CN103103205B - Gene for encoding recombinant porcine circovirus type 2 (PCV2) Cap protein and application of gene - Google Patents
Gene for encoding recombinant porcine circovirus type 2 (PCV2) Cap protein and application of gene Download PDFInfo
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- CN103103205B CN103103205B CN201310065186.4A CN201310065186A CN103103205B CN 103103205 B CN103103205 B CN 103103205B CN 201310065186 A CN201310065186 A CN 201310065186A CN 103103205 B CN103103205 B CN 103103205B
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Abstract
The invention provides a gene for encoding a recombinant porcine circovirus type 2 (PCV2) Cap protein and an application of the gene, and belongs to the field of molecular biology. The gene for encoding the recombinant PCV2 Cap protein contains a sequence as shown in SEQ ID NO:1. The invention also provides a recombinant expression vector containing the gene, and a composition containing the recombinant PCV2 Cap protein. The composition is prepared through the following steps of: culturing recombinant bacteria, breaking to obtain a supernatant of lysate, and then purifying to obtain the composition containing the recombinant PCV2 Cap protein. According to the invention, the Cap protein encoding gene is subjected to codon optimization, so the recombinant Cap protein can be high in yield, high in efficiency and suitable for soluble expression; and the composite contains peptidoglycan anchor protein (PA), so the subsequent purification is benefited. The recombinant PCV2 Cap protein provided by the invention has good activity, and the composite containing the recombinant protein can produce high-level specific antibodies of the PCV2 after a mouse is immunized by the composite.
Description
technical field
The invention belongs to biology field, be specifically related to the method for a kind of Recombinant Swine circovurus type 2 (PCV2) Cap albumen.
background technology
PCV2 contains two main open reading frame: ORFl and ORF2, and the ORFl two kinds of replicase proteins of encoding participate in virus replication; ORF2 coding nucleocapsid protein Cap is the main immunogenic protein of virus.McNeiUy etc. studies confirm that Cap protein immune animal can produce viral neutralizing antibody.ORF2 gene is as the key object of developing recombinant vaccine.
As everyone knows, the detection of PCV2 is the important component part of prevention and control PCV2 relative disease.Existing PCV2 detection method includes PCR, immunohistochemical methods, in situ hybridization and elisa technique etc.Elisa technique has high specific, susceptibility, and simple, convenient easy row is convenient to the advantage of a large amount of sample detection.Cap albumen, as the main immunogenic protein of PCV2, can be used as the diagnostic antigen that PCV2 ELISA detects.Therefore, obtaining highly purified Cap albumen is to set up the key that PCV2 ELISA detects.
But in prior art, Cap albumen adopts eukaryotic expression system to yield poorly, separation and purification difficulty; Carry out prokaryotic expression and mainly with the inclusion body form of non-activity, exist, so Cap albumen is difficult to large-scale production.
summary of the invention
The gene that the object of this invention is to provide a kind of restructuring carrying Cap gene of porcine circovirus type 2 of encoding, is easy to solubility expression.
Another object of the present invention is to provide the recombinant expression vector that contains said gene.
Another object of the present invention is to provide the recombinant bacterium that contains said gene, and the expression amount of target protein is high, and expression product is easy to purifying.
A further object of the present invention is to provide the mixture that contains the carrying Cap gene of porcine circovirus type 2 of recombinating, and this mixture has good immunogenicity, and production efficiency is high, is easy to purifying, and preparation method is simple, is easy to large-scale production.
Object of the present invention adopts following technical scheme to realize:
The gene of coding restructuring carrying Cap gene of porcine circovirus type 2, contains the sequence shown in SEQ ID NO:1.
Also contain the encoding gene of peptidoglycan grasp PA albumen.
The gene order of described coding restructuring carrying Cap gene of porcine circovirus type 2 is as shown in SEQ ID NO:3.
A kind of recombinant expression vector that contains said gene.
This recombinant expression vector is by gained between the Nde I of described gene insertion pET-32a and two restriction enzyme sites of Xho I.
The recombinant bacterium that contains said gene.
