CN107982528A - PCV2 recombinant baculovirus sample particle subunits vaccine and preparation method - Google Patents
PCV2 recombinant baculovirus sample particle subunits vaccine and preparation method Download PDFInfo
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Abstract
The invention discloses a kind of PCV2 recombinant baculovirus sample particle subunits vaccine and preparation method thereof, the vaccine is used as host cell using insect, transfection includes the carrier for expression of eukaryon of PCV2 recombinant baculovirus ORF2 genetic fragments, for the nucleotide sequence of the PCV2 recombinant baculovirus ORF2 genetic fragments as shown in SEQ ID NO.1, the carrier for expression of eukaryon is pAcGP67 A PCV2.The method utilizes recombinant baculovirus of the molecular biology method structure containing high immunogenicity PCV2ORF2 nucleotide sequences, the recombinant virus can in Sf9 cell lines high efficient expression PCV2 Cap, and the PCV2 virus-like particles with PCV2 surface antigens are assembled into kytoplasm.Filtration system processing and inactivation are carried out to the virus stock solution used of harvest, is aided with adjuvant, the PCV2 recombinant baculovirus sample particle subunits vaccines that immunity is high, security is good is made.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to a kind of PCV2 recombinant baculovirus sample particle subunits epidemic disease
Seedling and preparation method thereof.
Background technology
Pig circular ring virus (Porcine circovirus, PCV) belongs to circovirus section (Circoviridae) annulus disease
Poison belongs to (Circovirus), is nonencapsulated single stranded circle DNA virus, is to have now been found that minimum DNA virus.Formed according to gene
And virus antigenicity, it is two kinds of serotypes, PCV1 types and PCV2 types that PCV, which is divided to,.Wherein PCV1 is not pathogenic type, and PCV2 is pathogenic
Type.
Porcine circovirus desease (PCVD) is the pig transmissible disease as caused by PCV2 viruses, is the important inhibitive ability of immunity of pig
Disease, the disease are fallen ill seriously in swinery, cause huge economic loss to global pig breeding industry.PCV2 be considered as cause it is disconnected
The main pathogen of milk piglet multisystemic wasting syndrome (PWMS), it can not only cause weanling pig that exhaustion, death occurs, also
With piglet A2 types congenital tremors (AII, CT), farrowing sow miscarriage, Adult Pig dermatitis nephrotic syndrome (PDNS) and respiratory tract
Diseases complex (PRDS) is closely related, is that one of infectious disease for seriously endangering pig breeding industry is generally acknowledged in the whole world.In recent years, China PCV2
Virus infection is in rising trend.
PCV2 full-length genome 1768bp, there is 11 open reading frame, ORF1-ORF11.Wherein ORF1 and ORF2 is two
Main open reading frame.ORF1 is encoded and the relevant zymoprotein of virus replication, participation virus replication.The capsid of ORF2 coding viruses
Albumen, is the viral main immunogenic albumen.Research shows that under proper condition, ORF2 albumen can be assembled into virus-like
Particle (Virus-Like-Particles, VLPs), VLPs has similar profile with PCV2 virion, and does not contain nucleic acid.
Immune animal, which can induce, produces PCV2 virucidins, is the key object for developing recombinant vaccine.
Vaccinoprophylaxis is the most effective means for controlling Porcine circovirus desease (PCVD) caused by PCV2.Pig annulus at present
Viral vaccine mainly has inactivated virus vaccine, subunit vaccine and attenuated live vaccines.It is wherein domestic to be essentially inactivated vaccine.
PCV2 viruses are not easy to breed on cell, and production virus titer is low；In addition also there is the difficult inspection of inactivating efficacy for PCV2 inactivated vaccines
The problems such as survey, duration of immunity is short, needs multiple immunoprophylaxis, and production cost is far above other viral inactivation vaccines.Attenuated live vaccines are then
There is the potential danger such as virulence reversion and scattered poison, and immunogenicity is poor.
