant Piglet, and evaluation comparison its immune protective effect is immunized in mixed TBCap and Cap respectively.It is anti-with indirect ELISA measurement specificity Body, S/P value when as a result diluting 40 times with serum indicate.The results show that two immune groups are all first in addition to negative control group 14th day after immune is all in seropositivity, shows that two immune groups have different degrees of specific antibody to generate, wherein TBCap The antibody level of group immune group is slightly above Cap group；After enhancing is 2 weeks immune, the antibody level of two immune groups is gradually risen, The antibody level of TBCap group immune group is significantly higher than Cap group.
4. attacking piglet viremia virusemia after poison to detect
QPCR is carried out using the standard plasmid pMD-Ac of different dilutions, standard curve as shown in figure 14 is obtained, attacks poison After 28 days, piglet serum CRP is extracted as template, wherein viral level is measured for qPCR, has obtained the Ct value of each sample, Corresponding nucleic acid copies can be calculated according to standard curve.The viral level of two immune groups will be significantly lower than and attack as the result is shown The level of malicious control group, wherein TBCap group ratio Cap group is lower.This shows that immune group reduces lymph node to a certain extent Infection in tissue, and TBCap group effect is more preferable (Figure 15).
5. attacking piglet virus content detection after poison
QPCR is carried out using the standard plasmid pMD-Ac of different dilutions, has obtained standard curve as shown in figure 14.It attacks Poison cuts open after 28 days and kills all pigs and extract inguinal lymph nodes DNA as template, be used to measure wherein viral level, obtain The Ct value of each sample of different time points can calculate corresponding nucleic acid copies according to standard curve.With viremia virusemia result It is similar, the presence of virus can be detected in the lymph node of all experimental group pigs, but two immune groups will be significantly lower than and attack poison The level of control group, this shows to reduce the infection of the PCV2 in lymph node to a certain extent, but two group differences are little.As a result Show after attacking poison, although vaccine group cannot completely inhibit the generation of viremia virusemia, can largely drop Its low intensity (Figure 16).
6. Pathologic Observation
After the test, it cuts open and kills all pigs, observe the lesion situation of lymph node and lungs.Blank control group and vaccine group Lungs are normal, and the phenomenon that broadening interstitial, bleeding and consolidation occur in the lungs for attacking malicious control group.Lymph node in addition to attacking malicious control group Occur outside oedema, immune group and blank group are substantially all normal.After completing histotomy, pathological observation is carried out, as a result such as Figure 17 It is shown.Compared with immune group, the symptom that apparent interstitial pneumonia occurs in malicious control group is attacked, is mainly shown as that alveolar wall increases Thickness, interstitial are broadening and alveolar reduces, and inflammatory exudate increases.Immune group then more normal or only slight symptom.Attack poison The serious lymphocyte of all pigs of control group is exhausted, hemorrhagic lymphnoditis and erythremia, immune group do not find tissue The damage of pathology lymph, blank control group lymph node tissue have no obvious histologic lesion.The result shows that the Asia of this research preparation Subunit vaccine, which can reduce, infects caused lungs and lymph node pathological change by PCV2.
7. immunohistochemistry
IHC detection is carried out to lymph node tissue using PCV2 monoclonal antibody, testing result is shown in Figure 18.Attack poison group lymph group Knit middle there are more pale brown cytochrome, and immune group shows TBCap and Cap energy then without the pigmented cells of significant specificity The enough proliferation largely mitigated in PCV2 in lymph node, to provide protective effect to immune piglet.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.
