CN102911947A - Porcine circovirus (PCV) type 2 Cap protein and preparation method thereof - Google Patents
Porcine circovirus (PCV) type 2 Cap protein and preparation method thereof Download PDFInfo
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- CN102911947A CN102911947A CN2011102187803A CN201110218780A CN102911947A CN 102911947 A CN102911947 A CN 102911947A CN 2011102187803 A CN2011102187803 A CN 2011102187803A CN 201110218780 A CN201110218780 A CN 201110218780A CN 102911947 A CN102911947 A CN 102911947A
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Abstract
The invention provides a method for producing PCV2 Cap protein by using a silkworm bioreactor, and a Cap protein product. The method comprises the steps that: a coding gene sequence of the PCV2 Cap protein is cloned to a recombinant baculovirus; the recombinant insect baculovirus is used in direct injection infection of silkworm larva or silkworm pupa; and PCV2 Cap protein is recovered, separated, and purified. The method provided by the invention has the characteristics of simple operation and short period. A difficult and low-success-rate step which is virus homologous recombination plaque screening is avoided, such that an experiment period is greatly shortened. The operation is simple, the cost is low, and a post-extraction method is simple. The method provided by the invention is suitable for industrialized productions.
Description
Technical field
The present invention relates to a kind of carrying Cap gene of porcine circovirus type 2 and preparation method thereof, refer to especially a kind of carrying Cap gene of porcine circovirus type 2 and method thereof of using genetic engineering means manufacture in silkworm, belong to the genetically engineered field.
Background technology
Pig circular ring virus (Porcine circovirus, PCV) belongs to Circovirus, is that a kind of icosahedro is symmetrical, covalence closed, ring-type, Single-stranded DNA virus.According to pathogenic, the antigenicity of PCV and the difference of nucleotide sequence, PCV can be divided into pig circular ring virus 1 type (PCV1) and porcine circovirus 2 type (PCV2).Wherein, PCV1 is to the pig no pathogenicity, PCV2 is very large to clinical harm, it can cause multisystemic exhaustion syndrome (Postweaning multisystemic wastingsyndrome behind the weaned piglet, PMWS), the outburst of the scorching nephrotic syndrome (PDNS) of pigskin, PRDC (porcine respiratory disease complex) and the congenital various diseases such as tremble, brought huge financial loss for whole world pig industry.Research finds that PCV2 contains 11 open reading frame (Open reading frame, ORF), and ORF1 and ORF2 are topmost two reading frames.Having confirmed that now ORF2 is major structural protein and the capsid protein (Cap) of coding virus, had the antigen presentation site on the Cap albumen, had immunogenicity, is the basis of PCV2 specific serum detection method and subunit vaccine, nucleic acid vaccine.
Invention CN 101920012 A of Pulaike Biological Engineering Co., Ltd. disclose the method that a kind of silkworm biological reactor is produced carrying Cap gene of porcine circovirus type 2, take Bombyx mori nuclear polyhydrosis virus (BmNPV) as carrier, by the autographa california nuclear polyhedrosis virus in the transfer vector (AcNPV) and the mode of Bombyx mori nuclear polyhydrosis virus (BmNPV) homologous recombination the carrying Cap gene of porcine circovirus type 2 gene integration is carried out the target protein expression to silkworm caryogram polyhedron promotor, can obtain the extensive expression of PCV2 Cap albumen, be prepared into the subunit vaccine that contains the carrying Cap gene of porcine circovirus type 2 of recombinating.But the method need to be by using BmNPV and AcNPV cotransfection and the method by plaque screening, acquisition contains the recombinant bombyx mori nuclear polyhedrosis virus of PCV2 Cap albumen, and the probability of BmNPV and AcNPV homologous recombination is low, and there is the shortcoming that workload is large, success ratio is low in plaque screening; In addition, plaque screening need to be cultivated bombyx mori cell, need to contain the TC-100 substratum of foetal calf serum (FBS), and culture cycle is grown (being generally 7-8 days).Therefore, there is the shortcoming that workload is large, experimentation cost is high, the test period is long in this plaque screening method.
