CN109207441B

CN109207441B - Recombinant baculovirus expressed porcine circovirus type 3Cap protein and construction method and primer thereof

Info

Publication number: CN109207441B
Application number: CN201810912587.1A
Authority: CN
Inventors: 高崧; 程清如; 高清清; 王小波; 郇长超; 刘秀梵
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2018-08-12
Filing date: 2018-08-12
Publication date: 2022-05-27
Anticipated expiration: 2038-08-12
Also published as: CN109207441A

Abstract

The invention belongs to the technical field of vaccine manufacturing. The invention discloses a recombinant baculovirus expression porcine circovirus type 3Cap protein. The invention also discloses a primer for constructing the recombinant baculovirus expression porcine circovirus type 3Cap protein. The invention also discloses a construction method for the recombinant baculovirus to express the porcine circovirus type 3Cap protein, which comprises the following steps: 1. constructing pSK-sPCV3 containing PCV3 whole genome; 2. constructing a transfer vector; 3. constructing a shuttle plasmid; 4. obtaining a recombinant baculovirus; and (3) transfecting the positive shuttle plasmid Bacmid-Cap into an Sf9 cell to obtain a recombinant baculovirus rBac-Cap expressing the porcine circovirus type 3Cap gene. The Cap protein expressed by the invention has good biological activity, and the immune mouse finds that the Cap protein has good immunogenicity.

Description

Recombinant baculovirus expressed porcine circovirus type 3Cap protein and construction method and primer thereof

Technical Field

The invention relates to a construction method of a recombinant baculovirus expressing a Cap gene of porcine circovirus type 3 (PCV3), which is used for manufacturing vaccines.

Background

Porcine circovirus disease (PCVD) is one of the important diseases currently endangering the swine industry, of which PCV2 is the main causative agent. In 2016, Phan et al reported a novel porcine circovirus PCV3, which causes diseases such as Porcine Dermatitis and Nephrotic Syndrome (PDNS), porcine reproductive failure respiratory disease and digestive inflammation [ Phan TG, Giannitti F, Rosslow S, et al.detection of a novel circovirus PCV3 in pigs with cardiac and multi-system inflammation [ J TG].Virology Journal,2016,13(1):184.]. Through PCV3 whole genome genetic evolution analysis, PCV3 is found to be in different evolutionary branches with viruses of other two genotypes of PCV1 and PCV2, and the PCV3 isolates have evolutionary differences, so that the PCV3 is a novel porcine circovirus. Since PCV3 was reported, the virus was detected in many areas at home and abroad, and the harm of the virus is gradually regarded. In the beginning of 2017, PCV3 is detected for the first time in China when the cover is opened and the like. The current domestic PCV3 strains are mainly divided into three genotypes of 3a, 3B and 3c, wherein 3a and the American PCV3 reference strain are in the same evolutionary branch [ Fu X, Fan B, Ma J, et al].Transboundary and Emerging Diseases,2018,65(2):e296-e303.]. The serum of 14 commercial pig farm sows, piglets and finishing pigs in Poland was tested for PCV3 by Stadejek et al, confirming that PCV3 infection also exists in European areas [ Stadejek T,

niak A,

ek D,et al.First detection of porcine circovirus type 3 on commercial pig farms in Poland[J].Transboundary and Emerging Diseases,2017,64(5):1350-1353.]. To control the further prevalence of PCV3, the development of a commercial vaccine against this virus is urgently needed.

PCV3 is an enveloped virus like PCV1 and PCV2, and the virions are also icosahedral in structure, have a 2kb single-stranded closed circular negative-strand DNA genome, and contain the three most prominent open reading frames. ORF1 encodes related proteins involved in viral genome replication, whereas ORF2 encodes Cap nucleocapsid structural proteins responsible for immunogenicity, an ideal target gene for the development of PCV3 subunit genetic engineering vaccines [ Nawagitgul P, Morozov I, Bolin S R, et al open reading frame 2 of vaccine circular type2 codes a major capsid protein [ J ]. Journal of General Virology,2000,81(Pt 9): 2281-. PCV3 ORF3 encodes a functionally unknown protein consisting of 231 amino acids. PCV3 has lower amino acid homology with the Cap protein of PCV2, which is only 30%, and furthermore, the epitopes of the Cap proteins have no similarity, which suggests that the cross-protection of antigens between PCV3 and PCV2 strains may be very low, and the existing PCV2 vaccine can not effectively prevent PCV3 infection [ Zhanyang, Wanglong, and the like.

