CN103103205A - 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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- CN103103205A CN103103205A CN2013100651864A CN201310065186A CN103103205A CN 103103205 A CN103103205 A CN 103103205A CN 2013100651864 A CN2013100651864 A CN 2013100651864A CN 201310065186 A CN201310065186 A CN 201310065186A CN 103103205 A CN103103205 A CN 103103205A
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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 the Cap protein immune animal can produce the neutralizing antibody of virus.The 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 can be used as the main immunogenic protein of PCV2 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, the separation and purification difficulty; Carry out prokaryotic expression and exist mainly with the inclusion body form of non-activity, so Cap albumen is difficult to large-scale production.
Summary of the invention
The purpose of this invention is to provide a kind of gene of the restructuring carrying Cap gene of porcine circovirus type 2 of encoding, be 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 the preparation method is simple, is easy to large-scale production.
Purpose 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.
The encoding gene that also contains 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 with 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 the e. coli bl21 gained.
A kind of mixture that contains the carrying Cap gene of porcine circovirus type 2 of recombinating, adopt following method preparation: cultivate described recombinant bacterium, the broken rear lysate supernatant that obtains, after purifying, acquisition contains the mixture of the carrying Cap gene of porcine circovirus type 2 of recombinating.
Described purification process is: obtain Gram-positive after Lactococcus lactis is boiled with acid and strengthen matrix granule; Gram-positive is strengthened matrix granule add in the lysate supernatant and adsorb, get precipitation after centrifugal namely to get described mixture.Gram-positive strengthens matrix granule and is abbreviated as the GEM particle.
Described acid is hydrochloric acid; The add-on that Gram-positive strengthens matrix granule is: contain in the lysate supernatant of 0.5～2 milligram of albumen and add 2.5 * 10
9Individual Gram-positive strengthens matrix granule.
The application of a kind of described mixture aspect preparation 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 carried out the carrying Cap gene of porcine circovirus type 2 encoding gene codon optimized, so the restructuring carrying Cap gene of porcine circovirus type 2 can highly efficient and productive, soluble-expression, solved Cap albumen low in the eukaryotic expression amount, expressed the difficult problem of non-activity in prokaryotic cell prokaryocyte.Due to the 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 the 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, can produce high-caliber porcine circovurus type 2 specific antibody after containing the mixture immune mouse of this recombinant protein.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.
Description of drawings
Fig. 1 is peptidoglycan grasp PA protein gene pcr amplification product electrophorogram, and wherein M is DNA Marker, and 1 is the pure water contrast, and 2 is the gene amplification product of peptidoglycan grasp PA albumen.
Fig. 2 is that the 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; Swimming lane 2 is the full bacterium of recombinant strains BL21-Cap-PA of abduction delivering; Swimming lane 3 is the recombinant strains BL21-Cap-PA lysate supernatant of abduction delivering; Swimming lane 4 is the recombinant strains BL21-Cap-PA lysate precipitation of 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 the GEM particle.
Fig. 6 is the SDS-PAGE electrophorogram that contains the mixture of the carrying Cap gene of porcine circovirus type 2 of recombinating, and wherein M is albumen relative molecular weight Marker, and swimming lane 1 is for containing the mixture of the carrying Cap gene of porcine circovirus type 2 of recombinating, and swimming lane 2 is the GEM particle.
Fig. 7 (A) and Fig. 7 (B) are GEM particle and the Electronic Speculum figure that contains the mixture of the carrying Cap gene of porcine circovirus type 2 of recombinating, and wherein Fig. 7 (A) is the GEM particle, and Fig. 7 (B) is for containing the mixture of the carrying Cap gene of porcine circovirus type 2 of recombinating.
Fig. 8 is the antibody level of serum of respectively organizing after mouse immune.
Embodiment
Peptidoglycan grasp PA albumen designs as purification tag, can be in N end or the C end of restructuring carrying Cap gene of porcine circovirus type 2, on not impact of antibody horizontal after its expression amount, antigenicity and vaccine immunity.
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 (U17696.1) of Lactococcus lactis MG1363, 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 purpose fragment
Adopt ordinary method to cultivate Lactococcus lactis MG1363, the results thalline.Adopt RNAiso Plus(Takara) extract RNA, use the random primer reverse transcription and become cDNA as the PCR reaction template.
The system of reverse transcription and response procedures: (3 ~ 4ug) add random primer 1 μ l to every part of template, 11.75 μ l Rnase free H
2O, low speed wink from, then be incubated 10min under 70 ℃ of conditions; 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 ℃ act on 15min, and after ice bath ,-20 ℃ of preservations, standby as the cDNA template.