Described recombinant bacterium is that described recombinant expression vector is transformed to e. coli bl21 gained.
A mixture that contains the carrying Cap gene of porcine circovirus type 2 of recombinating, adopts preparation with the following method: cultivate described recombinant bacterium, the broken rear lysate supernatant that obtains, obtains the mixture that contains the carrying Cap gene of porcine circovirus type 2 of recombinating after purifying.
Described purification process is: after Lactococcus lactis is boiled with acid, obtain Gram-positive and strengthen matrix granule; Gram-positive is strengthened to matrix granule and add in lysate supernatant and adsorb, get precipitation after centrifugal to obtain described mixture.Gram-positive strengthens matrix granule and is abbreviated as GEM particle.
Described acid is hydrochloric acid; The add-on that Gram-positive strengthens matrix granule is: in the lysate supernatant that contains 0.5～2 milligram of albumen, add 2.5 × 10
9individual Gram-positive strengthens matrix granule.
A kind of described mixture is in the application of preparing aspect pig circular ring virus vaccine and pig circular ring virus diagnostic kit.
The present invention is with respect to the beneficial effect of art methods:
1. the present invention has been undertaken codon optimized by carrying Cap gene of porcine circovirus type 2 encoding gene, so restructuring carrying Cap gene of porcine circovirus type 2 can highly efficient and productive, soluble-expression, solve Cap albumen low in eukaryotic expression amount, in prokaryotic cell prokaryocyte, expressed a difficult problem for non-activity.Due to escherichia coli prokaryotic expression system comparative maturity, so simple to operate.
2. the present invention's carrying Cap gene of porcine circovirus type 2 of recombinating contains peptidoglycan grasp albumen PA, can be combined with GEM Particle Phase, is conducive to subsequent purification, and production cost is low, is easy to large-scale production.
3. Recombinant Swine circovurus type 2 Cap protein-active of the present invention is good, after the mixture immune mouse that contains this recombinant protein, can produce high-caliber porcine circovurus type 2 specific antibody.Therefore the mixture that, contains the carrying Cap gene of porcine circovirus type 2 of recombinating can be used for preparing diagnostic kit and the porcine circovirus type 2 subunit vaccine of porcine circovirus 2 type.
accompanying drawing explanation
Fig. 1 is peptidoglycan grasp PA protein gene pcr amplification product electrophorogram, and wherein M is DNA Marker, and 1 is pure water contrast, and 2 is the gene amplification product of peptidoglycan grasp PA albumen.
Fig. 2 is that recombinant plasmid enzyme is cut qualification result, and wherein M is DNA Marker, and swimming lane 1 is the double digestion product of plasmid PET-32a; The double digestion product of 2 positive recombinant plasmids.
Fig. 3 (A) and Fig. 3 (B) are the SDS-PAGE analysis chart of restructuring carrying Cap gene of porcine circovirus type 2; Wherein M is albumen relative molecular weight Marker; In Fig. 3 (A), swimming lane 1 and 2 is the lysate supernatant of the recombinant strains BL21-Cap-PA of abduction delivering; 3 is the lysate supernatant of the control strain of abduction delivering; In Fig. 3 (B), swimming lane 1 is the full bacterium of recombinant strains BL21-Cap-PA of not abduction delivering; The full bacterium of recombinant strains BL21-Cap-PA that swimming lane 2 is abduction delivering; The recombinant strains BL21-Cap-PA lysate supernatant that swimming lane 3 is abduction delivering; The recombinant strains BL21-Cap-PA lysate precipitation that swimming lane 4 is abduction delivering.
Fig. 4: the Western Blot qualification result of restructuring carrying Cap gene of porcine circovirus type 2, wherein M is albumen relative molecular weight Marker; 1 is the recombinant strains BL21-Cap-PA lysate supernatant of abduction delivering; 2 is the contrast bacterium lysate supernatant of abduction delivering.
Fig. 5 (A) and Fig. 5 (B) are the transmission electron microscope pictures of Lactococcus lactis MG1363 and GEM particle, and wherein Fig. 5 (A) is Lactococcus lactis MG1363, and Fig. 5 (B) is GEM particle.
