CN106399138B - Porcine Circovirus takes orally virus sample particle vaccines and its preparation method and application - Google Patents
Porcine Circovirus takes orally virus sample particle vaccines and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of kluyveromyces marxianus recombination engineering and its application in the oral Porcine Circovirus virus sample particle vaccines of production, the recombination engineering is to express Porcine Circovirus capsid protein by kluyveromyces marxianus expression vector, prepares Porcine Circovirus virus-like particle oral vaccine.The controlling element of kluyveromyces marxianus recombinant expression carrier provided by the invention derives from the kluyveromyces of aliment security level, has antibiotic-free resistant gene and Escherichia coli sequence signature.The present invention also provides a kind of methods for preparing Porcine Circovirus virus-like particle oral vaccine, utilize Porcine Circovirus virus-like particle oral vaccine provided by the invention, it can reduce and prevent to infect correlative clinical symptom caused by PCV2, have many advantages, such as that good immune effect, highly-safe, culture is easy to operate, yield is high, production cost is low and can amplify production on a large scale.
Description
Technical field
The present invention relates to a kind of oral vaccine and its preparation method and application more particularly to a kind of oral pig circular ring virus epidemic diseases
Seedling and its preparation method and application
Background technique
Pig circular ring virus (Porcine circovirus, PCV) is to find one of the smallest animal virus so far, belongs to circle
Circoviridae Circovirus is the sub-thread ring-type minus-strand dna virus of no cyst membrane, and 20 face bodies are symmetrical, diameter 17-20nm, it is known that
There are two serotype, i.e. pig annulus I type viral (PCV1) and pig annulus II type are viral (PCV2) for PCV tool, wherein PCV 1 is non-
Pathogenic virus, PCV2 are pathogenic virus.
Huge to the harm of animal husbandry although pig circular ring virus genome structure is simple, Porcine circovirus desease is close several
Year influences one of the most important viral infectious of world's pig breeding industry.
The infection of China's pig annulus II type viral (PCV2) has following three big features:
1) infection rate is high, infects wide: individual positive rate is 21.7-87.3%, and pig farm positive rate is 38.2-100%；
2) infection rate of PCV2 rises year by year: 2003 are 21.7%, and 2006 are 38.3%, and 2008 are 70.9%,
2012 are 83.2%；
3) PCV2 and the mixed infection of other cause of diseases are serious: in the serum of infection PCV2 virus, being often able to detect
The blue otopathy poison PRRSV of mixed infection, Pseudorabies virus PRV, parvovirus PPV, mycoplasma pneumoniae, pasteurella multocida,
Epidemic diarrhea virus PEDV, swine influenza virus SIV etc.；There is statistics report to show that PCV2 virus suprainfection rate is more than 50%,
With blue otopathy poison PRRSV coinfection rate be 51.9%, Pseudorabies virus PRV coinfection rate is 62.44%, parvovirus PPV is total
Infection rate is 56.9%.
More severe, it is early (delivery room suckling pig can infect) that infection age in days has been presented in the PVC2 in China's pig breeding industry
Situation.Therefore PCV2 has been considered as the important pathogen of world's pig breeding industry, can cause the immunosupress of pig, cause it is double or
Triple infection, morbidity and mortality are high, bring significant damage and serious economic loss to pig breeding industry.Currently, PCV2 vaccine
Have become the swine disease vaccine kind that dosage is maximum, dosage is most after the vaccines such as swine fever, aftosa, blue otopathy and pseudo- mad dog,
The huge market demand.
PCV2 vaccine is broadly divided into three classes: totivirus inactivated vaccine, PCV1-2 recombinant virus inactivated vaccine and viruslike particle
Subunit vaccine.Wherein, PCV2 subunit vaccine (mainly viruslike particle vaccine), it has already been proven that immune effect is better than entirely
Inactivation of virus seedling has significant improvement in safety, validity and manufacturing process than inactivated vaccine, is becoming a new generation
The new trend of recombinant vaccine development.