The 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, Taq enzyme (5U/ μ l) 0.2 μ l, the sterilization distilled water is supplied volume to 25 μ l.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.Substitute cDNA with pure water in contrast.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 the amplified production size is about 585bp.Reclaim amplified production, be cloned into the 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.
2.Cap the acquisition of protein gene fragment
(the GENEBANK accession number: KC153106.1), carried out codon optimizedly after 41 amino acid of amputation N end, obtained codon optimized Cap protein gene fragment, its sequence is as shown in SEQ ID NO:1 with PCV2 ORF2 gene order.
Add CATATG at codon optimized Cap protein gene fragment 5 ' end, add CTCGAG at 3 ' end, (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 ' end of codon optimized Cap protein coding gene, 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: fragment synthetic in the pcr amplification product of peptidoglycan grasp PA albumen and the present embodiment title 2 is connected through the T4 DNA ligase, obtains connecting product 1.
Carry out double digestion and connect with pET-32a connecting product 1 with Nde I, Xho I.
The 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.
The double digestion reaction system is acted on 4h under 37 ℃ of conditions, then cut product with agarose gel electrophoresis evaluation enzyme.
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 under 16 ℃ of conditions spend the night, obtain connecting product 2.
To connect product 2 transfection E.coli DH5 α competent cells, coating contains the LB flat board of penbritin, 37 ℃ of cultivations.Single bacterium colony on the picking flat board is cultivated in containing the LB substratum of penbritin, extracts plasmid, identifies through Nde I/Xho I double digestion, obtains Fig. 2 after electrophoresis.As can be seen from Figure 2, positive recombinant plasmid obtains the band of 5366bp and 1182bp two entries after double digestion.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. with positive recombinant plasmid transformed competence colibacillus e. coli bl21 (DE3)
PET-32a-Cap-PA transfection Escherichia coli BL21(DE3 with embodiment 1 acquisition), obtain recombinant strains BL21-Cap-PA.Empty plasmid pET-32a according to identical method transfection competence e. coli bl21 (DE3), is obtained control strain.
2. the abduction delivering of recombinant protein
(1) single bacterium colony of difference picking recombinant strains BL21-Cap-PA and control strain, be 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, approximately 3h is cultivated in jolting under 37 ℃, 200rpm condition, makes OD
600Reach 0.6～0.8.
(3) adding final concentration in the above-mentioned bacterium liquid is that the IPTG of 0.1mM carries out abduction delivering, and inducing temperature is 16 ℃, and induction time is 24h.
(4) with the fermented liquid after abduction delivering at 4 ℃, 6000
g Centrifugal 5min under condition collects thalline; Carry out resuspended after thalline is washed 2 times with PBS damping fluid (pH 7.2).
(5) thalline is placed in ice bath, uses the ultrasonic treatment thalline, power is 200W, ultrasonic degradation 5s, and pause 5s is until the clarification of bacterium liquid.
(6) with the thalline after ultrasonic degradation, at 4 ℃, 12000
g Centrifugal 10min under condition collects respectively cleer and peaceful lysate precipitation on lysate.Use with the isopyknic PBS damping fluid of supernatant the lysate precipitation 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 each 100 μ l of cleer and peaceful precipitation on its lysate, add 6 * albumen loading Buffer, 95 ℃ of sex change 5min after abundant mixing, instantaneous centrifugal before loading, draw the supernatant point sample and carry out the SDS-PAGE analysis.Can find out from Fig. 3 (A), compare with the contrast bacterium lysate supernatant of abduction delivering, the recombinant strains BL21-Cap-PA lysate supernatant of abduction delivering has an obvious band at the 46kDa place.Can find out from Fig. 3 (B), after abduction delivering, recombinant strains BL21-Cap-PA has expressed a large amount of target proteins; There is half to realize solubility expression in target protein.
The antigenicity of embodiment 3 restructuring carrying Cap gene of porcine circovirus type 2s is identified
1. the BL21-Cap-PA of inducing culture and the lysate supernatant of contrast bacterium are carried out the SDS-PAGE electrophoresis;
2. transfer printing: after the SDS-PAGE electrophoresis is complete, adopt half-dried transfer printing that Western blot is gone to pvdf membrane;
3. pvdf membrane is sealed with the PBST confining liquid that contains 5% skimming milk, be placed in 40min under room temperature condition;
4. the pvdf membrane after sealing immerses the porcine circovirus 2 type positive serum of 1:200 dilution (available from Veterinary Medical Research ﹠amp; Development, the U.S.), 4 ℃ of overnight incubation;
5. wash pvdf membrane 5 times with the PBST damping fluid, 5min/ time;
6. pvdf membrane is immersed the goat-anti pig IgG of 1:10000 dilution-HRP(Nanjing Sheng Xing Bioisystech Co., Ltd), incubated at room 1h;
7. wash pvdf membrane 5 times with the PBST damping fluid, 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 with the reaction of porcine circovirus 2 type positive serum, have good antigenicity.