Fig. 6 is that wherein M is albumen relative molecular weight Marker containing the SDS-PAGE electrophorogram of the mixture of restructuring carrying Cap gene of porcine circovirus type 2, and swimming lane 1 is the mixture containing restructuring carrying Cap gene of porcine circovirus type 2, and swimming lane 2 is GEM particle.
Fig. 7 (A) and Fig. 7 (B) are GEM particle and the Electronic Speculum figure that contains the mixture of restructuring carrying Cap gene of porcine circovirus type 2, and wherein Fig. 7 (A) is GEM particle, and Fig. 7 (B) is the mixture containing restructuring carrying Cap gene of porcine circovirus type 2.
Fig. 8 is the each group of antibody level of serum after mouse immune.
embodiment
Peptidoglycan grasp PA albumen designs as purification tag, can, in the N of restructuring carrying Cap gene of porcine circovirus type 2 end or C end, antibody horizontal after its expression amount, antigenicity and vaccine immunity not affected.
The structure of embodiment 1 peptidoglycan grasp PA albumen, Cap protein gene fragment and prokaryotic expression plasmid
1. the acquisition of peptidoglycan grasp PA protein gene fragment
(1) design of primers
According to the gene order of Lactococcus lactis MG1363 (U17696.1), design primer PAF(SEQ ID NO:4) and PAR(SEQ ID NO:5), the gene fragment of its peptidoglycan grasp PA albumen that increases.
The sequence of PAF is: 5 '-ACTACTTATACCGTCAAATC-3 ';
The sequence of PAR is: 5 '-TTTTATTCGTAGATACTGAC-3 '.
(2) pcr amplification object fragment
Adopt ordinary method to cultivate Lactococcus lactis MG1363, results thalline.Adopt RNAiso Plus(Takara) extract RNA, the reverse transcription of application random primer becomes cDNA as PCR reaction template.
The system of reverse transcription and response procedures: every part of template (3 ~ 4ug) adds random primer 1 μ l, 11.75 μ l Rnase free H
2o, low speed wink from, then under 70 ℃ of conditions, be incubated 10min; Ice bath 2min again, low speed wink from, add 2 μ l 5 × M – MLV buffer, 0.5 μ l dNTP Mix, 0.25 μ l RNase Inhibitor, 0.5 μ l Reverse Transcriptase, 42 ℃, 1h; Then 70 ℃ of effect 15min, after ice bath ,-20 ℃ of preservations, standby as cDNA template.
PCR reaction system is: primer PAF, PAR(10pmol/L) each 1 μ l, MgCl
2(25mmol/L) 1.5 μ l, dNTPs(2.5mmol/L) 2.0 μ l, 10 × PCR buffer, 2.5 μ l, cDNA template 2.0 μ l, (5U/ μ is 0.2 μ l l), and sterilizing distilled water is supplied volume to 25 μ l for Taq enzyme.PCR loop parameter: 95 ℃ of denaturation 5min; 94 ℃ of 45s, 54 ℃ of 45s, 72 ℃ of 45s, carry out 30 circulations; Last 72 ℃ are extended 10min.In contrast, with pure water, substitute cDNA.The agarose gel electrophoresis of employing 1% is identified the pcr amplification product (Fig. 1) of peptidoglycan grasp PA albumen.From Fig. 1, can find out that amplified production size is about 585bp.Reclaim amplified production, be cloned into pMD18-T carrier, carry out sequential analysis, the gene order of peptidoglycan grasp PA albumen (585 bp) as shown in SEQ ID NO:2.
Agents useful for same source TAKARA in reverse transcription and PCR reaction.
The acquisition of 2.Cap protein gene fragment
By PCV2 ORF2 gene order (GENEBANK accession number: KC153106.1), after 41 amino acid of amputation N end, carried out codon optimizedly, obtain codon optimized Cap protein gene fragment, its sequence is as shown in SEQ ID NO:1.