Current market sales of PCV-2Cap protide virus particle vaccine has two classes:
A) one kind is the PCV2 viruslike particle (prokaryotic expression) of Escherichia coli preparation, ORF2 gene (the Cap egg of PCV-2
It is white) it is inserted into Escherichia coli prokaryotic vector, using Bacillus coli expression PCV-2Cap albumen, prepare subunit vaccine.Large intestine
Bacillus expression system has the characteristics that the speed of growth is fast, easily culture, expression protein yield is high, but the albumen of its expression is mostly with nothing
Active inclusion bodies exist, after the sequence of operations such as albuminous degeneration renaturation, available soluble protein.But it is former
Nuclear expression, which is easy to appear not, has active inclusion body, needs to carry out protein renaturation to improve vaccine effect, at high cost, production is all
Phase long (every batch of time-consuming about 2-3 months), low efficiency, the protein immunogenic of acquisition are poor；Especially Bacillus coli expression
Although albumen is still possible to residual endotoxin by purifying, immune animal can generate side reaction.
B) another kind of that PCV2 viruslike particle vaccine processed is expressed by rod-shaped disease by baculoviral/insect cell expression system
Poison/insect cell expression system expression viruslike particle (eukaryotic expression), is inserted into the ORF2 gene (Cap protein) of PCV-2
To insect baculovirus, with Insect cellculture, obtain the PCV-2Cap albumen of recombination, it is purified, mixed with immunologic adjuvant after
It obtains, clinically immune with injection system, the technique of eukaryotic expression is more demanding, and baculoviral may be mixed in product
Grain generates side reaction, and there are certain bio-safety risks.
In addition, PCV2 vaccine can be used for oral vaccine there is not yet report based on injecting at present.
Summary of the invention
, side reaction low for production efficiency existing for current PCV2 vaccine is big, the defects of cannot taking orally, the present invention provides
A kind of orally available PCV2 vaccine.
First aspect of the present invention is to provide a kind of recombination kluyveromyces marxianus auxotrophic strain, is carried by recombination
Body converts the preparation of kluyveromyces marxianus auxotrophic strain.
By that will encode pig circular ring virus 2 virus capsid protein, (present invention is hereinafter also referred to as " pig circular ring virus 2 to the recombinant vector
Virus capsid protein Cap " or " Cap protein ") nucleotides inserted to kluyveromyces marxianus expression vector constructed by.
In an advantageous embodiment, the pig circular ring virus 2 virus capsid protein is preferably Porcine Circovirus viral capsid
Albumen.
The second aspect of the present invention is to provide a kind of method for preparing the Porcine Circovirus virus-like particle, packet
It includes:
The recombination kluyveromyces marxianus recombination engineering cultivation and fermentation, is assembled into pig circular ring virus II in the cell
Virus-like particle；
The bacterium of cultivation and fermentation is collected, Porcine Circovirus virus-like particle is obtained.
Or the method also includes constructing the recombination kluyveromyces marxianus recombination engineering as follows:
By the nucleotides inserted for encoding pig circular ring virus 2 virus capsid protein to kluyveromyces marxianus expression vector, weight is constructed
Group carrier；
Recombinant vector is converted into kluyveromyces marxianus auxotrophic strain, building obtains the recombination Marx gram
Tie up yeast recombination engineering in Shandong.
In an advantageous embodiment, the bacterium of collected cultivation and fermentation, can be separately as Porcine Circovirus disease
The virus-like particle of release is collected after malicious sample grain products, or rupture as Porcine Circovirus virus-like particle product,
Rupture after clasmatosis liquid or broken liquid desciccate as Porcine Circovirus virus-like particle product.
Third aspect of the present invention is to provide a kind of Porcine Circovirus virus-like particle oral drugs and/or vaccine,
Including the Porcine Circovirus virus-like particle.
