CN114214337A - Preparation method and application of porcine circovirus type 2a Cap protein - Google Patents
Preparation method and application of porcine circovirus type 2a Cap protein Download PDFInfo
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Abstract
The invention discloses a preparation method and application of porcine circovirus type 2a Cap protein, which comprises the steps of culturing a recombinant bacterium containing a nucleic acid molecule shown as SEQ ID NO.3, and inducing and expressing the porcine circovirus type 2a Cap protein by using isopropyl-beta-D-thiogalactoside. The nucleic acid molecule shown in SEQ ID NO.3 has obvious difference with nucleic acid molecules of other porcine circovirus serotypes, which encode porcine circovirus type 2a Cap protein, can be used for distinguishing PCV2b and PCV2a, and is expected to be applied to related aspects such as PCV2a antigen or antibody preparation and the like.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to a preparation method and application of porcine circovirus type 2a Cap protein.
Background
Porcine circovirus type 2 (PCV2) infects immune organs of pigs, easily causes mixed infection or secondary infection with other pathogens, thereby causing the death rate of the pigs to be increased and seriously threatening the healthy development of the pig industry.
PCV2 has 5 serosubtypes PCV2a, PCV2b, PCV2c, PCV2d and PCV2e, the dominant epidemic strain is mainly PCV2d at present, but other subtypes such as PCV2a have outbreak. PCV has no specific therapeutic drug and mainly takes vaccine immunity control as main treatment.
Therefore, the development of a detection method capable of accurately characterizing the specific subtype of PCV2 is of great significance for the prevention and control of PCV.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a preparation method and application of porcine circovirus type 2a Cap protein, the porcine circovirus type 2a Cap protein gene is obtained through a specific primer, and the porcine circovirus type 2a Cap protein gene can be efficiently expressed after being cloned into a pET28a vector, so that the preparation of related porcine circovirus type 2a antigen antibody is facilitated.
In a first aspect of the invention, an isolated nucleic acid molecule encoding a Cap protein of porcine circovirus type 2a is provided, the nucleotide sequence of which is shown in SEQ ID NO. 3.
The nucleotide sequence of the nucleic acid molecule is specifically as follows: 5'-ATGACGTATCCAAGGAGGCGTTTCCGCAGTCGAAGACACCGCCCCCGCAGCCATCTTGGCCAGATCCTCCGCCGCCGCCCCTGGCTCCTCCAACCCCGCCACCGTTACCGCTGGAGAAGGAAAAATGGCATCTTCAACACCCGCCTCTCCCGCACCTTCGGATATACTGTCAAGGCTACCACAGTCAGAACGCCCTCCTGGGCGGTGGACATGATGAGATTTAAAATTGACGACTTTGTACCCCCGGGAGGGGGGACCAACAAAATCTCTATACCCTTTGAATACTACAGAATAAGAAAGGTTAAGGTTGAATTCTGGCCCTGCTCCCCAATCACCCAGGGTGATAGGGGAGTGGGCTCCACTGCTGTTATTCTAGATGATAACTTTTTTCCAAAGTCCACAGCCCTAACCTATGACCCCTATGTAAACTACTCCTCCCGCCATACCATACCCCAGCCCTTCTCCTACCACTCCCGGTACTTTACCCCCAAACCTGTTCTTGATTCCACTATTGATTACTTCCAACCAAATAACAAAAGGAATCAGCTTTGGCTGAGGCTACAAACCTCTGCAAATGTGGACCACGTAGGCCTCGGCACTGCCTTCGAAAACAGTAAATACGACCAGGACTACAATATCCGTGTAACCATGTATGTACAATTCAGAGAATTTAATCTTAAAGACCCCCCACTTAAACCCTAA-3' (SEQ ID NO. 3).
The size of the nucleic acid molecule amplification product is 702 bp.
The inventor finds that the encoding genes of the Cap proteins of different serotypes of the porcine circovirus are different, and the Cap proteins as structural proteins and main immune proteins of the PCV have good immunogenicity and safety, and the difference can be well used as the research and development focus of developing novel targeted vaccines of different serotypes, so that the prevention and control effect of the PCV2 is effectively improved.
In a second aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the first aspect of the invention.
According to a second aspect of the invention, in some embodiments of the invention, the vector comprises a recombinant expression vector and/or a recombinant cloning vector.