Summary of the invention
In view of this, main purpose of the present invention is to put forward a kind of method of utilizing silkworm biological reactor to produce carrying Cap gene of porcine circovirus type 2, may further comprise the steps:
(1). the coding gene sequence of clone's carrying Cap gene of porcine circovirus type 2, change transfer vector over to, obtain to comprise the recombinant transfer vector of carrying Cap gene of porcine circovirus type 2 encoding gene;
(2). above-mentioned recombinant transfer vector is transformed in the competent escherichia coli cell that contains viral shuttle vectors, acquisition contains the recombinant shuttle vector of carrying Cap gene of porcine circovirus type 2 encoding gene, again this shuttle vectors is transfected in the insect cell, obtains recombinant baculovirus;
(3). recombinant baculovirus infectable infection silkworm larva or silkworm chrysalis;
(4). reclaim and obtain the silkworm larva of recombinant virus infection or the hemolymph of silkworm chrysalis;
(5). the restructuring carrying Cap gene of porcine circovirus type 2 in the separation and purification hemolymph.
Preferably, in the method for production carrying Cap gene of porcine circovirus type 2 of the present invention in the step (1) encoding sequence of carrying Cap gene of porcine circovirus type 2 comprise SEQ ID NO1 in the sequence table.
The expression of the gene that preferably, carrying Cap gene of porcine circovirus type 2 described in the step (2) is encoded in the method for production carrying Cap gene of porcine circovirus type 2 of the present invention is subjected to the control of polyhedron promotor.
Preferably, insect baculovirus described in the step (2) comprises the insect nuclear polyhedrosis virus in the method for production carrying Cap gene of porcine circovirus type 2 of the present invention.
Preferably, insect cell described in the step (2) comprises the Sf9 cell in the method for production carrying Cap gene of porcine circovirus type 2 of the present invention.
Preferably, step in the method for production carrying Cap gene of porcine circovirus type 2 of the present invention
(3) cultivated silkworm breed variety described in comprises european race: Luobendihong, Baohuang, Hungary; China seed: Dazao, Qiansanmian, Datuanyuan, Ankang, Lan 5; Japan plants: Ri110, Dongfei, Su16, Su14, YingHXZ, Haoyue etc.
Preferably, in the method for production carrying Cap gene of porcine circovirus type 2 of the present invention, the Recombinant Swine circovurus type 2 Cap protein sequence described in the step (3) comprises the SEQ ID NO2 in the sequence table.
Preferably, in the method for production carrying Cap gene of porcine circovirus type 2 of the present invention, the restructuring carrying Cap gene of porcine circovirus type 2 described in the step (3) is any one of following three peptide species:
1) contains the polypeptide of the SEQ ID NO2 sequence in the ordered list;
2) with 1) polypeptide of described polypeptide at least 80% homology;
3) 1) and 2) the immunogenicity part of described polypeptide.
Another object of the present invention is to provide the restructuring carrying Cap gene of porcine circovirus type 2 of aforesaid method production.
Technique effect
Can find out that from subsequent embodiment of the present invention the present invention has following advantage at least:
1, simple to operate, cycle weak point.The recombinant bombyx mori nuclear polyhedrosis virus that contains PCV2 Cap albumen, traditional method need to be selected recombinant virus by two kinds of viruses (BmNPV and AcNPV) cotransfection and by the method for plaque screening, operational cycle is long, a plaque screening cycle is about 10 days, also to just can obtain recombinant virus through several plaque screenings of taking turns in the situation smoothly, require simultaneously experiment operator to have skilled plaque screening technology.And the present invention has avoided viral homologous recombination plaque screening this has been difficult to operate and the low step of success ratio, has greatly shortened experimental period.
2, utilize silkworm larva and silkworm chrysalis to carry out on a large scale the secreting, expressing of Cap albumen, cost is low.Recombinant virus infects certain species silkworm larva or silkworm chrysalis by the mode of feed mulberry leaf or injection, gets hemolymph after certain hour is raised, and every silkworm or silkworm chrysalis on average can obtain about hemolymph 500 μ l.This process only needs that infected silkworm is carried out routine and raises, and do not need other consumption, so cost is very cheap; The last obtained hemolymph amount of every silkworm is very considerable, if seedling behind the postmenstruation purifying, the hemolymph of every silkworm can 10 parts of vaccine processed; Infect the hemolymph of virus first by behind high speed centrifugation and the ultracentrifugation, can obtain the PCV2 Cap albumen behind the purifying, whole purge process does not have the participation of chemical substance etc., and only rely on physical method, there is not the possibility of polluting sex change, and simple to operate, cost is low; If after the suitability for industrialized production maturation, every part vaccine cost can be controlled in 1 yuan, and every part market value of existing vaccine is all about 12 yuan, and the import vaccine price is all about 35 yuan.