The present invention the 4th aspect provides a kind of Porcine Circovirus virus-like particle drug and/or vaccine of preparing
Method especially prepares Porcine Circovirus and takes orally virus-like particle drug and/or vaccine (Porcine Circovirus virus is drawn
The oral drugs and/or vaccine of the disease of hair) method, including with after the bacterium of collected cultivation and fermentation or rupture obtain virus
Sample particle prepares Porcine Circovirus virus sample particle vaccines as active constituent or as one of active constituent.
In an advantageous embodiment, after collecting thallus, direct broken wall, products therefrom is as oral vaccine.
In an advantageous embodiment, releasing virus sample particle after the bacterium rupture of collected cultivation and fermentation, is added protection
Agent prepares Porcine Circovirus virus sample particle vaccines.
Wherein, the protective agent can be one of sucrose, lactose, trehalose or a variety of.
In another preferred embodiment, after the bacterium rupture of collected cultivation and fermentation, clasmatosis liquid is spray-dried
Prepare Porcine Circovirus virus sample particle vaccines.
The 6th aspect of the present invention is to provide a kind of Porcine Circovirus virus-like particle or pig circular ring virus II
The application of virus-like particle preferably prepares prevention and/or treats the drug for the disease that Porcine Circovirus virus causes
And/or vaccine.
In an advantageous embodiment, the pig circular ring virus 2 virus capsid protein amino acid sequence includes (preferably, being):
1) SEQ ID No.1 amino acid sequence；Or
2) SEQ ID No.1 amino acid sequence is by replacing, missing or adding one or several amino acid and having pig circle
The active protein as derived from SEQ ID No.1 amino acid sequence of circovirus virus nucleocapsid Cap protein.
In an advantageous embodiment, the 226th phenylpropyl alcohol in pig circular ring virus 2 virus capsid protein shown in the SEQ ID No.1
Propylhomoserin is replaced by leucine, such as include (and preferably): SEQ ID No.2 amino acid sequence.
In an advantageous embodiment, the nucleotide series for encoding the pig circular ring virus 2 virus capsid protein include, are simultaneously preferred
For sequence shown in SEQ ID No.3
In an advantageous embodiment, in the method, according to the codon preference of kluyveromyces marxianus, to volume
The nucleotide of the code pig circular ring virus 2 virus capsid protein carries out codon optimization, more preferably is guaranteeing to encode pig circular ring virus clothing
Glutelin sequence carries out codon optimization under the same conditions.
In an advantageous embodiment, the kluyveromyces marxianus expression vector includes kluyveromyces marxianus chrysanthemum
Powder enzyme promoters gene and terminator gene order, and preferably without containing non-resistant gene order, Escherichia coli starting duplication
Sequence.It is highly preferred that the kluyveromyces marxianus expression vector includes and preferably constitutes for core shown in SEQ ID No.6
Nucleotide sequence.
In an advantageous embodiment, the kluyveromyces marxianus expression vector auxotrophy riddled basins.
In a kind of preferred embodiment of the invention, the recombinant vector includes that kluyveromyces marxianus independently replicates
Sequence, kluyveromyces marxianus inulinase promoter gene sequence, the gene order for encoding pig circular ring virus 2 virus capsid protein, horse
Gram this kluyveromyces inulinase terminator gene order, auxotrophy riddled basins sequence；It is highly preferred that not containing nothing
Resistance gene sequences, Escherichia coli originate replication sequence.
The auxotrophic strain of kluyveromyces marxianus of the present invention knocks out part for kluyveromyces marxianus
Or obtained by whole specific nutrition genes.The specific nutrition gene is preferably selected from URA3 gene, HIS3 gene, in ADE2 gene
Any one or a few, and preferably URA3 gene.
Wherein, in a kind of more preferred embodiment, the kluyveromyces marxianus auxotrophic strain is in Uracil
It cannot be grown in auxotrophy culture medium.