At codon optimized Cap protein gene fragment 5 ' end, add CATATG, at 3 ' end, add CTCGAG, (designed restriction enzyme site, utilized follow-up connection and enzyme to cut), transfers to Shanghai Ying Jun Bioisystech Co., Ltd synthetic.
3. the structure of recombinant plasmid and evaluation
The gene of peptidoglycan grasp PA albumen is connected with 3 of codon optimized Cap protein coding gene ' end, the gene of the carrying Cap gene of porcine circovirus type 2 that obtains recombinating, its sequence is as shown in SEQ ID NO:3.Concrete grammar is: the pcr amplification product of peptidoglycan grasp PA albumen is connected through T4 DNA ligase with fragment synthetic in the present embodiment title 2, obtains connecting product 1.
To connecting product 1, carry out double digestion and be connected with pET-32a by Nde I, Xho I.
Double digestion system:
10×H buffer 3μl，
NdeⅠ 1μl，
XhoⅠ 1μl，
PET-32a or connection product 18 μ l,
DH
2o supplies volume to 30 μ l.
Double digestion reaction system is acted on to 4h under 37 ℃ of conditions, then with agarose gel electrophoresis evaluation enzyme, cut product.
Linked system:
10×T4 ligase buffer 2.5μl，
PET-32a double digestion fragment 4 μ l,
T4 DNA ligase 1μl，
DH
2o supplies volume to 25 μ l.
Linked system is connected and spent the night under 16 ℃ of conditions, obtain connecting product 2.
To connect product 2 transfection E.coli DH5 α competent cells, coating is containing the LB flat board of penbritin, 37 ℃ of cultivations.Single bacterium colony on picking flat board is cultivated in the LB substratum that contains penbritin, extracts plasmid, through Nde I/Xho I double digestion, identifies, obtains Fig. 2 after electrophoresis.As can be seen from Figure 2, positive recombinant plasmid, after double digestion, obtains the band of 5366bp and 1182bp two entries.Positive recombinant plasmid through check order correct after, called after pET-32a-Cap-PA.
The structure of embodiment 2 recombinant strains BL21-Cap-PA
1. by positive recombinant plasmid transformed competence colibacillus e. coli bl21 (DE3)
The pET-32a-Cap-PA transfection Escherichia coli BL21(DE3 that embodiment 1 is obtained), obtain recombinant strains BL21-Cap-PA.Empty plasmid pET-32a, according to identical method transfection competence e. coli bl21 (DE3), is obtained to control strain.
2. the abduction delivering of recombinant protein
(1) single bacterium colony of difference picking recombinant strains BL21-Cap-PA and control strain, is inoculated in the liquid LB substratum that contains penbritin (100 μ g/mL) 37 ℃ of incubated overnight.
(2) get above-mentioned cultured bacterium liquid 10 μ l and be inoculated in the LB liquid nutrient medium that 4ml contains penbritin, under 37 ℃, 200rpm condition, about 3h is cultivated in jolting, makes OD
600reach 0.6～0.8.
(3) to adding final concentration in above-mentioned bacterium liquid, be that the IPTG of 0.1mM carries out abduction delivering, inducing temperature is 16 ℃, and induction time is 24h.
(4) by the fermented liquid after abduction delivering at 4 ℃, 6000
g centrifugal 5min under condition, collects thalline; After PBS damping fluid for thalline (pH 7.2) is washed to 2 times, carry out resuspended.
(5) thalline is placed in to ice bath, uses ultrasonic treatment thalline, power is 200W, ultrasonic degradation 5s, and pause 5s, until the clarification of bacterium liquid.
(6) by the thalline after ultrasonic degradation, at 4 ℃, 12000
g centrifugal 10min under condition, collects respectively cleer and peaceful lysate precipitation on lysate.Lysate precipitation is used with the isopyknic PBS damping fluid of supernatant resuspended.