Vaccines are still the current primary means of controlling PCVD and therefore vaccines against the novel PCV3 infection will naturally be the hotspot of worldwide research. Unlike prokaryotic expression systems, yeast and mammalian cell eukaryotic expression systems, baculovirus expression systems, one of eukaryotic expression systems, have many advantages such as large foreign insert gene capacity, high protein expression level, simple operation and post-translational processing modification, and are widely used [ Lin SY, Chen GY, Hu YC. Recent Patents on the bacterial systems [ J ]. Recent Patents on Biotechnology,2011,5(1):1-11 ]. Currently, the production of subunit vaccines for PCV2 is based mainly on insect cell/baculovirus expression systems, such as the Circumvent subunit vaccine developed by integwort corporation [ Baech NM, Meng xj. efficiency and future promoters of commercial available and experimental vaccines against bacteria vaccine type2(PCV2). Virus Res,2012(164):33-42 ]. At present, little report about PCV3 vaccine exists, and the PCV3Cap protein expression is carried out by constructing recombinant baculovirus, so that a foundation can be laid for developing PCV3 subunit vaccine.

Disclosure of Invention

The invention designs a primer for amplifying PCV3 full length by screening PCV3 strains; specific primers are designed aiming at PCV3 full-length Cap genes, and based on a Bac-to-Bac system, the recombinant baculovirus expressing the porcine circovirus type 3Cap protein is obtained. The method can conveniently and quickly construct the recombinant baculovirus expressing the PCV3Cap protein, and can determine that the expressed Cap protein has good biological activity and immune mice find that the expressed Cap protein has good immunogenicity.

The first purpose of the invention is to provide a recombinant baculovirus expressing porcine circovirus type 3Cap protein, wherein the recombinant baculovirus rBac-Cap is preserved in 2018, 5, 10 days and is named by classification suggested by the China general microbiological culture Collection center: the recombinant baculovirus expressing the 3-type Cap protein of the porcine circovirus has the preservation number of CGMCC No. 15692.

The second purpose of the invention is to provide a primer for constructing recombinant baculovirus expression porcine circovirus type 3Cap protein, which is independently designed according to PCV3 sequence published on GenBank and Cap gene full-length sequence, and the primer is shown in Table 1:

TABLE 1 primers and cleavage sites required

The restriction enzyme site of the primer Xho I-3F isCTCGAG；

The restriction enzyme site of the primer EcoR I-3R isGAATTC；

The restriction enzyme cutting site of the primer Cap-F isGGATCC；

The restriction enzyme cutting site of the primer Cap-R isAAGCTT。

The third purpose of the invention is to provide a construction method of the 3-type Cap protein of the recombinant baculovirus expression porcine circovirus, and screening of PCV3 strain; amplifying the full length of PCV3 to preserve PCV3 genome; designing a full-length primer for expressing PCV3Cap gene to amplify PCV3Cap gene full length; transfer vector pFastBac^TMConstructing HT A-Cap; obtaining shuttle plasmid Bacmid-Cap; obtaining recombinant baculovirus rBac-Cap; identifying the expression of PCV3Cap protein; observing the virus-like particles by an electron microscope; and (4) an immune mouse test. Finally obtaining the recombinant baculovirus rBac-Cap which can be used for industrial production and expresses the porcine circovirus type 3Cap gene; the method specifically comprises the following steps:

1. construction of pSK-sPCV3 containing the entire genome of PCV 3:

using PCV3 DNA as a template, and amplifying PCV3 whole genome by using the primer Xho I-3F and the primer EcoR I-3R in the claim 1; recovering PCV3 whole genome glue, performing double enzyme digestion on the recovered PCV3 whole genome glue and a pBluescript SK + vector stored in a laboratory by using Xho I and EcoR I, purifying and recovering two enzyme digestion products, and connecting the two enzyme digestion products according to a molar ratio of 3:1 to obtain a recombinant plasmid pSK-sPCV 3; identifying the recombinant plasmid by Xho I and EcoR I double digestion, and carrying out electrophoresis on the digestion product by 0.7% agarose gel to obtain bands with sizes of 2958bp and 2000bp respectively;