In a preferred embodiment of the invention, above-mentioned kluyveromyces marxianus is preferably CGMCC No.10621.It should
Bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution address: Beijing's southern exposure
No. 3 Institute of Microorganism, Academia Sinica, institute of area North Star West Road 1, the deposit date is on March 13rd, 2015.
In one preferred embodiment of the invention, the conversion can be any one in electrotransformation or chemical conversion
Or it is several.
In one preferred embodiment of the invention, glucose, sulphur are contained in the recombination yeast culture used medium
Sour ammonium, magnesium sulfate, potassium dihydrogen phosphate.
Wherein, the Porcine Circovirus virus-like particle or Porcine Circovirus virus sample particle vaccines are preferably
It is used to prepare prevention and/or treats the drug and/or vaccine of the disease that Porcine Circovirus virus causes.
The application preferably comprises but is not limited to be used as oral vaccine, as in feed addictive, drinking water additive
Any one or a few.
Wherein, the disease that the Porcine Circovirus virus causes includes but is not limited to postweaning multisystemic failure
Any one or a few in syndrome, pigskin inflammation nephrotic syndrome, piglet congenital tremors and sow breeding difficulty.
In one preferred embodiment of the invention, any vaccine can be preferably solid pharmaceutical preparation, liquid preparation,
Any one or a few in semisolid preparation, gaseous formulation, and preferably solid pharmaceutical preparation.
Wherein, any vaccine can be but be not limited to dry powder, particle, tablet, capsule, paste, aerosol, is sprayed
Agent, solution, suspension, any one or a few in lotion.
In one preferred embodiment of the invention, any vaccine also may include auxiliary material, and auxiliary material preferably can be with
Be but be not limited to corrigent, preservative, moisturizer, disintegrating agent, lubricant, wetting agent, filler, adhesive or caking inhibiter,
Suspending agent, pigment, release retarding agent, diluent, solubilizer, solvent, flocculant or deflocculant, defoaming agent or foaming agent, increasing
Thick dose, plasticizer, buffer, pH adjusting agent, any one or a few in emulsifier.
In one preferred embodiment of the invention, any vaccine also may include adjuvant, and adjuvant preferably can be with
It is but is not limited to oil-in-water, Water-In-Oil, any one or a few in W/O/W adjuvant.
Above-mentioned various aspects of the invention and each embodiment can be combined with each other by those skilled in the art, various
Combination is also within the scope of the content of present invention.
It is Porcine Circovirus virus-like particle or Porcine Circovirus virus sample particle vaccines provided by the invention, excellent
It is selected as oral vaccine, can reduce and prevents to infect correlative clinical symptom caused by PCV2, there is orally available, immune effect
It is good, highly-safe, culture is easy to operate, yield is high, production cost is low and can amplify on a large scale the advantages that production.
Detailed description of the invention
Fig. 1 is the pcr amplified DNA electrophoretogram of Cap gene；
Fig. 2 is Cap protein kluyveromyces marxianus recombinant vector pUKD-N125/Cap schematic diagram；
Fig. 3 is expression of the pig circular ring virus II Cap protein in kluyveromyces marxianus；Wherein, swimming lane 1:Fim-
1ura3Δ；The full cell pyrolysis liquid of swimming lane 2:Fim-1ura3 Δ-pUKD-N125/Cap recombination yeast；Swimming lane 3:Fim-1ura3
Δ-pUKD-N125/Cap recombination yeast solution liquid supernatant；
Fig. 4 is the immunoblotting Western Blot inspection that kluyveromyces marxianus expresses pig circular ring virus II Cap protein
Survey reaches；Wherein, swimming lane 1:Fim-1ura3 Δ；Swimming lane 2:Fim-1ura3 Δ-pUKD-N125/Cap recombination yeast；
Fig. 5 is that kluyveromyces marxianus expresses PCV virus-like particle electron microscope observation；
Fig. 6 is Fim-1ura3 Δ-pUKD-N125/Cap recombination yeast pilot scale fermentation growth curve；
Fig. 7 is Cap protein under identical bacterium amount during Fim-1ura3 Δ-pUKD-N125/Cap recombination yeast pilot scale fermentation
Expression；
Fig. 8 is oral circovirus vaccine and the horizontal ratio of Cap IgG antibody in serum after injection import vaccine immune mouse
Compared with；
The Cap IgA antibody that Fig. 9 is generated in the excrement for the oral circovirus vaccine of mouse feeding is horizontal.