At present, PCV2 virus-like particles (Virus-Like-Particle, VLP) vaccine in PCV2 subunit vaccines is
Develop the emphasis of recombinant vaccine.VLP vaccines not only have good immunogenicity, but also security is good, can induce body production
Raw humoral immunity and cellular immunity.Although escherichia expression system has the advantages that easily culture, safe, expression it is outer
Source gene protein is insoluble and lacks bioactivity and immunogenicity.Insect baculovirus expression system is in virus-like particle
(VLP) structure and application aspect has obvious advantage.Insect baculovirus expression system can carry out letter to foreign protein
The processing modification such as the excision of number peptide, glycosylation；With strong promoter, the transcription and translation of destination protein can be strengthened；A 10kb left sides can be accommodated
Right foreign gene, and the expression of multiple exogenous sequences can be carried out at the same time；With obvious host's boundary, to vertebrate without cause
Characteristic of disease, it is safe.The CircoFlex vaccines of the Circumvent-PCVM and Bo Linge companies of Merck companies use insect
Baculovirus expression PCV2 Cap, then it is prepared into vaccine.It is few that the vaccine is proven to have dosage, immune effect
The advantages that good, great function has been played to the PCV2 virus controls in Europe and North America.Although baculovirus expression system there is
The advantages that production cost is low, expressing quantity is high, but due to its height to production technology, production technology and production equipment etc.
It is required that limit popularization and use of the expression system in China's PCV2 production of vaccine.
Therefore, a kind of immunity is high, security is good, can industrialized production PCV2 recombinant baculovirus sample particle subunits
Vaccine is that domestic PC V2 production of vaccine is required.
The content of the invention
It is contemplated that at least solve one of problems of the prior art.
One of the technical problem to be solved in the present invention be to solve the problems, such as it is domestic lack high immunogenicity PCV2 vaccines, be
The country provide a kind of immunity is high, security is good, can industrialized production PCV2 recombinant baculovirus sample particle subunits vaccines
Preparation method.
In order to solve the above technical problems, the present invention provide a kind of PCV2 recombinant baculovirus sample particle subunits vaccine and its
Preparation method.
For the PCV2 recombinant baculovirus sample particle subunits vaccine using insect as host cell, transfection includes PCV2 weights
The carrier for expression of eukaryon of group baculoviral ORF2 genetic fragments.
The nucleotide sequence of the PCV2 recombinant baculovirus ORF2 genetic fragments is as shown in SEQ ID NO.1.
The carrier for expression of eukaryon is pAcGP67-A-PCV2, is by PCV2 recombinant baculovirus ORF2 gene fragment clones
To obtained from the EcoR1/BglII double enzyme sites in pAcGP67-A carriers；The insect is the Sf9 cell lines in insect.
The method utilizes restructuring of the molecular biology method structure containing high immunogenicity PCV2ORF2 nucleotide sequences
Baculoviral, the recombinant virus can in Sf9 cell lines high efficient expression PCV2 Cap, and tool is assembled into kytoplasm
There are the PCV2 virus-like particles of PCV2 surface antigens, specifically comprise the following steps：
(1) BD BaculoGold baculovirus expression systems are used, by PCV2 recombinant baculovirus ORF2 nucleotide sequences
It is connected to by EcoR1/BglII double digestions in pAcGP67-A shuttle vectors, BD BaculoGoldTMLinearized baculovirus dna
Recombinant baculovirus is obtained with pAcGP67-A shuttle vector cotransfection Sf9 cells, screening high titre recombinant baculovirus is as life
Production seed culture of viruses；
(2) full suspension free serum culture is carried out to Sf9 cells using bioreactor, when cell density culture extremely
1.5-2×106Cells/ml, is infected, suspend serum-free entirely using the high titre recombinant baculovirus of screening in step (1)
Culture 5-7 days, harvest virus, protein production amount can form virus-like particle up to 200~300mg/l, expressing protein；
(3) filtration system processing and inactivation are carried out to the virus stock solution used of harvest, is aided with adjuvant, immunity height, peace is made
The PCV2 recombinant baculovirus sample particle subunits vaccines of good perfection.