SEQUENCE LISTING
<110>Agricultural University Of South China
<120>a kind of 2 type Cap protein of recombinant porcine circovirus and its application of Dominant Epitopes of connecting
<130> 1
<160> 22
<170> PatentIn version 3.3
<210> 1
<211> 378
<212> PRT
<213> Artificial
<220>
<223>a kind of 2 type Cap protein of recombinant porcine circovirus for Dominant Epitopes of connecting
<400> 1
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Cys His Ile Glu Lys Ala Lys Gly Thr Asp Gln
20 25 30
Gln Asn Lys Glu Tyr Cys Ser Lys Glu Gly Gly Lys Trp Trp Asp Gly
35 40 45
Tyr His Gly Glu Glu Val Val Val Ile Asp Asp Phe Tyr Gly Trp Gly
50 55 60
Gly Thr Val Arg Thr Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn
65 70 75 80
Ile Asn Asp Phe Val Pro Pro Gly Gly Gly Gly Gly Gln Gly Asp Arg
85 90 95
Gly Val Gly Ser Thr Ala Val Ile Leu Asp Asp Asn Phe Val Thr Gly
100 105 110
Gly Ser Thr Ile Asp Tyr Phe Gln Pro Asn Asn Lys Arg Gly Gly Asn
115 120 125
Val Asp His Val Gly Leu Gly Ile Ala Phe Gly Gly Thr Tyr Pro Arg
130 135 140
Arg Arg Phe Arg Arg Arg Arg His Arg Pro Arg Ser His Leu Gly Leu
145 150 155 160
Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro Arg His Arg Tyr Arg
165 170 175
Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg Leu Ser Cys Thr Phe
180 185 190
Gly Tyr Thr Val Lys Ala Thr Thr Val Arg Thr Pro Ser Trp Ala Val
195 200 205
Asp Met Met Arg Phe Asn Ile Asn Asp Phe Val Pro Pro Gly Gly Gly
210 215 220
Thr Asn Glu Ile Ser Ile Pro Phe Glu Tyr Tyr Arg Ile Arg Lys Val
225 230 235 240
Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr Gln Gly Asp Arg Gly
245 250 255
Val Gly Ser Thr Ala Val Ile Leu Asp Asp Asn Phe Val Thr Arg Ala
260 265 270
Thr Ala Leu Thr Tyr Gly Pro Tyr Val Asn Tyr Ser Ser Arg His Thr
275 280 285
Ile Pro Gln Pro Phe Ser Tyr His Ser Arg Tyr Phe Thr Pro Lys Pro
290 295 300
Val Leu Asp Ser Thr Ile Asp Tyr Phe Gln Pro Asn Asn Lys Arg Asn
305 310 315 320
Gln Leu Trp Leu Arg Leu Gln Thr Ser Ala Asn Val Asp His Val Gly
325 330 335
Leu Gly Ile Ala Phe Glu Asn Ser Thr Tyr Asp Gln Asp Tyr Asn Ile
340 345 350
Arg Val Thr Met Tyr Val Gln Phe Arg Glu Phe Asn Leu Lys Asp Pro
355 360 365
Pro Leu Lys Pro His His His His His His
370 375
<210> 2
<211> 1137
<212> DNA
<213> Artificial
<220>
<223>nucleotide sequence of the 2 type Cap protein of recombinant porcine circovirus of above-mentioned series connection Dominant Epitopes is encoded
<400> 2
atgaaattct tagtcaacgt tgcccttgtt tttatggtcg tatacatttc ttacatctat 60
gcgtgtcaca tcgaaaaggc taagggcacc gaccagcaga acaaggagta ctgtagcaag 120
gagggtggca agtggtggga cggttaccac ggtgaagaag tggttgtgat cgacgacttc 180
tacggctggg gtggcacagt gcgtactcct agctgggctg tggacatgat gcgtttcaac 240
atcaacgact tcgtccctcc tggtggtggc ggcggccagg gtgaccgtgg cgtgggcagc 300
actgctgtga tcctggacga caacttcgtg accggcggct ccactatcga ctacttccag 360
ccaaacaaca agcgtggtgg taacgtggac cacgtgggcc tcggcatcgc tttcggcggt 420
acatacccac gtcgtcgttt ccgtcgtcgc cgccaccgtc ctcgttccca cctgggtctc 480
atcctgcgtc gtcgcccctg gctggtgcac cctcgccacc gttaccgctg gcgccgcaag 540
aacggcatct tcaacacccg cctgtcctgt actttcggtt acactgtcaa ggccaccacc 600
gtgcgtaccc cttcctgggc tgtggacatg atgcgtttca acatcaacga cttcgtgcct 660
cctggtggcg gcactaacga aatctccatc ccattcgagt actaccgtat ccgtaaggtg 720
aaggtggaat tctggccatg tagccctatc acacagggtg accgcggtgt gggtagcacc 780
gccgtgatcc tggacgacaa cttcgttacc cgtgccaccg ctctgaccta cggtccttac 840
gtgaactact cctcccgcca cacaatccct cagccattca gctaccactc ccgctacttc 900
acccctaagc ctgtgctgga cagcaccatc gactacttcc agcctaacaa caagcgtaac 960
cagctgtggc tgcgcctgca gaccagcgcc aacgtggacc acgtgggtct gggcatcgct 1020
ttcgagaact ccacctacga ccaggactac aacatccgtg tgaccatgta cgtgcagttc 1080