2. construction of transfer vector:

using pSK-sPCV3 recombinant plasmid as template, using primer Cap-F and primer Cap-R described in claim 1 to amplify PCV3Cap full length, purifying PCR product, and mixing with transfer vector pFastBac stored in this laboratory^TMHT A carries out double enzyme digestion of BamH I and Hind III together; carrying out conventional ligation reaction after secondary purification; extraction of recombinant plasmid pFastBac by Axygen plasmid small-amount DNA extraction kit^TMHT A-Cap, respectively utilizing single enzyme digestion identification of restriction enzymes BamH I and HindIII and double enzyme digestion identification of BamH I-HindIII; the enzyme products are electrophoresed by 0.7 percent agarose gel, and BamH I and Hind III are used for respectively singly cutting pFast Bac^TMHT A-Cap plasmid, then 5501bp single band is obtained; BamH I-HindIII double digestion recombinant plasmid pFastBac^TMHT A-Cap, two bands of 4856bp and 645bp are obtained;

3. construction of shuttle plasmid:

the correct recombinant vector pFastBac with two bands of 4856bp and 645bp is obtained by double enzyme digestion identification of BamH I-HindIII^TMHT A-Cap is transformed into DH10 alpha Bac competent cells; white single colonies with successful transposition are screened by the blue white spots, corresponding PCR identification is carried out by referring to Bac-to-Bac baculovirus expression system specifications of Invirtogen company, and after the identification is correct, recombinant shuttle plasmid Bacmid-Cap is extracted; a band of about 3075bp can be amplified by using a primer pUC/M13; amplifying a target band of about 2500bp by using a primer pUC/M13F + CapR; amplifying a 1000bp band by using a primer Cap F + pUC/M13R; respectively amplifying 645bp bands by using primers Cap F/R;

4. obtaining of recombinant baculovirus:

and (3) transfecting the positive shuttle plasmid Bacmid-Cap into an Sf9 cell to obtain a recombinant baculovirus rBac-Cap expressing the porcine circovirus type 3Cap gene.

Compared with the prior art, the invention has the following beneficial effects:

the invention first obtainsThe PCV3 complete genome is obtained, and then a conventional ligation reaction is carried out to realize the construction of a single copy of PCV 3. Meanwhile, the PCV3 full-length Cap gene is obtained, and the gene is proved to be capable of being expressed by an insect baculovirus expression system to obtain a high-titer recombinant baculovirus, and the titer of the amplified recombinant baculovirus can reach 10^8.3TCID₅₀More than mL, the His-tag mouse monoclonal antibody and mouse anti-PCV 3 positive serum are used as primary antibodies to carry out Western Blot identification, the expressed protein is found to have good immunoreactivity, and an immune mouse test finds that the mouse can be stimulated to generate a specific antibody aiming at PCV3, so that the recombinant baculovirus rBac-Cap which is completely suitable for industrial production and expresses the porcine circovirus type 3Cap gene is finally obtained. Can be used as a genetic engineering subunit vaccine and lays a material foundation for the deep research of the functions and the immune mechanism of the PCV3 virus-related genes.

Drawings

FIG. 1 is an electrophoretogram of a double digestion of a whole genome single-copy recombinant plasmid pSK-sPCV3 containing PCV 3.

FIG. 2 is a transfer vector pFastBac^TMAnd (3) carrying out enzyme digestion electrophoresis diagram on HT A-Cap.

FIG. 3 is a PCR identification electrophoretogram of shuttle vector Bacmid-Cap.

FIG. 4 is a PCR identification electrophoresis chart for extracting recombinant baculovirus rBac-Cap.

FIG. 5 is a total RNA electrophoresis chart for extracting recombinant baculovirus rBac-Cap.

FIG. 6 is an electrophoretogram of PCR identification after RT-PCR.

FIG. 7-1 is an IFA map of Sf9 cells seeded with recombinant baculovirus rBac-Cap.

FIG. 7-2 is a comparison graph of IFA of Sf9 cells inoculated with wild-type baculovirus.

FIGS. 7-3 are IFA profiles of healthy Sf9 cells.

FIG. 8 is an SDS-PAGE identification picture of the expression of porcine circovirus type 3Cap protein.

FIG. 9 is a Western Blot analysis of expression of porcine circovirus type 3Cap protein (His-tag mouse monoclonal antibody as primary antibody).

FIG. 10 is a Western Blot identification chart of the expression of porcine circovirus type 3Cap protein (mouse anti-PCV 3 positive serum was used as the primary antibody).

FIG. 11 is an SEM image of PCV3Cap VLPs.