In some preferred embodiments of the present invention, the vector is a recombinant expression vector pET28a-Cap, the recombinant expression vector pET28a-Cap comprises a T7 promoter, BamH I, SEQ ID NO.3 and Sal I in sequence, and the plasmid length is 6052 bp.
According to a second aspect of the invention, in some embodiments of the invention, the vector is derived from the nucleic acid molecule by replacing the SalI and BamHI double-cleaved fragments of pET28 a.
In a third aspect of the present invention, there is provided a recombinant bacterium comprising the nucleic acid molecule according to the third aspect of the present invention.
According to a third aspect of the invention, in some embodiments of the invention, the recombinant bacterium is obtained by transforming E.coli with the vector of the second aspect of the invention.
In some preferred embodiments of the invention, the E.coli is E.coli BL21(DE 3).
In a fourth aspect of the present invention, a method for preparing a Cap protein of porcine circovirus type 2a is provided, which comprises the following steps:
culturing the recombinant strain of the third aspect of the invention, and inducing and expressing the 2a type Cap protein of the porcine circovirus by using isopropyl-beta-D-thiogalactoside.
In a fifth aspect of the present invention, there is provided a use of the reagent for detecting the nucleic acid molecule according to the first aspect of the present invention in the preparation of a reagent for extracting the Cap protein of porcine circovirus type 2 a.
According to a fifth aspect of the invention, in some embodiments of the invention, the reagent for detecting the nucleic acid molecule according to the first aspect of the invention comprises a porcine circovirus type 2a Cap gene detection primer.
According to a fifth aspect of the invention, in some embodiments of the invention, the nucleotide sequence of the primer for detecting a Cap gene type 2a of porcine circovirus is:
an upstream primer F: 5'-GCGGATCCGTGACGTATCCAAG-3' (SEQ ID NO. 1);
a downstream primer R: 5'-CACTAGTATAGGGGTTAAGTGGGG-3' (SEQ ID NO. 2).
Wherein, the 5' end of the upstream primer is also connected with a restriction enzyme cutting site (GTCGAC, italic part below) and an HIS tag sequence (CATCATCACCATCACCAT, underlined part below) in sequence. The method specifically comprises the following steps: 5' -GTCGACATCATCACCATCACCATCGCGGATCCGTGACGTATCCAAG-3'。
The invention has the beneficial effects that:
1. the invention provides a nucleic acid molecule for coding porcine circovirus type 2a Cap protein, which has obvious difference with nucleic acid molecules for coding porcine circovirus type 2a Cap protein of other porcine circovirus serotypes, can be used for distinguishing PCV2b and PCV2a, and is expected to be applied to relevant aspects such as PCV2a antigen or antibody preparation and the like.
2. The invention provides a preparation method of porcine circovirus type 2a Cap protein, which is based on a recombinant expression vector containing nucleic acid molecules for encoding the porcine circovirus type 2a Cap protein shown in SEQ ID NO.3, can stably express the porcine circovirus type 2a Cap protein through IPTG induction, and provides favorable method support for the application of the porcine circovirus type 2a Cap protein.
Drawings
FIG. 1 is a graph comparing PK15 cells (A) inoculated with PCV2a with normal PK15 cells (B) in the example of the present invention, with a field magnification of 100 ×;
FIG. 2 is an electron microscope view of PCV2a in an embodiment of the invention;
FIG. 3 is an electrophoretogram of an amplification product (Cap gene) amplified in the example of the present invention;
FIG. 4 is a comparative graph of Cap gene evolution analysis of PCV2a in an example of the present invention;
FIG. 5 shows the results of the double restriction enzyme digestion of the recombinant expression vector pET28a-Cap according to the present invention;
FIG. 6 shows the Coomassie blue staining results after expression of Cap protein obtained before and after purification in the examples of the present invention.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.
Preparation of porcine circovirus type 2a virus material
In this example, the inventors isolated porcine circovirus type 2a virus from a sample of suspected porcine inguinal lymph node of a porcine infected with porcine circovirus collected from a pig farm in Hunan province for subsequent experiments.