PCV2ORF2 nucleotides sequences are classified as GenBankAIT11738.1 sequences in the step (1), are that PCV2 restructuring is rod-shaped
The plasmid pUC57-PCV2 of virus O RF2 nucleotide sequences exists respectively according to shuttle vector pAcGP67-A multiple cloning sites sequences
ORF2 sequences 5 ' are held and 3 ' ends add EcoR1 and BglII restriction enzyme sites；The PCV2 recombinant baculovirus ORF2 nucleotide total lengths
713bp, particular sequence is as shown in SEQ ID NO.1.
Serum free medium is EX-CELL in the step (2)TM420 serum free mediums；Sf9 cells are in cell biological
Condition of culture in reactor is：Cell inoculation amount is 2-5 × 105Cells/ml, bioreactor rotating speed are 80-
90rpm, cultivation temperature are 26-28 DEG C, oxygen dissolving value 60%-80%, pH value 6.2-6.5；High titre recombinant baculovirus is inoculated with
Amount MOI is 1-10, and cultivation temperature is 26-28 DEG C, pH value 6.2-6.5.
Further, in the step (2) expressing protein be recombinant baculovirus expression PCV2 Cap, specifically
Amino acid sequence is as shown in Seq ID No.2.
The inactivator that PCV2 recombinant baculovirus sample particle subunits vaccine uses in the step (3) is sub- for divinyl
Amine；Inactivation step is 5mM binary ethylenimines when 37 DEG C of inactivations 72 are small.
Further, PCV2 recombinant baculovirus sample particle subunits vaccine antigens adjuvant is selected in the step (3)5984EP polymer, final concentration of 01.~0.5mg/ml.
Wherein, Sf9 cells, purchased from U.S. ATCC；EX-CELLTM420 serum free mediums, purchased from Sigma Co., USA.
Also a kind of carrier for expression of eukaryon of the present invention, the carrier for expression of eukaryon is pAcGP67-A-PCV2, is by PCV2 weights
Obtained from group baculoviral ORF2 gene fragment clones to the EcoR1/BglII double enzyme sites in pAcGP67-A carriers.
Compared with existing disclosed technical solution, the present invention have the advantage that for：
1st, the present invention uses shape virus-insect cell expressioning system, is suspended entirely using macro-organism retort
Large-scale production.
2nd, the antigen protein of Expression product is soluble protein in the present invention, utilizes simple filtration system, you can is reduced
Vaccine impurity, vaccine purity are high.
3rd, the antigen protein content of Expression product is high in the present invention.200~300mg/ of immune protein amount of every milliliter of production
Ml, needs not move through the techniques such as concentration, cost-effective, flow is simple.
4th, PCV2 antigen proteins security is good in the present invention, using free serum culture technology, reduces vaccine since serum draws
The anaphylactogen risen.Water phase adjuvant, reaction of animals are gentle.Without allergic reaction after immune animal, no body temperature rise, feeding declines and shadow
Weightening phenomenon is rung, injection site is without adverse reactions such as red and swollen, suppurations.
5th, vaccine antigen protein immunogenicity builds well recombinant baculovirus and selects high immunogenic sequences, vaccine in the present invention
After immune, body immune system capable of fast starting, produces high-level antibody, viral infection resisting.
6th, in the present invention, the baculovirus-insect expression system that production of vaccine uses is a very safe system, table
The product reached is the memebrane protein of virus, without any virus activity, will not produce any influence and harm to human body and environment.
Brief description of the drawings
Fig. 1 show shuttle vector pAcGP67-A and MCS sequence diagram in the embodiment of the present invention 1；
Fig. 2 show PCV2ORF2 destination proteins SDS-PAGE interpretation of result figures in the embodiment of the present invention 1；
Fig. 3 show PCV2ORF2 destination proteins Western-blot interpretation of result figures in the embodiment of the present invention 1；
Fig. 4 show recombinant baculovirus expression Porcine circovirus type 2 ORF2 protein negative staining electron microscope figure in the embodiment of the present invention 2.