Specific embodiment
Embodiment 1, the building of PCV capsid protein recombinant expression carrier pUKD-N125/Cap
In the present embodiment, coding pig circular ring virus 2 virus capsid protein (is also referred to as " pig circular ring virus capsid Cap egg in context
It is white " or " Cap protein ") amino acid sequence gene have SEQ ID No.3 sequence.According to kluyveromyces marxianus
Codon preference, codon optimization is carried out to Cap gene order, and the Cap gene order after optimization is manually closed
At.
Encoded Cap protein amino acid sequence can be as shown in SEQ ID No.1 or 226 in SEQ ID No.1
The phenylalanine of position substitutes (such as SEQ ID No.2) by leucine.
Using the method for PCR amplification, with PCVN125-F (5'-TTTTTTTGTTAGATCCGCGGATGACATATCCAAGG
) and PCVN125-R (5'-AGCTTGCGGCCTTAACTAGTTCA AGGCGTTTC-3'
It CTTAGGGTTAAGTGGAGGGTCCTTAAG-3') is primer amplification Cap gene.It is solidifying with DNA after 1% agarose gel electrophoresis
Glue kit recycles the segment (such as Fig. 1) of 700kb or so.
It provides pUKD-N125 carrier (SEQ ID No.6), the carrier is by PUKD-N118 carrier (referring to Chinese invention patent
Apply for PUKDN118 in 201510562564.9 embodiments) it constructs, construction method is as follows:
Forward primer 5'-ACTAGTTAAGGCCGCAAGCTTTGATCTGATCTGC-3' and reverse primer 5'-TCCCCCG
CGGATCTAACAAAAAAAAAATTAAATG-3' carries out PCR amplification to PUKDN118 plasmid, includes inulin in amplified fragments
Enzyme terminator, KmURA3 promoter, KmURA3ORF, yeast autonomously replicating sequence and inulin enzyme promoters；
It is carried out using the more amplified fragments of Taq enzyme plus A is handled, then connect recovery product with PMD18-T carrier；Sequencing
Afterwards, SacII and SpeI carries out digestion and obtains pUKD-N125 carrier.
In pUKD-N125 carrier, including kluyveromyces marxianus autonomously replicating sequence, kluyveromyces marxianus inulin
The auxotrophy screening of enzyme promoters sequence, kluyveromyces marxianus inulinase terminator sequence, kluyveromyces marxianus
Marker gene URA3 (KmURA3ORF) does not contain Escherichia coli sequence.
Using two restricted digestion pUKD-N125 carriers of Sac II and Spe II, 1% agarose gel electrophoresis uses DNA
Gel reagents box recycles the carrier segments of 8kb or so
The method that the connection of Cap genetic fragment and carrier segments uses Gibson Assembly, with NEB company
Gibson Assembly traceless linker system (product number E2611S/L) constructs recombinant vector pUKD-N125/Cap, such as Fig. 2
Shown, the gene order (PCV2CAP) for encoding Cap protein is inserted into carrier.
Embodiment 2, PCV capsid protein Cap protein is in kluyveromyces marxianus genetic engineering bacterium Fim-1ura3 Δ-
The building of pUKD-N125/Cap and Cap expression analysis
Kluyveromyces marxianus uracil auxotrophy bacterial strain Fim-1ura3 Δ, the preparation method of the bacterial strain are provided
It can refer to the construction method of the bacterial strain of Fim-1 (ura3 Δ) disclosed in Chinese patent publication document CN105112313A embodiment 1.