cgtgaattca acctgaagga ccctcctctg aagcctcatc atcaccatca ccattaa 1137
<210> 3
<211> 259
<212> PRT
<213> Artificial
<220>
<223>amino acid sequence of PCV2-Cap albumen
<400> 3
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile
1 5 10 15
Ser Tyr Ile Tyr Ala Thr Tyr Pro Arg Arg Arg Phe Arg Arg Arg Arg
20 25 30
His Arg Pro Arg Ser His Leu Gly Leu Ile Leu Arg Arg Arg Pro Trp
35 40 45
Leu Val His Pro Arg His Arg Tyr Arg Trp Arg Arg Lys Asn Gly Ile
50 55 60
Phe Asn Thr Arg Leu Ser Cys Thr Phe Gly Tyr Thr Val Lys Ala Thr
65 70 75 80
Thr Val Arg Thr Pro Ser Trp Ala Val Asp Met Met Arg Phe Asn Ile
85 90 95
Asn Asp Phe Val Pro Pro Gly Gly Gly Thr Asn Glu Ile Ser Ile Pro
100 105 110
Phe Glu Tyr Tyr Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys
115 120 125
Ser Pro Ile Thr Gln Gly Asp Arg Gly Val Gly Ser Thr Ala Val Ile
130 135 140
Leu Asp Asp Asn Phe Val Thr Arg Ala Thr Ala Leu Thr Tyr Gly Pro
145 150 155 160
Tyr Val Asn Tyr Ser Ser Arg His Thr Ile Pro Gln Pro Phe Ser Tyr
165 170 175
His Ser Arg Tyr Phe Thr Pro Lys Pro Val Leu Asp Ser Thr Ile Asp
180 185 190
Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln
195 200 205
Thr Ser Ala Asn Val Asp His Val Gly Leu Gly Ile Ala Phe Glu Asn
210 215 220
Ser Thr Tyr Asp Gln Asp Tyr Asn Ile Arg Val Thr Met Tyr Val Gln
225 230 235 240
Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro Leu Lys Pro His His His
245 250 255
His His His
<210> 4
<211> 780
<212> DNA
<213> Artificial
<220>
<223>nucleotide sequence of PCV2-Cap albumen
<400> 4
atgaaattct tagtcaacgt tgcccttgtt tttatggtcg tatacatttc ttacatctat 60
gcgacctacc ctcgccgccg cttccgtcgt cgtcgtcacc gtcctcgtag ccacctgggt 120
ctgatcctgc gtcgtcgtcc ttggctggtg caccctcgcc accgttaccg ctggcgccgt 180
aagaacggta tcttcaacac ccgtctgagc tgtaccttcg gttacacagt gaaggctacc 240
accgtgcgta cccctagctg ggccgtggac atgatgcgtt tcaacatcaa cgacttcgtg 300
cctcctggcg gcggtaccaa cgaaatcagc atccctttcg agtactaccg tatccgcaag 360
gtgaaggtgg agttctggcc ttgctcccct atcacccagg gtgaccgtgg tgtgggctcc 420
actgctgtga tcctggacga caacttcgtc acccgtgcta ccgctctgac ctacggtcct 480
tacgtgaact actcctcccg ccacaccatc ccacagcctt tcagctacca cagccgctac 540
ttcaccccta agcctgtgct ggactccacc atcgactact tccagcctaa caacaagcgc 600
aaccagctgt ggctccgcct gcagactagc gctaacgtgg accacgtggg tctgggcatc 660
gctttcgaaa actccaccta cgaccaggac tacaacatcc gtgtgaccat gtacgtgcag 720
ttccgtgaat tcaacctgaa ggaccctcct ctgaagcctc atcatcacca tcaccattaa 780
<210> 5
<211> 34
<212> DNA
<213> Artificial
<220>
<223>primer Cap-p10-F
<400> 5
ctcgagatga aattcttagt caacgttgcc cttg 34
<210> 6
<211> 34
<212> DNA
<213> Artificial
<220>
<223>primer Cap-p10-R
<400> 6
gctagcttaa tggtgatggt gatgatgagg cttc 34
<210> 7
<211> 34
<212> DNA
<213> Artificial
<220>
<223>primer Cap-pH-F
<400> 7
ggatccatga aattcttagt caacgttgcc cttg 34
<210> 8
<211> 34
<212> DNA
<213> Artificial
<220>
<223>primer Cap-pH-R
<400> 8
gagctcttaa tggtgatggt gatgatgagg cttc 34
<210> 9
<211> 26
<212> DNA
<213> Artificial
<220>
<223>primer TBCap-p10-F
<400> 9
ctcgagatga aattcttagt caacgt 26
<210> 10
<211> 26
<212> DNA
<213> Artificial
<220>
<223>primer TBCap-p10-R
<400> 10
gctagcttaa tggtgatggt gatgat 26
<210> 11
<211> 26
<212> DNA
<213> Artificial
<220>
<223>primer TBCap-pH-F
<400> 11
ggatccatga aattcttagt caacgt 26
<210> 12
<211> 26
<212> DNA
<213> Artificial
<220>
<223>primer TBCap-pH-R
<400> 12
gagctcttaa tggtgatggt gatgat 26
<210> 13
<211> 20
<212> DNA
<213> Artificial
<220>
<223>primer pFBD-1F
<400> 13
tattccggat tattcatacc 20
<210> 14
<211> 19
<212> DNA
<213> Artificial
<220>
<223>primer pFBD-1R
<400> 14
acaaatgtgg tatggctga 19
<210> 15
<211> 20
<212> DNA
<213> Artificial
<220>
<223>primer pFBD-2F
<400> 15
tttgttcgcc caggactcta 20
<210> 16