3.SDS-PAGE electrophoretic analysis
In order to analyze the target protein expression level of recombinant strains BL21-Cap-PA and control strain, get respectively the each 100 μ l of cleer and peaceful precipitation on its lysate, add 6 × albumen loading Buffer, fully mix rear 95 ℃ of sex change 5min, instantaneous centrifugal before loading, draw supernatant point sample and carry out SDS-PAGE analysis.From Fig. 3 (A), can find out, compare with the bacterium lysate supernatant that contrasts of abduction delivering, the recombinant strains BL21-Cap-PA lysate supernatant of abduction delivering has an obvious band at 46kDa place.From Fig. 3 (B), can find out, after abduction delivering, recombinant strains BL21-Cap-PA has expressed a large amount of target proteins; In target protein, there is half to realize solubility expression.
1. the lysate supernatant of the BL21-Cap-PA of inducing culture and contrast bacterium is carried out to SDS-PAGE electrophoresis;
2. transfer printing: after SDS-PAGE electrophoresis, adopt half-dried transfer printing that Western blot is gone to pvdf membrane;
3. by the PBST confining liquid sealing that contains 5% skimming milk for pvdf membrane, be placed in 40min under room temperature condition;
4. the pvdf membrane after sealing is immersed to the porcine circovirus 2 type positive serum (purchased from Veterinary Medical Research & Development, the U.S.) of 1:200 dilution, 4 ℃ of overnight incubation;
5. with PBST damping fluid, wash pvdf membrane 5 times, 5min/ time;
6. pvdf membrane is immersed to goat-anti pig IgG-HRP(Nanjing Sheng Xing Bioisystech Co., Ltd of 1:10000 dilution), incubated at room 1h;
7. with PBST damping fluid, wash pvdf membrane 5 times, 5min/ time;
8. with DAB staining kit (green skies biotechnology research institute) colour developing, the results are shown in Figure 4.Western Blot result shows, the restructuring carrying Cap gene of porcine circovirus type 2 of expression can react with porcine circovirus 2 type positive serum, has good antigenicity.
The recombinate preparation of carrying Cap gene of porcine circovirus type 2 great expression and mixture thereof of embodiment 4
1. restructuring carrying Cap gene of porcine circovirus type 2 great expression
Press inductive condition in embodiment 2, abduction delivering BL21-Cap-PA 200ml, centrifugal collection thalline, adds the resuspended bacterial sediment of 50ml PBS damping fluid, centrifugal after ultrasonic degradation, collects lysate supernatant.
2. the preparation of GEM particle
(1) under anaerobic cultivate Lactococcus lactis MG1363, substratum is the GM17 substratum that adds 0.5% glucose, and culture temperature is 30 ℃, and incubation time is 16 ~ 18 hours.
(2) centrifugal collection thalline: by fermented liquid 4000
g under condition, centrifugal 5min, gets thalline, uses distilled water wash 1 time.
(3) the resuspended thalline of hydrochloric acid with the pH 1.0 of former volume of culture 1/5 by Lactococcus lactis MG1363, boils 30min, and centrifuging and taking precipitation obtains GEM particle.
(4) with PBS(pH 7.2) resuspended after washing GEM particle 3 times, with blood counting chamber counting, with the concentration packing (1U=2.5*10 of 10U
9individual/ml) ,-70 ℃ save backup.
Lactococcus lactis MG1363 and GEM particle are fixed by 2.5% glutaraldehyde respectively, with transmission electron microscope observing evaluation GEM particle, as shown in Fig. 5 (A) and Fig. 5 (B).From Fig. 5 (A), Lactococcus lactis MG1363 core has nucleic acid in district; From Fig. 5 (B), the tenuigenin of GEM particle is homogeneous, and nucleic acid and albumen in former nuclear zone are removed.
3. the mixture that preparation contains the carrying Cap gene of porcine circovirus type 2 of recombinating
In the BL21-Cap-PA lysate supernatant of abduction delivering, add GEM particle, the amount adding is according to adding 2.5 × 10 in the lysate supernatant that contains 1mg albumen
9individual GEM particle.Incubated at room 30min, 12000
g collecting precipitation after centrifugal 10min under condition, obtains containing the mixture of carrying Cap gene of porcine circovirus type 2 of recombinating.This mixture is carried out to SDS-PAGE analysis, the results are shown in Figure 6.As can be seen from Figure 6, on mixture swimming lane, there is the band of a 46kDa, illustrate and in mixture, contain restructuring carrying Cap gene of porcine circovirus type 2.