3, because silkworm can adopt feed to raise, therefore be subjected to seasonal effect little.And raise on a large scale silkworm and can adopt the modes such as the mulberry leaf of feeding, the silkworm infection rate can reach more than 99.8%.Whole silkworm rearing and infectious cycle are about 1 month, and the later stage purifying also only needed get final product in several days.Use the method to carry out PCV2 Cap protein expression, only need carry out the conventional insect cell in 1-2 cycle and cultivate, about 1 week of time.After obtaining a certain amount of virus quantity, carry out the infection of next step silkworm larva or silkworm chrysalis.There is not difficulty in whole experimentation, and can carry out at Routine Test Lab.
Therefore, the present invention can utilize autographa californica nuclear polyhedrosis virus (AcNPV) direct infection silkworm larva and silkworm chrysalis, carries out PCV2 Cap target protein and expresses, and can produce on a large scale PCV2 Cap albumen.
Description of drawings
Fig. 1 is PCV2 ORF2 gene amplification figure;
Fig. 2 is PCV2 Cap protein SDS-PAGE electrophorogram;
Fig. 3 is PCV2 Cap albumen Western blot figure.
Embodiment
Insect cell has used Sf9 cell (Spodoptera frugiperda (fall army worm) gonad cell) in the embodiment of the invention, available from Invitrogen company.The insect cell of other types, such as the silkworm embryos cell, silkworm gonad cell (BmN) etc. as long as can infect transfection virus of the present invention, can use the present invention's method to produce carrying Cap gene of porcine circovirus type 2.
The cultivated silkworm breed variety that uses in the embodiment of the invention is for making greatly (Dazao), other with it consistent cultivated silkworm breed variety, and cultivated silkworm breed variety as described comprises european race: Luobendihong, Baohuang, Hungary; China seed: Qiansanmian, Datuanyuan, Ankang, Lan5; Japan plants: Ri110, Dongfei, Su16, Su14, YingHXZ, Haoyue etc., also can use the present invention's method to produce carrying Cap gene of porcine circovirus type 2.
Carrying Cap gene of porcine circovirus type 2 of the present invention, can use this area pharmaceutical carrier commonly used such as ISA206 adjuvant etc., or water-based adjuvant or the oil adjuvant of this area other types, and this area other emulsification methods commonly used, preparation carrying Cap gene of porcine circovirus type 2 subunit vaccine.
Carrying Cap gene of porcine circovirus type 2 of the present invention can use method well known in the art to prepare test kit or direct as the infection of detection reagent for detection of porcine circovirus 2 type.
The restructuring autographa californica nuclear polyhedrosis virus (AcNPV) that makes up in the present embodiment, with complete PCV2 Cap protein gene, it is lower to be positioned at the control of silkworm caryogram polyhedrosis gene promotor, polyhedrosis gene is replaced by PCV2 Cap protein gene, has the utmost point and expresses and the high advantage of expression amount late period.In silkworm larva and silkworm chrysalis, expression product identifies that through SDS-PAGE and Western blot the result shows that the recombinant bombyx mori nuclear polyhedrosis virus of structure can efficiently express PCV2 Cap albumen, and size is about 28kDa.
The restructuring autographa californica nuclear polyhedrosis virus that makes up in the present embodiment, that use is Bac-to-Bac baculovirus expression system (Bac-to-Bac Baculovirus Expression System, Invitrogen), the expression system of any other type and transfer vector, expression vector, as long as can realize the Cap protein gene cloning to recombinant virus, and can infected silkworm and silkworm chrysalis and express Cap albumen, can use the identical method of the present invention to produce carrying Cap gene of porcine circovirus type 2.
Adopt intestinal bacteria DH10Bac (being purchased from Invitrogen) to transform in the present embodiment, any intestinal bacteria that contain shuttle plasmid can use the identical method of the present invention to produce carrying Cap gene of porcine circovirus type 2.
The restructuring PCV2 Cap is the polypeptide of sequence SEQ ID NO2 in the embodiment of the invention; Use the present invention's method also can produce following restructuring PCV2 Cap: with the polypeptide of described polypeptide at least 80% homology that is selected from sequence SEQ ID NO2 and the immunogenicity part of aforementioned polypeptides, such as the Cap albumen of truncation type (namely, although aminoacid sequence is removed part, still keep the Cap albumen of immunity).The embodiment method of preparation aforementioned polypeptides is identical with process and the embodiment of the invention, and the below is not repeated.
For making the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention that NM concrete experimental technique in the following example should carry out according to the normal experiment method.