<211> 20
<212> DNA
<213> Artificial
<220>
<223>primer pFBD-2R
<400> 16
acggaccttt aattcaaccc 20
<210> 17
<211> 26
<212> DNA
<213> Artificial
<220>
<223>primer M13F
<400> 17
cccagtcacg acgacgttgt aaaacg 26
<210> 18
<211> 23
<212> DNA
<213> Artificial
<220>
<223>primer M13R
<400> 18
agcggataac aatttcacac agg 23
<210> 19
<211> 18
<212> DNA
<213> Artificial
<220>
<223>primer Ac-F
<400> 19
agatgcgaca cagatgga 18
<210> 20
<211> 20
<212> DNA
<213> Artificial
<220>
<223>primer Ac-R
<400> 20
tcaacaagaa tggaccgaat 20
<210> 21
<211> 20
<212> DNA
<213> Artificial
<220>
<223>primer AcMK107-F
<400> 21
gaaattacaa ccgtccaact 20
<210> 22
<211> 20
<212> DNA
<213> Artificial
<220>
<223>primer AcMK107-R
<400> 22
cagcgacact gactaccata 20

Claims (10)

1. a kind of 2 type Cap protein of recombinant porcine circovirus for Dominant Epitopes of connecting, it is characterised in that its amino acid sequence such as SEQ Shown in ID No.1.
2. encoding the nucleotide sequence of the 2 type Cap protein of recombinant porcine circovirus of series connection Dominant Epitopes described in claim 1 such as Shown in SEQ ID No.2.
3. a kind of recombinant baculovirus transfer vector, it is characterised in that include coding as claimed in claim 2 series connection Dominant Epitopes 2 type Cap protein of recombinant porcine circovirus nucleotide sequence.
4. recombinant baculovirus transfer vector according to claim 3, it is characterised in that:
The carrier that sets out of the recombinant baculovirus transfer vector is pFastBacTMDual carrier.
5. recombinant baculovirus transfer vector according to claim 4, it is characterised in that:
The recombinant baculovirus transfer vector is respectively in pFastBacTMRight is cloned under Dual carrier P10, PH promoter to want The nucleotide sequence of the 2 type Cap protein of recombinant porcine circovirus of coding series connection Dominant Epitopes described in asking 2.
6. a kind of recombinant baculovirus, it is characterised in that carried by the described in any item recombinant baculovirus transfers of claim 3~5 Body converts DH10Bac Escherichia coli, is recombinated by swivel base, obtains recombinant baculovirus plasmid；Finally utilize cationic-liposome Cellfectin II after recombinant baculovirus plasmid transfection insect cell, will collect supernatant and obtain.
7. recombinant baculovirus according to claim 6, it is characterised in that:
The insect cell is sf9 cell or High-five cell.
8. a kind of PCV2 subunit vaccine, it is characterised in that the inoculation insect of recombinant baculovirus described in claim 6 or 7 is thin Born of the same parents after expanding culture, collect destination protein, destination protein and immunologic adjuvant are mixed to get.
9. the preparation method of PCV2 subunit vaccine according to claim 8, characterized by comprising the steps of:
Recombinant baculovirus described in claim 6 or 7 is inoculated with to the High-Five insect cell for the culture that suspends, is received after infection Collect culture, centrifugation removal culture supernatant；Sonicated cells, centrifugation removal cell fragment, collect ultrasonic supernatant；It will be in ultrasound Destination protein and immunologic adjuvant emulsify or are mixed to prepare PCV2 subunit vaccine in clear.
10. the 2 type Cap protein of recombinant porcine circovirus of series connection Dominant Epitopes described in claim 1 is in preparation PCV2 vaccine Application.
CN201910610744.8A 2019-07-08 2019-07-08 Tandem dominant epitope recombinant porcine circovirus type 2 Cap protein and application thereof Active CN110423269B (en)

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