FIG. 12 is a graph of experimental mouse antibody levels.

Detailed Description

Firstly, constructing a recombinant baculovirus rBac-Cap for expressing a porcine circovirus type 3Cap gene:

the primer sequences designed for amplification were as follows:

TABLE 1 primers and cleavage sites required

Note: the underlined sections are the respective cleavage sites

1. Construction of pSK-sPCV3 containing the entire genome of PCV 3:

the extracted PCV3 DNA is used as a template, and a PCV3 whole genome is amplified by a primer Xho I-3F and a primer EcoR I-3R; recovering PCV3 whole genome glue, performing double enzyme digestion with Xho I and EcoR I simultaneously with a pBluescript SK (+) vector stored in a laboratory, purifying and recovering two enzyme digestion products, and connecting according to a molar ratio of 3:1 to finally obtain a recombinant plasmid pSK-sPCV 3. Recombinant plasmids were identified by double restriction with Xho I and EcoR I, and the restriction products were electrophoresed on 0.7% agarose gel to obtain bands of about 3000bp and 2000bp, respectively, as shown in FIG. 1. In fig. 1, M: DL5000 DNA Marker; 1: the pSK-sPCV3 plasmid is subjected to double enzyme digestion by Xho I enzyme and EcoR I enzyme to obtain fragments of about 2958bp and 2000 bp.

2. Construction of transfer vector:

using pSK-sPCV3 recombinant plasmid as a template, amplifying PCV3Cap full length by using primer Cap-F and primer Cap-R, purifying PCR product, and then mixing with transfer vector pFastBac stored in laboratory^TMHT A carries out double enzyme digestion of BamH I and Hind III together; after further purification, the ligation reaction was carried out conventionally. Axygen plasmid small-amount DNA extraction kitSmall extraction of recombinant plasmid pFastBac^TMAnd HT A-Cap, single enzyme cutting identification by using restriction enzymes BamH I and HindIII and double enzyme cutting identification by using BamH I-HindIII respectively. The enzyme products are electrophoresed by 0.7 percent agarose gel, and BamH I and Hind III are used for respectively singly cutting pFast Bac^TMThe 5501bp single band can be obtained from the HT A-Cap plasmid. BamH I-HindIII double digestion recombinant plasmid pFastBac^TMHT A-Cap, two bands of 4856bp and 645bp were obtained, as shown in FIG. 2. In fig. 2, M: DL5000 DNA Marker; 1: enzyme digestion of pFastBac by BamH I^TMObtaining a fragment of about 5501bp from the HT A-Cap plasmid; 2: hind III enzyme digestion pFastBac^TMObtaining a fragment of about 5501bp from the HT A-Cap plasmid; 3: double enzyme digestion pFastBac of BamH I enzyme and Hind III enzyme^TMFragments of about 4856bp and 6_45bp were obtained from the HT A-Cap plasmid.

4. Construction of shuttle plasmid:

the correct recombinant vector, pFastBac, will be identified^TMHT A-Cap is transformed into DH10 alpha Bac competent cells; and (3) screening a white single colony with successful transposition by using a blue-white spot, and extracting the recombinant shuttle plasmid Bacmid-Cap after the PCR identification is correct. The primer pUC/M13 can be used for amplifying a band about 300 bp; a 3075bp band can be amplified by using the primers pUC/M13F + CapR; the primer Cap F + pUC/M13R can be used for amplifying a target band of about 2500 bp; the primer Cap F/R can amplify about 645bp bands respectively. As shown in fig. 3. In fig. 3, M: DL5000 DNA Marker; 1: pUC/M13 amplified the product of about 300bp of wild type shuttle plasmid Bacmid; 2: pUC/M13 amplified the product of about 3000bp of the recombinant shuttle plasmid Bacmid-Cap; 3: M13F + CapR amplifying the product of about 2500bp of the recombinant shuttle plasmid Bacmid-Cap; 4: CapF + M13R amplifies a product of about 1000bp of the recombinant shuttle plasmid Bacmid-Cap; 5: amplifying a product of about 645bp of the recombinant shuttle plasmid Bacmid-Cap by the CapF + CapR; 6, Negative control.

4. Obtaining of recombinant baculovirus:

And (3) identification: and extracting DNA of the recombinant baculovirus rBac-Cap, and respectively carrying out PCR identification by using a primer Cap F/R. Electrophoresis was performed on a 0.7% agarose gel, as shown in FIG. 4: m: DL5000 DNA Marker; 1: a Negative control; 2: and the Cap F/R amplifies a target fragment of about 645bp of rBac-Cap.