The preparation of embodiment 4 restructuring carrying Cap gene of porcine circovirus type 2 great expression and mixture thereof
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, and is centrifugal after ultrasonic degradation, collects the lysate supernatant.
2. the preparation of GEM particle
(1) under anaerobic cultivate Lactococcus lactis MG1363, substratum is for adding the GM17 substratum of 0.5% glucose, and culture temperature is 30 ℃, and incubation time is 16 ~ 18 hours.
(2) centrifugal collection thalline: with fermented liquid 4000
g Under condition, centrifugal 5min, get thalline, uses distilled water wash 1 time.
(3) with the hydrochloric acid resuspended thalline of Lactococcus lactis MG1363 with the pH 1.0 of former volume of culture 1/5, boil 30min, the centrifuging and taking precipitation namely obtains the GEM particle.
(4) with PBS(pH 7.2) resuspended after washing GEM particle 3 times, with the 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 with 2.5% glutaraldehyde respectively, identify the GEM particle with transmission electron microscope observing, as shown in Fig. 5 (A) and Fig. 5 (B).From Fig. 5 (A) as seen, in Lactococcus lactis MG1363 core district, nucleic acid is arranged; From Fig. 5 (B) as seen, the tenuigenin of GEM particle is homogeneous, and nucleic acid and albumen in former nuclear zone are removed.
3. preparation contains the mixture of the carrying Cap gene of porcine circovirus type 2 of recombinating
Add the GEM particle in the BL21-Cap-PA lysate supernatant of abduction delivering, the amount that adds is according to adding 2.5 * 10 in the lysate supernatant that contains 1mg albumen
9Individual GEM particle.Incubated at room 30min is 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 SDS-PAGE analyze, the results are shown in Figure 6.As can be seen from Figure 6, the band of a 46kDa is arranged on the mixture swimming lane, illustrate and contain the restructuring carrying Cap gene of porcine circovirus type 2 in mixture.
After containing the suitable dilution of mixture of the carrying Cap gene of porcine circovirus type 2 of recombinating, use BCA protein detection kit (green skies biotechnology research institute) and measure protein concentration ,-4 ℃ or-20 ℃ save backup.This recombinant C ap expressing quantity 1.5g/L.
The GEM particle is actually the shell that peptidoglycan forms, so contain the surface that the restructuring carrying Cap gene of porcine circovirus type 2 of peptidoglycan grasp PA albumen can be combined in the GEM particle.
4. electron microscopic observation contains the mixture of the carrying Cap gene of porcine circovirus type 2 of recombinating
With GEM particle and the mixture that contains the carrying Cap gene of porcine circovirus type 2 of recombinating, after fixing with 2.5% glutaraldehyde respectively, use transmission electron microscope observing, result such as Fig. 7 (A) and Fig. 7 (B).Figure can find out from this two width, and the 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 mouse immuning test of embodiment 5 restructuring carrying Cap gene of porcine circovirus type 2s
The mixture that will contain the carrying Cap gene of porcine circovirus type 2 of recombinating is mixed with PBS damping fluid (pH=7.2 ~ 7.4) complex solution that protein concentration is 0.5mg/mL, and wherein the concentration of GEM particle is 1.25 * 10
9Individual/mL.Preparation GEM particle solution, in this solution, the GEM granule content is identical with complex solution.Complex solution and GEM particle solution are mixed with aluminium glue adjuvant equal-volume respectively, obtain compound vaccine and GEM particle control vaccine after emulsification.
Chose for 5 ages in week and clean 30 of level ICR mouse, 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 the blank group.Immunization method: every mouse, the subcutaneous multi-point injection 0.2mL vaccine at the back.
Rear 21 days of immunity, with every eyeball of mouse blood sampling separation of serum, (Bioisystech Co., Ltd before the section of Wuhan) detects antibody level of serum with porcine circovirus 2 type ELISA detection kit.During detection, the goat-anti pig IgG-HRP in test kit is replaced with the sheep anti-mouse igg of 1:10000 dilution-HRP(Nanjing Sheng Xing Bioisystech Co., Ltd).
Calculate the average serum antibody horizontal of respectively organizing mouse, concrete outcome is seen 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, perhaps for the preparation of porcine circovirus type 2 subunit vaccine.