The specific separation steps are as follows:
under aseptic conditions, grinding a proper amount of collected inguinal lymph node sample in a mortar subjected to autoclaving, adding 2mL of sterile PBS (phosphate buffer solution) after grinding, collecting the grinding solution in a 2mL centrifuge tube, placing the centrifuge tube in a-20 ℃ for repeated freeze thawing for 3 times, centrifuging to obtain a supernatant, and filtering the supernatant through a 0.22 mu m filter to obtain the virus solution. The virus liquid can be placed in a refrigerator at the temperature of 70 ℃ below zero for standby.
The obtained virus solution was inoculated into PK15 cells (porcine kidney cells) grown to a cell density of about 80% in a monolayer, and the cells were incubated at 37 ℃ with 5% CO2Culturing in a cell culture box, subculturing for 3 times, and collecting cell sediment after the cells have pathological changes for subsequent detection.
Identification of porcine circovirus type 2a
To further verify whether the virus solution contains porcine circovirus type 2a, the inventors performed the following test using the following method.
(1) Peroxidase immunostaining method (IPMA):
the obtained virus solution was inoculated to PK15 cells grown to a cell density of about 80% in a monolayerIn the presence of 5% CO at 37 deg.C2After incubation in a cell incubator for 72 hours, the cells were fixed in PBS buffer containing 33% (v/v) acetone, and then subjected to IPMA test using PCV2 positive serum (IPMA staining was performed using peroxidase activity detection kit (purchased from Sigma-Aldrich) according to the staining protocol), and the virus infection was measured.
Normal PK15 cells that were not inoculated with virus fluid were used as negative controls.
The results are shown in FIG. 1.
It can be found that after the isolated virus solution is inoculated with PK15 cells and subcultured, the cell nucleus is stained into a brownish red color according to the detection characteristics of PCV2 by using a peroxidase immunostaining method (IPMA), and the healthy PK15 cells are not stained, which indicates that the virus in the isolated virus solution is PCV 2.
(2) And (3) virus morphology observation:
the PK15 cells inoculated with the virus liquid in the above example are taken, freeze thawing is carried out for 3 times repeatedly, then ultrasonic wave is used for crushing, and centrifugation is carried out for 5min at 4 ℃ and 10000 r/min. And taking the supernatant, adding 2% phosphotungstic acid for negative staining for 2min, collecting the negative staining supernatant, and observing the virus particle morphology by using a projection electron microscope.
The results are shown in FIG. 2.
As can be seen from FIG. 2, after negative staining, the particles with diameters of about 17-20 nm can be seen by transmission electron microscopy, which conforms to the characteristics of PCV2, and the virus isolated in the above example is PCV 2.
Amplification of porcine circovirus type 2a Cap protein Gene
The above diseased cells were taken, and DNA in the cells was extracted using a DNA extraction kit (purchased from OMEGA), and PCR amplification was performed using the following primer pairs to recover the amplified product.
The nucleotide sequence of the PCV2a Cap protein gene amplification primer is as follows:
an upstream primer F: 5'-GCGGATCCGTGACGTATCCAAG-3' (SEQ ID NO. 1);
a downstream primer R: 5'-CACTAGTATAGGGGTTAAGTGGGG-3' (SEQ ID NO. 2).
Wherein, the 5' end of the upstream primer is also sequentially connected with enzymeCleavage site (GTCGAC, italics below) and HIS tag sequence (CATCATCACCATCACCAT, lower underlined). The method specifically comprises the following steps: 5' -GTCGACATCATCACCATCACCATCGCGGATCCGTGACGTATCCAAG-3'。
The amplification system is as follows:
| 10nM forward primer F | 0.5μL |
| 10nM downstream primer R | 0.5μL |
| Extaq enzyme | 8μL |
| Diseased cell DNA | 2μL |
| Water (W) | Make up to 20 mu L |
The reaction procedure is as follows: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 45s, extension at 72 ℃ for 1min, 35 cycles of amplification, and extension at 72 ℃ for 7 min.
And (5) running glue to verify whether the amplification product is the porcine circovirus type 2a Cap protein.
The judgment standard is as follows:
when the size of the amplification product is 702bp, the PCV2a Cap protein is contained in the amplification product;
when the size of the amplification product is not 702bp, the PCV2a Cap protein is not contained in the amplification product.
The electrophoretogram is shown in FIG. 3.