After the mixture that contains the carrying Cap gene of porcine circovirus type 2 of recombinating is suitably diluted, application BCA protein detection kit (green skies biotechnology research institute) is measured protein concentration, and-4 ℃ or-20 ℃ save backup.This recombinant C ap expressing quantity 1.5g/L.
GEM particle is actually the shell of peptidoglycan composition, so the restructuring carrying Cap gene of porcine circovirus type 2 that contains peptidoglycan grasp PA albumen can be combined in the surface of GEM particle.
4. the mixture that electron microscopic observation contains the carrying Cap gene of porcine circovirus type 2 of recombinating
By GEM particle and the mixture that contains the carrying Cap gene of porcine circovirus type 2 of recombinating, after fixing by 2.5% glutaraldehyde respectively, with transmission electron microscope observing, result is as Fig. 7 (A) and Fig. 7 (B).From this two width, figure can find out, GEM particle surface can be in conjunction with the recombinate mixture of carrying Cap gene of porcine circovirus type 2 of a large amount of containing.
The recombinate mouse immuning test of carrying Cap gene of porcine circovirus type 2 of embodiment 5
To contain the mixture of the carrying Cap gene of porcine circovirus type 2 of recombinating, be mixed with the complex solution that protein concentration is 0.5mg/mL with PBS damping fluid (pH=7.2 ~ 7.4), wherein the concentration of GEM particle is 1.25 × 10
9individual/mL.Preparation GEM particle solution, in this solution, GEM granule content is identical with complex solution.Complex solution and GEM particle solution are mixed with aluminium glue adjuvant equal-volume respectively, after emulsification, obtain compound vaccine and GEM particle control vaccine.
Choose and clean 30 of level ICR mouse 5 week age, be divided at random 3 groups, 10/group.First group, immune complex vaccine; Second group, immune GEM particle control vaccine; The 3rd group: unavoidably as blank group.Immunization method: every mouse, the subcutaneous multi-point injection 0.2mL vaccine at back.
Latter 21 days of immunity, by every eyeball of mouse blood sampling separation of serum, with porcine circovirus 2 type ELISA detection kit (Wuhan Ke Qian Bioisystech Co., Ltd) detection antibody level of serum.During detection, the goat-anti pig IgG-HRP in test kit is replaced with to sheep anti-mouse igg-HRP(Nanjing Sheng Xing Bioisystech Co., Ltd of 1:10000 dilution).
The average serum antibody horizontal that calculates each group of mouse, concrete outcome is shown in Fig. 8.As can be seen from Figure 8, compare with the mouse of immune GEM particle control vaccine with control group, the mixture that contains the carrying Cap gene of porcine circovirus type 2 of recombinating significantly inducing mouse produce high-caliber specific antibody (
p< 0.01).Therefore, this mixture can be for detection of porcine circovirus 2 type, or for the preparation of porcine circovirus type 2 subunit vaccine.