1, clone pig circovurus type 2 ORF2 gene changes the pFastBacI transfer vector over to, obtains to comprise the recombinant transfer vector of porcine circovirus 2 type ORF2 gene:
Extract PCV2-SH strain (preserving number is CGMCC No.2389) DNA as the template of amplification Cap protein gene, be stored in-20 ℃ for subsequent use; According to the PCV2 complete genome sequence of GenBank, the design primer adds BamHI and XholI restriction enzyme site at upstream and downstream primer 5` end respectively.
Upstream primer: 5`-CGCGGATCCATGACGTATCCAAGGAGGC-3`;
Downstream primer: 5`-CCGCTCGAGGGGTTTAAGTGGGGGGTCT-3`;
Utilize the primer amplification PCV2 ORF2 gene fragment of design, agarose electrophoresis reclaims and identifies PCV2 ORF2 gene band.Be the PCR electrophorogram such as Fig. 1, wherein 1 is the purpose band, and M is DNA Marker2000.
Band to recovery carries out BamHI and XholI double digestion evaluation recovery, simultaneously pBacPAK8 plasmid (available from Clontech company) is carried out equally double digestion and identifies recovery with BamHI and XholI.The purpose fragment that reclaims is carried out the T4 ligase enzyme connect with the pBacPAK8 plasmid, be converted in the DH5a intestinal bacteria, screening positive clone extracts plasmid, and with positive plasmid called after pBacPAK8-ORF2.With pcr amplification and order-checking, sequencing result is consistent with the porcine circovirus 2 type ORF2 gene of GenBank registration.Therefore, by sequencing result as can be known, contain in the PCV2 Cap protein gene recombinant transfer vector and contain the ORF2 gene order.
2, will contain PCV2 Cap protein gene recombinant transfer vector is transformed in the competent escherichia coli cell that contains shuttle vectors, acquisition comprises the shuttle vectors of PCV2 ORF2 gene, again its shuttle vectors is transfected in the insect cell, obtains recombinant baculovirus vBac-ORF2:
PBacPAK8-ORF2 is transformed the intestinal bacteria DH10Bac (available from Invitrogen company) that contains viral shuttle plasmid Bacmid, obtain recombinant plasmid Bacmid-ORF2; Positive plasmid is extracted in the PCR screening, prepares positive recombinant plasmid Bacmid-ORF2 DNA.Adopt Cellfectin Reagent transfection Sf9 cell (available from Invitrogen company), after pathology appears in cell, carry out recombinant virus plaque purification and virus amplification, then by using primer M13F and M13R to carry out PCR checking purpose restructuring ORF2 gene.Purifying obtains recombinant baculovirus, called after vBac-ORF2.Recombinant baculovirus vBac-ORF2 is as kind of a poison in amplification, is undertaken after the titration in 4 ℃ of preservations by plaque ethods.
3, recombinant baculovirus vBac-ORF2 infected silkworm larva produces PCV2 Cap albumen;
Recombinant baculovirus vBac-ORF2 is with 1.0 * 10
5The infective dose of pfu (plaque forming unit)/head is injected in the silkworm hemolymph chamber in silkworm larva abdominal segment place with microsyringe.Behind 72h, 84h, 96h, 108h, 120h, 132h, 144h and 156h after infecting, results infect the larva of recombinant baculovirus, utilize tissue mashing machine to carry out the fragmentation of silkworm body, and 10000rpm is centrifugal, reclaims supernatant, and the silkworm liquid of haemolymph.Then the PBS (pH7.4,0.01M) that adds 100 times of volumes utilizes the ultrafiltration and concentration machine to carry out ultrafiltration and goes out foreigh protein removing, utilizes SDS-PAGE electrophoresis and Western blot to carry out the evaluation of PCV2 Cap albumen.
Qualification result is seen Fig. 2,3, as seen from the figure, obvious Cap protein band is arranged all among SDS-PAGE electrophoresis and the Western blot, illustrates that PCV2 Cap expresses in Silkworm, Bombyx mori.
Fig. 2 is the SDS-PAGE electrophorogram, and wherein 1 is the protein induced front sample of PCV2 Cap; 2 is the protein induced rear sample of PCV2 Cap; M is albumen Marker; Fig. 3 is Western blot evaluation figure, and wherein 1 is the protein induced rear sample of PCV2 Cap; 2 is the protein induced front sample of PCV2 Cap; M is the double-colored albumen Marker that dyes in advance.