As can be seen from fig. 4: and (3) identifying a Cap target band with the size of about 645bp in the PCR product by electrophoresis, and indicating that the recombinant baculovirus rBac-Cap is obtained.

The recombinant baculovirus rBac-Cap is preserved in the general microbiological center of China Committee for culture Collection of microorganisms (address: No.1 Xilu-Beijing, Chaoyang, Beijing) for 5 months and 10 days in 2018, and the suggested classification is named as: the recombinant baculovirus expressing the 3-type Cap protein of the porcine circovirus has the preservation number of CGMCC No. 15692.

II, biological characteristic identification of the recombinant baculovirus expressing the porcine circovirus type 3Cap gene:

materials: recombinant baculovirus rBac-Cap, healthy Sf9 cells.

Pig source anti-PCV 3 positive serum (self-made serum), mouse source anti-PCV 3 positive serum (self-made serum), and commercial His-tag monoclonal antibody.

1. cDNA detection of recombinant baculovirus expressing porcine circovirus type 3Cap gene

F is to be₂The generation of recombinant baculovirus rBac-Cap was inoculated to healthy Sf9 cells respectively, and after 72h, total RNA was extracted by TRIzol lysis, as shown in FIG. 5. After genome DNA is removed, RT-PCR is carried out, the transcription condition of the target gene is detected through PCR, and a PCV3Cap gene specific band is detected through the PCR method, as shown in FIG. 6.

In fig. 5, M: DL5000 DNA Marker; 1: rBac-Cap total RNA.

In fig. 6, M: 200bp DNA Marker; 1: 645bp bands are generated after reverse transcription of rBac-Cap total RNA; 2: negative control.

2. Indirect Immunofluorescence (IFA) detection of recombinant baculovirus expressing porcine circovirus type 3Cap gene:

porcine anti-PCV 3 positive serum is used as a primary antibody, FITC fluorescence labeled goat anti-porcine IgG is used as a secondary antibody, and obvious specific fluorescence appears in Sf9 cells of recombinant baculovirus rBac-Cap which is inoculated with and expresses porcine circovirus type 3Cap gene (shown in figure 7-1); and no fluorescence appears in inoculated wild type baculovirus (shown in figure 7-2) and normal control cells (shown in figure 7-3), so that cells infected with recombinant baculovirus rBac-Cap expressing the porcine circovirus type 3Cap gene can all express PCV3Cap protein.

2. SDS-PAGE detection of recombinant baculovirus expressing porcine circovirus type 3Cap gene

After Sf9 cells are inoculated with F2 generation recombinant baculovirus rBac-Cap for 96h, protein samples are collected and boiled, and then are used for SDS-PAGE electrophoretic analysis, and observed after Coomassie brilliant blue staining. The desired band (FIG. 8) is seen at the expected size, and the desired protein band of about 30kD is seen.

3. Western Blot identification of recombinant baculovirus expressing porcine circovirus type 3Cap gene

Western Blot identification is carried out by using His-tag mouse monoclonal antibody (figure 9) and anti-PCV 3 mouse positive serum (figure 10) respectively, and whether the expressed full-length Cap protein or the truncated delta Cap protein with the nuclear localization signal removed is in the correct position of a target band in SDS-PAGE, so that the target protein is successfully expressed.

4. TCID of recombinant baculovirus expressing porcine circovirus type 3Cap gene₅₀Measurement of

The well-grown Sf9 cells are subpackaged into 96-well plates, and the recombinant baculovirus solution expressing the porcine circovirus type 3Cap gene is diluted by 10 times by using Grace culture medium (10)^-1～10^-10Dilution). After inoculation of 96-well plates, 5% CO at 37 ℃₂The culture was continued for 96h after 1.5 h of adsorption in the incubator. Discarding the culture solution, washing with PBS, drying, fixing with cold methanol-20 for 20min, discarding the fixing solution, washing with PBS for 3 times, 5min each time, and drying on absorbent paper. 50 μ L/well of swine PCV3 positive serum (1:500) diluted with PBS was added, incubated at 37 ℃ for 1h, washed with PBS, and patted dry. mu.L of a 1:200 diluted FITC-labeled goat anti-porcine fluorescent secondary antibody (IgG) was added in the dark, incubated for 45min in the dark, washed with PBS, and patted dry. Performing indirect Immunofluorescence (IFA) visualization, and virus TCID₅₀Calculated according to the Reed-Muench two-law method.