It can be found that the size of the amplification product is 702bp (repeated 2 times) according to the expected result by running gel verification.
And (3) carrying out gene sequencing on the amplification product, and finding that the nucleotide sequence of the amplification product is as follows: 5'-ATGACGTATCCAAGGAGGCGTTTCCGCAGTCGAAGACACCGCCCCCGCAGCCATCTTGGCCAGATCCTCCGCCGCCGCCCCTGGCTCCTCCAACCCCGCCACCGTTACCGCTGGAGAAGGAAAAATGGCATCTTCAACACCCGCCTCTCCCGCACCTTCGGATATACTGTCAAGGCTACCACAGTCAGAACGCCCTCCTGGGCGGTGGACATGATGAGATTTAAAATTGACGACTTTGTACCCCCGGGAGGGGGGACCAACAAAATCTCTATACCCTTTGAATACTACAGAATAAGAAAGGTTAAGGTTGAATTCTGGCCCTGCTCCCCAATCACCCAGGGTGATAGGGGAGTGGGCTCCACTGCTGTTATTCTAGATGATAACTTTTTTCCAAAGTCCACAGCCCTAACCTATGACCCCTATGTAAACTACTCCTCCCGCCATACCATACCCCAGCCCTTCTCCTACCACTCCCGGTACTTTACCCCCAAACCTGTTCTTGATTCCACTATTGATTACTTCCAACCAAATAACAAAAGGAATCAGCTTTGGCTGAGGCTACAAACCTCTGCAAATGTGGACCACGTAGGCCTCGGCACTGCCTTCGAAAACAGTAAATACGACCAGGACTACAATATCCGTGTAACCATGTATGTACAATTCAGAGAATTTAATCTTAAAGACCCCCCACTTAAACCCTAA-3' (SEQ ID NO. 3).
Comparison by gene evolution analysis showed PCV2a (fig. 4).
The results show that the PCV2a Cap protein gene is successfully amplified.
In order to further embody the specificity of the amplified product, the inventor adopts the primer to amplify the PCV2b DNA sample under the same condition, finds that the size of the amplified product is 708bp after running gel, and sequences the amplified product to find that the nucleotide sequence is as follows: 5'-ATGACGTATCCAAGGAGGCGTTACCGGAGAAGAAGACACCGCCCCCGCAGCCATCTTGGCCAGATCCTCCGCCGCCGCCCCTGGCTCGTACACCCCCGCCACCGTTACCGCTGGAGAACGAAAAATGGCATCTTCAACGCCCGCCTCTCCCGCACCTTCGGATATACTATCAAGCGAGCCACAGTCAAAACGCCCTCCTGGGCGGTGGACATGATGAGATTCAATATTAATGACTTTCTTCCCCCAGGAGGGGGCTCAAACCCCCGCTCTGTGCCCTTTGAATACTACAGAATAAGAAAGGTTAAGGTTGAATTCTGGCCCTGCTCCCCGATCACCCAGGGTGACAGGGGAGTGGGCTCCAGAGCTGCTATTCTAGATGATAACTTTGTAACAAAGGCCACAGCCCTCACCTATGACCCCTATGTAAACTACTCCTCCCGCCATACCATAACCCAACCCTTCTCCTACCACTCCCGCTACTTTACCCCCAAACCTGTCCTAGATTCCACTATTGATTACTTCCAACCAAACAACAAAAGAAATCAGCTGTGGCTGAGGCTACAAACTGCTGGAAATGTAGACCACGTAGGCCTCGGCACTGCGTTCGAAAACAGTATATACGACCAGGAATACAATATCCGTGTAACCATGTATGTACAATTCAGAGAATTTAATCTTAAAGACCCCCCACTTAACCTTAATGAATAA-3' (SEQ ID NO. 4).
The amplified product of PCV2b is obviously different from the amplified product of PCV2a, and the sequence identity is low, so that the amplified product of PCV2b can be used as an effective specific target for distinguishing PCV2b from PCV2a, and is expected to be applied to related aspects such as PCV2a antigen or antibody preparation.