The above only is preferred embodiment of the present invention, and is in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. a method of utilizing silkworm biological reactor to produce carrying Cap gene of porcine circovirus type 2 is characterized in that, may further comprise the steps:
(1). the coding gene sequence of clone's carrying Cap gene of porcine circovirus type 2, change transfer vector over to, obtain to comprise the recombinant transfer vector of carrying Cap gene of porcine circovirus type 2 encoding gene;
(2). above-mentioned recombinant transfer vector is transformed in the competent escherichia coli cell that contains viral shuttle vectors, acquisition contains the recombinant shuttle vector of carrying Cap gene of porcine circovirus type 2 encoding gene, again this shuttle vectors is transfected in the insect cell, obtains recombinant baculovirus;
(3). recombinant baculovirus infectable infection silkworm larva or silkworm chrysalis;
(4). reclaim and obtain the silkworm larva of recombinant virus infection or the hemolymph of silkworm chrysalis;
(5). the restructuring carrying Cap gene of porcine circovirus type 2 in the separation and purification hemolymph.
2. method according to claim 1 is characterized in that, the encoding sequence of carrying Cap gene of porcine circovirus type 2 comprises the SEQ ID NO1 in the sequence table in the described step (1).
3. method according to claim 1 is characterized in that, the expression of the gene of the carrying Cap gene of porcine circovirus type 2 coding described in the described step (2) is subjected to the control of polyhedron promotor.
4. method according to claim 1 is characterized in that, the insect baculovirus described in the step (2) comprises the insect nuclear polyhedrosis virus.
5. method according to claim 1 is characterized in that, the insect cell described in the step (2) comprises the Sf9 cell.
6. method according to claim 1 is characterized in that, described in the step (3)
Cultivated silkworm breed variety comprises Dazao, Luobendihong, Baohuang, Hungary, Qiansanmian, Datuanyuan, Ankang, Lan 5, Ri110, Dongfei, Su16, Su14, YingHXZ, Haoyue.
7. method according to claim 1 is characterized in that, the Recombinant Swine circovurus type 2 Cap protein sequence described in the step (3) comprises the SEQ ID NO2 in the sequence table.
8. method according to claim 1 is characterized in that, the restructuring carrying Cap gene of porcine circovirus type 2 described in the step (3) is any one of following three peptide species:
1) contains the polypeptide of the SEQ ID NO2 sequence in the ordered list;
2) with 1) polypeptide of described polypeptide at least 80% homology;
3) 1) and 2) the immunogenicity part of described polypeptide.
9. the restructuring carrying Cap gene of porcine circovirus type 2 produced of the method for any one according to claim 1～8.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103255171A (en) * | 2013-03-07 | 2013-08-21 | 江苏省农业科学院 | Recombinant virus-like particles of porcine circovirus 2 type codon optimized OFRF2 gene |
| WO2014113855A1 (en) * | 2013-01-25 | 2014-07-31 | Fundação De Amparo À Pesquisa Do Estado De Minas Gerais - Fapemig | Recombinant antigens of porcine circovirus 2 (pcv-2) for vaccine formulations, diagnostic kit and use thereof |
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| CN101920012A (en) * | 2010-07-22 | 2010-12-22 | 洛阳普莱柯生物工程有限公司 | Method for producing porcine circovirus type II recombinant capsid protein subunit vaccine by utilizing silkworm bioreactor and products thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101920012A (en) * | 2010-07-22 | 2010-12-22 | 洛阳普莱柯生物工程有限公司 | Method for producing porcine circovirus type II recombinant capsid protein subunit vaccine by utilizing silkworm bioreactor and products thereof |
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| 《中国抗生素杂志》 20030531 黄亚东 等 "天蚕抗菌肽AD 基因在AcNPV载体系统中的表达研究" 第304-307、313页 1-9 第28卷, 第5期 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014113855A1 (en) * | 2013-01-25 | 2014-07-31 | Fundação De Amparo À Pesquisa Do Estado De Minas Gerais - Fapemig | Recombinant antigens of porcine circovirus 2 (pcv-2) for vaccine formulations, diagnostic kit and use thereof |
| US9717785B2 (en) | 2013-01-25 | 2017-08-01 | Fundação De Amparo À Pesquisa Do Estado De Mg-Fapemig | Recombinant antigens of porcine circovirus 2 (PCV-2) for vaccine formulations, diagnostic kit and use thereof |
| CN103255171A (en) * | 2013-03-07 | 2013-08-21 | 江苏省农业科学院 | Recombinant virus-like particles of porcine circovirus 2 type codon optimized OFRF2 gene |
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Application publication date: 20130206 |