SEQUENCE LISTING
<110> Jiangsu Province Agriculture Science Institute
Gene and the application thereof of <120> coding restructuring carrying Cap gene of porcine circovirus type 2
<130> 20130228
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 579
<212> DNA
<213> artificial
<220>
The Cap protein gene that <223> is codon optimized
<400> 1
aatggtattt tcaatactcg tcttagtcgt actattggct acactgtcaa gaaaaccact 60
gttcgtactc ctagttggaa tgttgatatg atgcgtttca atattaatga ctttcttccg 120
cctggcggtg gtagtaatcc tcttacagtt ccgttcgaat actatcgtat tcgtaaagtt 180
aaggttgagt tttggccgtg tagtcctatt acacaaggtg accgtggcgt tggcagtaca 240
gctgtcattc ttgatgataa tttcgttact aaagcaaatg ctcttacata cgacccttat 300
gtgaattact ctagccgtca cactatcacc caaccgttct cctaccacag ccgttacttt 360
acacctaaac ctgtcttaga tcgtacaatt gactatttcc agcctaataa taaacgtaat 420
cagctttggc ttcgtcttca aaccacaggc aatgttgatc atgtaggtct tggtactgca 480
tttgaaaata gtatttacga ccaggattac aatattcgta ttacaatgta cgtgcaattc 540
cgtgaattta atcttaaaga tcctccattg aatccgtaa 579
<210> 2
<211> 585
<212> DNA
<213> Lactococcus lactis MG1363
<400> 2
actacttata ccgtcaaatc tggtgatact ctttggggaa tctcacaaag atatggaatt 60
agtgtcgctc aaattcaaag tgcgaataat cttaaaagta ccattatcta cattggtcaa 120
aaacttgtac tgacaggttc agcttcttct acaaattcag gtggttcaaa caattccgca 180
agcactactc caaccacttc tgtgacacct gcaaaaccaa cttcacaaac aactgttaag 240
gttaaatccg gagataccct ttgggcgcta tcagtaaaat ataaaactag tattgctcaa 300
ttgaaaagtt ggaatcattt aagttcagat accatttata ttggtcaaaa tcttattgtt 360
tcacaatctg ctgctgcttc aaatccttcg acaggttcag gctcaactgc taccaataac 420
tcaaactcga cttcttctaa ctcaaatgcc tcaattcata aggtcgttaa aggagatact 480
ctctggggac tttcgcaaaa atctggcagc ccaattgctt caatcaaggc ttggaatcat 540
ttatctagcg atactatttt aattggtcag tatctacgaa taaaa 585
<210> 3
<211> 1164
<212> DNA
<213> artificial
<220>
The gene of <223> restructuring carrying Cap gene of porcine circovirus type 2
<400> 3
aatggtattt tcaatactcg tcttagtcgt actattggct acactgtcaa gaaaaccact 60
gttcgtactc ctagttggaa tgttgatatg atgcgtttca atattaatga ctttcttccg 120
cctggcggtg gtagtaatcc tcttacagtt ccgttcgaat actatcgtat tcgtaaagtt 180
aaggttgagt tttggccgtg tagtcctatt acacaaggtg accgtggcgt tggcagtaca 240
gctgtcattc ttgatgataa tttcgttact aaagcaaatg ctcttacata cgacccttat 300
gtgaattact ctagccgtca cactatcacc caaccgttct cctaccacag ccgttacttt 360
acacctaaac ctgtcttaga tcgtacaatt gactatttcc agcctaataa taaacgtaat 420
cagctttggc ttcgtcttca aaccacaggc aatgttgatc atgtaggtct tggtactgca 480
tttgaaaata gtatttacga ccaggattac aatattcgta ttacaatgta cgtgcaattc 540
cgtgaattta atcttaaaga tcctccattg aatccgtaaa ctacttatac cgtcaaatct 600
ggtgatactc tttggggaat ctcacaaaga tatggaatta gtgtcgctca aattcaaagt 660
gcgaataatc ttaaaagtac cattatctac attggtcaaa aacttgtact gacaggttca 720
gcttcttcta caaattcagg tggttcaaac aattccgcaa gcactactcc aaccacttct 780
gtgacacctg caaaaccaac ttcacaaaca actgttaagg ttaaatccgg agataccctt 840
tgggcgctat cagtaaaata taaaactagt attgctcaat tgaaaagttg gaatcattta 900
agttcagata ccatttatat tggtcaaaat cttattgttt cacaatctgc tgctgcttca 960
aatccttcga caggttcagg ctcaactgct accaataact caaactcgac ttcttctaac 1020
tcaaatgcct caattcataa ggtcgttaaa ggagatactc tctggggact ttcgcaaaaa 1080
tctggcagcc caattgcttc aatcaaggct tggaatcatt tatctagcga tactatttta 1140
attggtcagt atctacgaat aaaa 1164
<210> 4
<211> 20
<212> DNA
<213> artificial
<220>
<223> PAF
<400> 4
actacttata ccgtcaaatc 20
<210> 5
<211> 20
<212> DNA
<213> artificial
<220>
<223> PAR
<400> 5
ttttattcgt agatactgac 20
Claims (9)
1. the gene of coding restructuring carrying Cap gene of porcine circovirus type 2, is characterized in that: its sequence is as shown in SEQ ID NO:3.