Expression of porcine circovirus type 2a Cap protein
Cloning the amplification product (porcine circovirus type 2a Cap protein gene, SEQ ID NO.3) obtained in the above example into an expression vector pET28a, specifically comprising the following steps:
the expression vector pET28a (purchased from Youbao Bio Inc.) and the amplified Cap protein gene were subjected to double digestion using SalI and BamHI, respectively, in a 50. mu.L digestion system: mu.g DNA, 2. mu.L SalI, 2. mu.L BamHI, 5. mu.Lbuffer, water supplemented to 50. mu.L.
The enzyme was digested in 37 ℃ constant temperature water bath for 4h, and the vector fragment was recovered after 1% gel electrophoresis purification (repeated 4 times as shown in FIG. 5).
Ligation was performed using T4 ligase (purchased from NEB) as follows: 1 μ L T4 ligase, 1 μ L T4 ligase buffer, 1 μ L pET28a vector cleavage fragment and 7 μ L SEQ ID NO. 3.
And (3) incubating for 24h at 16 ℃ to obtain a recombinant expression plasmid pET28a-Cap (containing a T7 promoter, BamH I, SEQ ID NO.3 and Sal I in sequence, wherein the length of the plasmid is 6052 bp).
Escherichia coli BL21(DE3) was added to the recombinant expression plasmid pET28a-Cap prepared in the above example, and after 30min in ice bath, the mixture was heat-shocked at 42 ℃ for 90s, immediately placed back on ice and ice-cooled for 2 min. Adding LB culture medium, and culturing for 45-60min by slowly shaking in 37C shaking table; LB solid medium containing ampicillin (100pg/mL) was used for overnight culture to select recombinant bacteria with successful transformation.
The protein of interest was extracted by ultrasonication using isopropyl-beta-D-thiogalactoside (IPTG) at a concentration of 1mM for 20 hours at 37 ℃ and then the expressed Cap protein was purified using a Ni-Agarose HIS tag protein purification kit (available from Beijing Soilebao technologies, Ltd.), and the protein was eluted with imidazole at a concentration of 200mM and identified.
The identification method adopts Coomassie brilliant blue dyeing, and specifically comprises the following steps: the Cap proteins obtained before and after purification are respectively boiled for 10min, and are separated by SDS-PAGE and then stained by Coomassie brilliant blue.
The results are shown in fig. 6, and the Cap proteins obtained before and after purification are consistent, which indicates that the Cap proteins are successfully expressed and the purification effect is good.
In conclusion, based on the method in the embodiment, the recombinant plasmid with large protein expression amount and stable expression can be obtained, the obtained protein is easy to purify, the preparation cost is low, and an effective material basis is provided for further research on the PCV-2a novel vaccine.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> Guangzhou Gotty Biotechnology Ltd
<120> preparation method and application of porcine circovirus type 2a Cap protein
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213> Artificial sequence
<400> 1
gcggatccgt gacgtatcca ag 22
<210> 2
<211> 24
<212> DNA
<213> Artificial sequence
<400> 2
cactagtata ggggttaagt gggg 24
<210> 3
<211> 702
<212> DNA
<213> PCV2
<400> 3
atgacgtatc caaggaggcg tttccgcagt cgaagacacc gcccccgcag ccatcttggc 60
cagatcctcc gccgccgccc ctggctcctc caaccccgcc accgttaccg ctggagaagg 120
aaaaatggca tcttcaacac ccgcctctcc cgcaccttcg gatatactgt caaggctacc 180
acagtcagaa cgccctcctg ggcggtggac atgatgagat ttaaaattga cgactttgta 240
cccccgggag gggggaccaa caaaatctct ataccctttg aatactacag aataagaaag 300
gttaaggttg aattctggcc ctgctcccca atcacccagg gtgatagggg agtgggctcc 360
actgctgtta ttctagatga taactttttt ccaaagtcca cagccctaac ctatgacccc 420
tatgtaaact actcctcccg ccataccata ccccagccct tctcctacca ctcccggtac 480
tttaccccca aacctgttct tgattccact attgattact tccaaccaaa taacaaaagg 540
aatcagcttt ggctgaggct acaaacctct gcaaatgtgg accacgtagg cctcggcact 600