Embodiment
The embodiment and effect of the method for the present invention are described in detail below in conjunction with specific embodiment.It should be noted that
It is that the combination of the technical characteristic or technical characteristic described in following embodiments is not construed as isolated, they can be with
It is mutually combined so as to reach superior technique effect.
Below in conjunction with specific embodiment, the present invention is described in detail.
The acquisition of embodiment 1PCV2ORF2 recombinant baculovirus
Artificial synthesized plasmid pUC57-PCV2 (the raw works containing codon PCV2 recombinant baculovirus ORF2 nucleotide sequences
Bioengineering (Shanghai) limited company synthesizes).PCV2ORF2 nucleotides sequences are classified as GenBankAIT11738.1 sequences.Root
According to shuttle vector pAcGP67-A multiple cloning sites sequences, EcoR1 and BglII enzymes are added at the end of ORF2 sequences 5 ' and 3 ' ends respectively
Enzyme site.The PCV2 recombinant baculovirus ORF2 full length gene 713bp synthesized in the present invention, particular sequence such as SEQ IDNO.1 institutes
Show.
Recombinant plasmid pUC57-PCV2 is subjected to EcoR1/BglII double digestions, purifying recycling purpose fragment.The purpose of recycling
Fragment is cloned into shuttle vector pAcGP67-A (BD Biosciences by EcoR1/BglII double enzyme sites
Pharmingen, San Diego, CA, as shown in Figure 1) on, obtain recombinant shuttle vector pAcGP67-A-PCV2.It is right
PAcGP67-A-PCV2 carries out PCR detections and gene sequencing, and sequencing result shows that target gene is correctly cloned into shuttle vector
On pAcGP67-A.
By BD BaculoGoldTMLinearized baculovirus dna and recombinant shuttle vector pAcGP67-A-PCV2 cotransfections Sf9
Insect cell (Invitrogen), obtains high titre PCV2 recombinant baculovirus, is named as RD-AcMNPV.
Using the recombinate shape virus infection Sf9 cells of acquisition, expand numerous, collected supernatant when 72 is small after transfection,
As original seed culture of viruses, -80 DEG C are stored in.
The supernatant of original seed culture of viruses is taken to carry out SDS-PAGE and Western-blot protein expression analysis, such as Fig. 2 and Fig. 3 institutes
Show (wherein：In Fig. 2, Marker is PageRuler Prestained Protein Ladder, and 1,2 represent recombinant baculovirus
The albumen of expression, 1 applied sample amount are 40 μ l, and 2 applied sample amounts are 20 μ l；1 represents Sf9 cell culture supernatants in Fig. 3, right for feminine gender
According to 2 be PCV2 virus proteins, is positive control, and 3,4 represent the ORF2 destination proteins of recombinant baculovirus expression), restructuring is rod-shaped
The albumen of viral great expression about 30kDa or so；The albumen can specifically be reacted with rabbit-anti PCV2 hyper-immune serums.The PCV2 of structure
Target structure albumen is about 30kDa, and can great expression.
The preparation of embodiment 2PCV2 recombinant baculovirus sample particles
1st, prepared by cell
The Sf9 working cardial cells storehouse cell frozen is taken, cell is distributed to 75cm2Cell bottle in, add EX-CELLTM
420 serum free mediums.When 27 DEG C of cultures 24 are small, by 1:5 dispersion rates pass to cell in blake bottle, add new serum-free
Culture medium, continues 27 DEG C of cultures, collects cell.Passed in the same way, expand culture, it is thin until expanding to what is needed
Born of the same parents' quantity.With 4 × 105Cells/ml cell densities access bioreactor carries out full suspension free serum culture, 27 DEG C of trainings
It is 2 × 10 to support to cell density6During cells/ml, for connecing poison.
2nd, virus inoculation, culture and harvest
By production seed culture of viruses with the Sf9 of 2 × 106cells/ml of inoculum concentration inoculation bioreactor Nei Miduda of MOI=1
Work storehouse cell, using full suspension free serum culture virus.Bioreactor rotating speed is 80rpm, and cultivation temperature is 27 DEG C, dissolved oxygen
Value 60%, pH value 6.2.After culture 5 days, cell culture supernatant, mark, 2~8 DEG C of preservations are harvested.