2. a recombinant expression vector that contains gene described in claim 1.
3. recombinant expression vector according to claim 2, is characterized in that: this recombinant expression vector is that described gene is inserted to gained between the Nde I of pET-32a and two restriction enzyme sites of Xho I.
4. contain the recombinant bacterium of gene described in claim 1.
5. recombinant bacterium according to claim 4, is characterized in that: described recombinant bacterium is that recombinant expression vector described in claim 3 is transformed to e. coli bl21 gained.
6. a mixture that contains the carrying Cap gene of porcine circovirus type 2 of recombinating, it is characterized in that adopting preparation with the following method: recombinant bacterium described in cultivation claim 5, after broken, obtain lysate supernatant, after purifying, obtain the mixture that contains the carrying Cap gene of porcine circovirus type 2 of recombinating.
7. contain according to claim 6 the mixture of the carrying Cap gene of porcine circovirus type 2 of recombinating, it is characterized in that described purification process is: after Lactococcus lactis acid is boiled, obtain Gram-positive and strengthen matrix granule; Gram-positive is strengthened to matrix granule and add in lysate supernatant and adsorb, get precipitation after centrifugal to obtain described mixture.
8. contain according to claim 7 the mixture of the carrying Cap gene of porcine circovirus type 2 of recombinating, it is characterized in that described acid is hydrochloric acid; The add-on that Gram-positive strengthens matrix granule is: in the lysate supernatant that contains 0.5～2 milligram of albumen, add 2.5 × 10
9individual Gram-positive strengthens matrix granule.
Described in a claim 6 mixture in the application of preparing aspect pig circular ring virus vaccine and pig circular ring virus diagnostic kit.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310065186.4A CN103103205B (en) | 2013-03-01 | 2013-03-01 | Gene for encoding recombinant porcine circovirus type 2 (PCV2) Cap protein and application of gene |
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| CN201310065186.4A CN103103205B (en) | 2013-03-01 | 2013-03-01 | Gene for encoding recombinant porcine circovirus type 2 (PCV2) Cap protein and application of gene |
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| Publication Number | Publication Date |
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| CN103103205A CN103103205A (en) | 2013-05-15 |
| CN103103205B true CN103103205B (en) | 2014-04-30 |
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| CN104098657B (en) * | 2014-07-07 | 2017-06-27 | 南京农业大学 | A kind of porcine circovirus 2 type immunoprotection polypeptide and vaccine |
| CN107337718B (en) * | 2017-06-20 | 2021-06-01 | 山东省农业科学院畜牧兽医研究所 | Gene for coding porcine circovirus type 2Cap protein and application thereof |
| CN107460173B (en) * | 2017-09-08 | 2020-09-22 | 江苏省农业科学院 | Method for purifying porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus or avian influenza virus |
| CN114317345B (en) * | 2021-12-28 | 2023-12-01 | 龙岩学院 | Preparation method of lactococcus lactis GEM particles |
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| CN101948849A (en) * | 2010-09-02 | 2011-01-19 | 洛阳普莱柯生物工程有限公司 | Preparation and use of truncated Cap protein of porcine circovirus type 2 |
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| Title |
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| ADB28033.1;EMBL;《EMBL》;20100118 * |
| EMBL.ADB28033.1.《EMBL》.2010, |
| 猪圆环病毒2型Cap蛋白表达及其间接ELISA诊断试剂盒研制;董林等;《畜牧与兽医》;20101231;第42卷(第7期);71-76 * |
| 董林等.猪圆环病毒2型Cap蛋白表达及其间接ELISA诊断试剂盒研制.《畜牧与兽医》.2010,第42卷(第7期),71-76. |
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