gccttcgaaa acagtaaata cgaccaggac tacaatatcc gtgtaaccat gtatgtacaa 660
ttcagagaat ttaatcttaa agacccccca cttaaaccct aa 702
<210> 4
<211> 708
<212> DNA
<213> Artificial sequence
<400> 4
atgacgtatc caaggaggcg ttaccggaga agaagacacc gcccccgcag ccatcttggc 60
cagatcctcc gccgccgccc ctggctcgta cacccccgcc accgttaccg ctggagaacg 120
aaaaatggca tcttcaacgc ccgcctctcc cgcaccttcg gatatactat caagcgagcc 180
acagtcaaaa cgccctcctg ggcggtggac atgatgagat tcaatattaa tgactttctt 240
cccccaggag ggggctcaaa cccccgctct gtgccctttg aatactacag aataagaaag 300
gttaaggttg aattctggcc ctgctccccg atcacccagg gtgacagggg agtgggctcc 360
agagctgcta ttctagatga taactttgta acaaaggcca cagccctcac ctatgacccc 420
tatgtaaact actcctcccg ccataccata acccaaccct tctcctacca ctcccgctac 480
tttaccccca aacctgtcct agattccact attgattact tccaaccaaa caacaaaaga 540
aatcagctgt ggctgaggct acaaactgct ggaaatgtag accacgtagg cctcggcact 600
gcgttcgaaa acagtatata cgaccaggaa tacaatatcc gtgtaaccat gtatgtacaa 660
ttcagagaat ttaatcttaa agacccccca cttaacctta atgaataa 708
Claims (10)
1. An isolated nucleic acid molecule encoding a porcine circovirus type 2a Cap protein, wherein the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID No. 3.
2. A vector comprising the nucleic acid molecule of claim 1.
3. The vector of claim 2, wherein the vector comprises a recombinant expression vector and/or a recombinant cloning vector.
4. The vector of claim 2 or 3, wherein said vector is obtained by double digestion of said nucleic acid molecule in place of SalI and BamHI fragments of pET28 a.
5. A recombinant bacterium comprising the nucleic acid molecule of claim 1.
6. The recombinant bacterium according to claim 5, wherein the recombinant bacterium is obtained by transforming Escherichia coli with the vector according to any one of claims 2 to 4.
7. A preparation method of porcine circovirus type 2a Cap protein comprises the following steps:
culturing the recombinant strain of claim 5 or 6, and inducing and expressing the porcine circovirus type 2a Cap protein by using isopropyl-beta-D-thiogalactoside.
8. Use of a reagent for detecting the nucleic acid molecule of claim 1 in the preparation of a reagent for extracting the Cap protein of porcine circovirus type 2 a.
9. The use of claim 8, wherein the reagent for detecting the nucleic acid molecule of claim 1 comprises a primer for detecting the Cap gene of porcine circovirus type 2 a.
10. The use of claim 8, wherein the nucleotide sequence of the primer for detecting the porcine circovirus type 2a Cap gene is as follows:
an upstream primer F: 5'-GCGGATCCGTGACGTATCCAAG-3' (SEQ ID NO. 1);
a downstream primer R: 5'-CACTAGTATAGGGGTTAAGTGGGG-3' (SEQ ID NO. 2).
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| CN202111646582.7A Pending CN114214337A (en) | 2021-12-29 | 2021-12-29 | Preparation method and application of porcine circovirus type 2a Cap protein |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174086A (en) * | 2011-03-07 | 2011-09-07 | 南京农业大学 | Porcine circovirus type 2 recombinant cap protein and subunit vaccine |
| CN103451196A (en) * | 2013-09-13 | 2013-12-18 | 江苏省农业科学院 | Codon optimized porcine circovirus type 2 Cap protein coding gene and application thereof |
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- 2021-12-29 CN CN202111646582.7A patent/CN114214337A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174086A (en) * | 2011-03-07 | 2011-09-07 | 南京农业大学 | Porcine circovirus type 2 recombinant cap protein and subunit vaccine |
| CN103451196A (en) * | 2013-09-13 | 2013-12-18 | 江苏省农业科学院 | Codon optimized porcine circovirus type 2 Cap protein coding gene and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| SAHA, D.等: "Porcine circovirus 2 isolate 390 capsid protein (cap) and replication associated protein (rep) genes, complete cds", GENBANK, pages 1 - 2 * |
| 高宇等: "山西省部分地区猪圆环病毒 2 型基因遗传变异分析", 山西农业科学, vol. 49, no. 5, pages 644 - 648 * |
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