3rd, negative staining electron microscope detects
Negative staining electron microscope detection is carried out to the vial supernatant of collection.As shown in figure 4, the Porcine circovirus type 2 ORF2 protein of expression being capable of shape
Into virus like particle, size is about 17nm, similar to real PCV2 morphology of virus size, it was demonstrated that the PCV2 Cap of expression
Virus-like particle can be properly formed.
The preparation of 3 PCV2 recombinant baculovirus sample particle subunits vaccines of embodiment
1st, the acquisition of virus stock solution used
Sf9 work storehouse cells are subjected to full suspension free serum culture, when cell density is up to 1.5 × 106Cells/ml is carried out
Recombinate shape virus infection, virus inoculation amount are MOI=2.Continue full suspension free serum culture 6 days, directly harvest cell culture
Liquid, is virus stock solution used.
2nd, virus liquid filters
By the cell culture supernatant of harvest, successively by 1.0 μm of filter membrane systems and 0.22 μm of filter membrane system filtration sterilization,
2~8 DEG C of preservation filtered fluids.
3rd, inactivation of virus
In order to ensure the security of PCV2 recombinant baculovirus sample particle subunits vaccines, we are to the restructuring in virus liquid
Baculoviral is inactivated.Inactivator uses binary ethylenimine BEI, 5mM BEI when 37 DEG C of inactivations 72 are small, continuous during inactivation
Agitated liquid, BEI in the sodium thiosulfate of final concentration of 5mM and unnecessary is added after reaction terminating.
IFA detections are carried out to the cell culture supernatant after inactivation, show weight if detection specificity fluorescent does not occur
Group baculoviral inactivation is complete.
5th, immunologic adjuvant
In order to ensure the immunogenicity of vaccine, stability and security, PCV2 recombinant baculovirus sample particles in the present invention
Subunit vaccine uses5984EP polymer is as antigen adjuvant.Every batch of is inactivated into complete cells and supernatant
Liquid and preparation5984EP polymer solutions are sufficiently stirred mixing, and final vaccine antigen content answers >=8 μ g/ml,The final concentration of 0.1mg/ml of 5984EP polymer.
6th, dispense
After vaccine is sufficiently mixed, it is distributed into the dose aseptic of 10ml or 50ml in injection bottle.Seal bottleneck, and label adhering
Label.
7th, vaccine valence is examined
6 week old female Balb/c mouse are carried out with 200 μ l/ of subcutaneous inoculation injection only, heart on the 28th is adopted after immune
Blood, separates serum, carries out ELISA antibody titer detections.According to testing result, laboratory vaccine can induce mice produced high titers
PCV2 antibody.
Although having been presented for some embodiments of the present invention herein, it will be appreciated by those of skill in the art that
Without departing from the spirit of the invention, the embodiments herein can be changed.Above-described embodiment is only exemplary, no
Restriction that should be using the embodiments herein as interest field of the present invention.
Claims (10)
- A kind of 1. PCV2 recombinant baculovirus sample particle subunits vaccine, it is characterised in that the PCV2 recombinant baculovirus sample For particle subunits vaccine using insect as host cell, transfection includes the eucaryon table of PCV2 recombinant baculovirus ORF2 genetic fragments Up to carrier.
- 2. a kind of PCV2 recombinant baculovirus sample particle subunits vaccine according to claim 1, it is characterised in that described The nucleotide sequence of PCV2 recombinant baculovirus ORF2 genetic fragments is as shown in SEQ ID NO.1.
- A kind of 3. PCV2 recombinant baculovirus sample particle subunits vaccine according to claim 1 or 2, it is characterised in that The carrier for expression of eukaryon is pAcGP67-A-PCV2, is to arrive PCV2 recombinant baculovirus ORF2 gene fragment clones Obtained from EcoR1/BglII double enzyme sites in pAcGP67-A carriers；The insect is the Sf9 cell lines in insect.