SEQUENCE LISTING
＜110〉Jiangsu Province Agriculture Science Institute
＜120〉gene and the application thereof of coding restructuring carrying Cap gene of porcine circovirus type 2
<130> 20130228
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<170> PatentIn version 3.3
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Claims (11)
1. the gene of coding restructuring carrying Cap gene of porcine circovirus type 2, is characterized in that: contain the sequence shown in SEQ ID NO:1.
2. the gene of coding restructuring carrying Cap gene of porcine circovirus type 2 according to claim 1, is characterized in that: the encoding gene that also contains peptidoglycan grasp PA albumen.
3. the gene of coding restructuring carrying Cap gene of porcine circovirus type 2 according to claim 2, it is characterized in that: its sequence is as shown in SEQ ID NO:3.
4. recombinant expression vector that contains one of claim 1-3 described gene.
5. recombinant expression vector according to claim 4 is characterized in that: this recombinant expression vector is that described gene is inserted gained between the Nde I of pET-32a and two restriction enzyme sites of Xho I.
6. the recombinant bacterium that contains one of claim 1-3 described gene.
7. recombinant bacterium according to claim 6, it is characterized in that: described recombinant bacterium is that the described recombinant expression vector of claim 5 is transformed the e. coli bl21 gained.
8. mixture that contains the carrying Cap gene of porcine circovirus type 2 of recombinating, it is characterized in that adopting following method preparation: cultivate the described recombinant bacterium of claim 7, obtain the lysate supernatant after broken, obtain to contain the mixture of the carrying Cap gene of porcine circovirus type 2 of recombinating after purifying.
9. the mixture that contains according to claim 8 the carrying Cap gene of porcine circovirus type 2 of recombinating is characterized in that described purification process is: obtain Gram-positive after Lactococcus lactis is boiled with acid and strengthen matrix granule; Gram-positive is strengthened matrix granule add in the lysate supernatant and adsorb, get precipitation after centrifugal namely to get described mixture.
10. the mixture that contains according to claim 9 the carrying Cap gene of porcine circovirus type 2 of recombinating is characterized in that described acid is hydrochloric acid; The add-on that Gram-positive strengthens matrix granule is: contain in the lysate supernatant of 0.5～2 milligram of albumen and add 2.5 * 10
9Individual Gram-positive strengthens matrix granule.
11. the application of the described mixture of claim 8 aspect preparation pig circular ring virus vaccine and pig circular ring virus diagnostic kit.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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 |
Publications (2)
| Publication Number | Publication Date |
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| CN103103205A true CN103103205A (en) | 2013-05-15 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104098657A (en) * | 2014-07-07 | 2014-10-15 | 南京农业大学 | Porcine circovirus 2 immune protection polypeptide and vaccine comprising same |
| CN107337718A (en) * | 2017-06-20 | 2017-11-10 | 山东省农业科学院畜牧兽医研究所 | A kind of gene for encoding carrying Cap gene of porcine circovirus type 2 and its application |
| CN107460173A (en) * | 2017-09-08 | 2017-12-12 | 江苏省农业科学院 | A kind of method for purifying Porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus or avian influenza virus |
| CN114317345A (en) * | 2021-12-28 | 2022-04-12 | 龙岩学院 | Preparation method of lactococcus lactis GEM particles |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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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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Patent Citations (1)
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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 |
Non-Patent Citations (2)
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| EMBL: "ADB28033.1", 《EMBL》, 18 January 2010 (2010-01-18) * |
| 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》, vol. 42, no. 7, 31 December 2010 (2010-12-31), pages 71 - 76 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104098657A (en) * | 2014-07-07 | 2014-10-15 | 南京农业大学 | Porcine circovirus 2 immune protection polypeptide and vaccine comprising same |
| CN107337718A (en) * | 2017-06-20 | 2017-11-10 | 山东省农业科学院畜牧兽医研究所 | A kind of gene for encoding carrying Cap gene of porcine circovirus type 2 and its application |
| 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 |
| CN107460173A (en) * | 2017-09-08 | 2017-12-12 | 江苏省农业科学院 | A kind of method for purifying Porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus or avian influenza virus |
| 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 |
| CN114317345A (en) * | 2021-12-28 | 2022-04-12 | 龙岩学院 | Preparation method of lactococcus lactis GEM particles |
| CN114317345B (en) * | 2021-12-28 | 2023-12-01 | 龙岩学院 | Preparation method of lactococcus lactis GEM particles |
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| CN103103205B (en) | 2014-04-30 |
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