Through detection, the expression of the 3 type Cap group of the porcine circovirusTCID of recombinant baculovirus of factor₅₀Is 10^8.3More than mL, meets the requirement of preparing the vaccine.

5. Observation by transmission electron microscope

Sf9 cells inoculated with the recombinant baculovirus and the culture were repeatedly frozen and thawed 3 times, and centrifuged at 4000rpm at 4 ℃ for 20min to remove cell debris. The virus solution was applied to a sucrose cushion, centrifuged at 4 ℃ and ultra high speed, and then suspended and precipitated with an appropriate amount of PBS. Dropping one drop of heavy suspension on a copper net, and carrying out negative dyeing by using 2% phosphotungstic acid. Sucking excessive dye solution, drying, and observing the formation condition of virus-like particles by a transmission electron microscope, as shown in figure 11: virus-like particles with uniform shapes, which are round and have the diameter of about 20nm, are observed under a transmission electron microscope after phosphotungstic acid is negatively stained.

6. Animal immunization test

6 weeks old BALB/c mice 20 were randomized into 4 groups, of which group 1 was injected with inactivated rBac-Cap (10)^6.5TCID₅₀Group 2 injection inactivated rBac-Cap (10)^7.5TCID₅₀Group 3 and group 4 were injected with inactivated wild-type baculovirus-infected Sf9 cell culture supernatant and PBS, respectively, and each group was completely emulsified with an equal amount of freund's complete adjuvant and then subjected to primary immunization, and after two weeks, emulsified with incomplete freund's adjuvant and then subjected to secondary booster immunization. Blood was collected from infraorbital sinus before immunization and after the first immunization every week, and serum was isolated and stored.

Performing prokaryotic expression PCV3 on Cap protein coated ELISA plate without nuclear localization signal, coating at 100 mu L/hole overnight at 4 ℃, washing for 3 times and 6 min/time by PBST, and drying on absorbent paper; adding 200 mu L of sealing liquid into each hole, and sealing for 3h at 37 ℃; washing the plate for 3 times again, adding mouse serum diluted by PBS at a proper multiple ratio, incubating for 1h at the temperature of 37 ℃ by 100 mu L/hole, and taking the mouse PCV3 positive serum as a positive control; diluting HRP-labeled goat anti-mouse IgG secondary antibody with 4% FBS-containing PBS at a ratio of 1:8000, 100 μ L/well, incubating at 37 ℃ for 1 h; adding 200 μ L of TMB color developing solution into each well, and developing at 37 deg.C for 15 min; the reaction was stopped by adding 50. mu.L of a stop solution to each well, and then absorbance was measured at 450nm using a microplate reader.

The results of indirect ELISA for detecting PCV3 specific antibody levels are shown in fig. 12: PCV3 specific antibody production can be detected in the second week after the first immunization, the antibody level in the third week rises rapidly, the antibody level in the fourth week rises continuously, and the titer of the high-dose immunization group can reach about 1: 4000.

<110> Yangzhou university

<120> porcine circovirus type 3Cap protein expressed by recombinant baculovirus, and construction method and primer thereof

<160>6

SEQ ID NO.1

<210>1

<211>34

<212>DNA

<213> Artificial sequence

<400>1

TTTCTCGAGA TTGGCGAAGA TTCCTCTTCG GGTA 34

SEQ ID NO.2

<210>2

<211>33

<212>DNA

<213> Artificial sequence

<400>2

CCGGAATTCG TAATCCCCCT CTTTCTTGCA ATA 33

SEQ ID NO.3

<210>3

<211>31

<212>DNA

<213> Artificial sequence

<400>3

CGCGGATCCC ATGAGACACA GAGCTATATT C 31

SEQ ID NO.4

<210>4

<211>31

<212>DNA

<213> Artificial sequence

<400>4

CCCAAGCTTT TAGAGAACGG ACTTGTAACG A 31

SEQ ID NO.5

<210>5

<211>17

<212>DNA

<213> Artificial sequence

<400>5

GTTTTCCCAG TCACGAC 17

SEQ ID NO.6

<210>6

<211>17

<212>DNA

<213> Artificial sequence

<400>6

CAGGAAACAG CTATGAC 17

Claims

1. The recombinant baculovirus expressing the 3-type Cap protein of the porcine circovirus has the preservation number of CGMCC No. 15692; primers for the construction of recombinant baculovirus expressing porcine circovirus type 3Cap protein, said primers are shown in table 1:

TABLE 1 primers and cleavage sites required

The restriction enzyme site of the primer Xho I-3F isCTCGAG；

The restriction enzyme site of the primer EcoR I-3R isGAATTC；

The restriction enzyme cutting site of the primer Cap-F isGGATCC；

The restriction enzyme cutting site of the primer Cap-R isAAGCTT。

2. The method for constructing a recombinant baculovirus expressing porcine circovirus type 3Cap protein of claim 1, wherein the method comprises the following steps:

(1) constructing pSK-sPCV3 containing PCV3 whole genome:

using PCV3 DNA as a template, and amplifying PCV3 whole genome by using the primer Xho I-3F and the primer EcoR I-3R in the claim 1; recovering PCV3 whole genome glue, performing double enzyme digestion with Xho I and EcoR I simultaneously with a pBluescript SK + vector, purifying and recovering two enzyme digestion products, and connecting to obtain a recombinant plasmid pSK-sPCV 3;

(2) and constructing a transfer vector:

using the pSK-sPCV3 recombinant plasmid as a template, the primer Cap-F and primer of claim 1Amplifying PCV3Cap full length by the substance Cap-R, purifying PCR product and transferring carrier pFastBac^TMHT A carries out double enzyme digestion of BamH I and Hind III together; purifying again and then carrying out conventional ligation reaction to obtain recombinant plasmid pFastBac^TMHT A-Cap; extraction of recombinant plasmid pFastBac^TMHT A-Cap, respectively utilizing single enzyme digestion identification of restriction enzymes BamH I and HindIII and double enzyme digestion identification of BamH I-HindIII; the enzyme products are electrophoresed by 0.7 percent agarose gel, and BamH I and Hind III are used for respectively singly cutting pFast Bac^TMThe HT A-Cap plasmid obtains a 5501bp single band; BamH I-HindIII double digestion recombinant plasmid pFastBac^TMHT A-Cap, obtaining two bands of 4856bp and 645 bp;

(3) construction of shuttle plasmid:

the correct recombinant vector pFastBac with two bands of 4856bp and 645bp is obtained by double enzyme digestion identification of BamH I-HindIII^TMHT A-Cap is transformed into DH10 alpha Bac competent cells; white single colonies with successful transposition are screened by the blue white spots, and after the PCR identification is correct, the recombinant shuttle plasmid Bacmid-Cap is extracted; amplifying a band of 2430bp plus the size of the inserted target fragment by using a primer pUC/M13, namely a band of 3075 bp; identifying a recombinant Bacmid DNA pattern diagram according to PCR, speculatively calculating the size of an amplified band obtained by combining an upstream primer and a downstream primer of pUC/M13 with an upstream primer and a downstream primer of a target fragment, and amplifying a 2500bp target band of a Bacmid-Cap recombinant shuttle plasmid by using the primers pUC/M13F + CapR; similarly, a 1000bp band is amplified by using a primer Cap F + pUC/M13R; respectively amplifying 645bp bands by using primers Cap F/R;

(4) obtaining of recombinant baculovirus:

and transfecting Sf9 cells with the correctly identified recombinant shuttle plasmid Bacmid-Cap to obtain the recombinant baculovirus rBac-Cap expressing the porcine circovirus type 3Cap gene.

3. The construction method according to claim 2, wherein the PCV3 whole genome adhesive is recovered, then is subjected to double enzyme digestion with Xho I and EcoR I simultaneously with pBluescript SK + vector, and the two enzyme digestion products are purified and recovered and then are connected according to the molar ratio of 3: 1.

4. The method of claim 2, wherein the recombinant plasmid pSK-sPCV3 is identified by double restriction with Xho I and EcoR I, and the restriction products are electrophoresed on 0.7% agarose gel to obtain bands of 2958bp and 2000bp, respectively.

5. The method according to claim 2, wherein the recombinant plasmid pFastBac is extracted^TMHT A-Cap was performed using Axygen plasmid miniprep DNA extraction kit.

6. The construction method according to claim 2, wherein the PCR identification in step 4 is carried out by referring to the instruction of Bac-to-Bac baculovirus expression system of Invirtogen.