- 4. a kind of carrier for expression of eukaryon, it is characterised in that the carrier for expression of eukaryon is pAcGP67-A-PCV2, is to recombinate PCV2 Obtained from baculoviral ORF2 gene fragment clones to the EcoR1/BglII double enzyme sites in pAcGP67-A carriers.
- 5. a kind of preparation method of PCV2 recombinant baculovirus sample particle subunits vaccine, it is characterised in that the method utilizes Molecular biology method builds the recombinant baculovirus containing high immunogenicity PCV2ORF2 nucleotide sequences, the recombinant virus Can in Sf9 cell lines high efficient expression PCV2 Cap, and the PCV2 with PCV2 surface antigens is assembled into kytoplasm Virus-like particle, specifically comprises the following steps：(1) BD BaculoGold baculovirus expression systems are used, PCV2 recombinant baculovirus ORF2 nucleotide sequences are passed through EcoR1/BglII double digestions are connected in pAcGP67-A shuttle vectors, BD BaculoGoldTMLinearized baculovirus dna with PAcGP67-A shuttle vector cotransfection Sf9 cells obtain recombinant baculovirus, and screening high titre recombinant baculovirus is as production Use seed culture of viruses；(2) full suspension free serum culture is carried out to Sf9 cells using bioreactor, when cell density culture to 1.5-2 ×106Cells/ml, is infected, suspend free serum culture entirely using the high titre recombinant baculovirus of screening in step (1) 5-7 days, harvest virus, protein production amount can form virus-like particle up to 200~300mg/l, expressing protein；(3) filtration system processing and inactivation are carried out to the virus stock solution used of harvest, is aided with adjuvant, immunity height, security is made Good PCV2 recombinant baculovirus sample particle subunits vaccines.
- 6. a kind of preparation method of PCV2 recombinant baculovirus sample particle subunits vaccine according to claim 5, it is special Sign is that PCV2ORF2 nucleotides sequences are classified as GenBankAIT11738.1 sequences in the step (1), is that PCV2 restructuring is rod-shaped The plasmid pUC57-PCV2 of virus O RF2 nucleotide sequences exists respectively according to shuttle vector pAcGP67-A multiple cloning sites sequences ORF2 sequences 5 ' are held and 3 ' ends add EcoR1 and BglII restriction enzyme sites；The PCV2 recombinant baculovirus ORF2 nucleotide total lengths 713bp, particular sequence is as shown in SEQ ID NO.1.
- 7. a kind of preparation method of PCV2 recombinant baculovirus sample particle subunits vaccine according to claim 5, it is special Sign is that serum free medium is EX-CELL in the step (2)TM420 serum free mediums；Sf9 cells are in cell biological Condition of culture in reactor is：Cell inoculation amount is 2-5 × 105Cells/ml, bioreactor rotating speed are 80- 90rpm, cultivation temperature are 26-28 DEG C, oxygen dissolving value 60%-80%, pH value 6.2-6.5；High titre recombinant baculovirus is inoculated with Amount MOI is 1-10, and cultivation temperature is 26-28 DEG C, pH value 6.2-6.5.
- 8. a kind of preparation method of PCV2 recombinant baculovirus sample particle subunits vaccine according to claim 5 or 7, its Be characterized in that, in the step (2) expressing protein be recombinant baculovirus expression PCV2 Cap, specific amino acid sequence As shown in Seq ID No.2.
- 9. a kind of preparation method of PCV2 recombinant baculovirus sample particle subunits vaccine according to claim 5, it is special Sign is that the inactivator that PCV2 recombinant baculovirus sample particle subunits vaccine uses in the step (3) is binary ethylenimine； Inactivation step is 5mM binary ethylenimines when 37 DEG C of inactivations 72 are small.
- 10. a kind of preparation method of PCV2 recombinant baculovirus sample particle subunits vaccine according to claim 5 or 9, It is characterized in that, PCV2 recombinant baculovirus sample particle subunits vaccine antigen adjuvant is selected in the step (3) 5984EP polymer, final concentration of 01.~0.5mg/ml.
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