Fim-1ura3 Δ is inoculated in the teat glass of the culture medium of YEPD containing 3mL, and 30 DEG C of shaking tables are incubated overnight to OD600
For 12-15.Collect thallus, LiAc-TE solution (100mM LiAc, 10mM Tris-HCl, 1mM EDTA) washing.
Carrier DNA, recombinant vector pUKD-N125/Cap, PEG solution (40%PEG are sequentially added in thallus
4000,100m M LiAc, 10mM Tris-HCl pH 7.5,1mM EDTA) and final concentration of 10mM DTT, after mixing well,
30 DEG C of water-bath 15min, 47 DEG C of water-bath 15min, 8000rpm winks are from abandoning supernatant adds 100 μ L sterile water suspension thallines, applies SD plate
(0.67% without amino yeast nitrogen YNB, 2% glucose, 2% agar), 30 DEG C are cultivated 2-4 days until Clone formation.
The identification of recombinant bacterium by extract genome, with Cap gene specific PCVN125-F and PCVN125-R primer into
PCR verifying, the band for amplifying 700bp or so is positive colony, is as transferred to the gene of pUKD-N125/Cap recombinant vector
Engineering bacteria Fim-1ura3 Δ-pUKD-N125/Cap.
Embodiment 3, kluyveromyces marxianus genetic engineering bacterium Fim-1ura3 Δ-pUKD-N125/Cap express Cap egg
The assembling of bletilla virus-like particle
The positive clone of PCR verifying is inoculated in the YP culture medium (1% yeast extract (Yeast equipped with 50ml
Extract), 2% glucose) triangle shake bottle in, 30 DEG C, 220rpm culture 96h after cell is collected by centrifugation, use is high-pressure homogeneous
Method smudge cells, cell pyrolysis liquid carry out polyacrylamide gel protein electrophoresis SDS-PAG (polyacrylamide gel
Electrophoresis, PAGE) and PCV antibody immunoblotting Western Blot detection pig circular ring virus (PCV) Cap protein
In the expression of kluyveromyces.
It is compared with bacterium Fim-1ura3 Δ (the 1st swimming lane) is compareed, genetic engineering bacterium Fim-1ura3 Δ-pUKD-N125/Cap
Full cell pyrolysis liquid and supernatant (the 2nd and the 3rd swimming lane) in the more one article of protein band (such as Fig. 3) of 35-40kD size, the albumen
Band is the Cap protein of pig circular ring virus, and obtains solubility expression.
Further utilize the Cap protein of Western Blot verifying Fim-1ura3 Δ-pUKD-N125/Cap expression.Experiment
Steps are as follows:, will using the Trans-Blot of Bio-Rad company by cell pyrolysis liquid sample after protein electrophorese SDS-PAGE
On protein delivery to nitrocellulose filter (220mA, 100min), then by film containing 5% skimmed milk power TBST buffer
Incubation at room temperature 2h in (20mM Tris-HCl pH 8.0,137mM NaCl, 0.1%Tween 20), anti-PCV antibody Swine serum
Mostly anti-1 ︰ 400 dilution, after 4 degree are incubated overnight, TBST is washed 3 times, 2500 times of 1 ︰ of goat-anti pig HRP ELIAS secondary antibody dilutions, incubation at room temperature
After 1.5h, TBST wash 3 times, the developing solution of pig circular ring virus antibody A, B mixed in equal amounts is added, observes result.
As a result: Fim-1ura3 Δ (the 1st swimming lane) does not have an immunoblotting band, and Fim-1ura3 Δ-pUKD-N125/Cap
The Strip Immunoblot of 25-35kD, almost the same with the theoretical molecular weight 28kD of Cap protein, it was demonstrated that egg in sample (the 2nd swimming lane)
Informal voucher band is the Cap protein (Fig. 4) of circovirus.
Using Electron Microscope images technology, the Cap of Fim-1ura3 Δ-pUKD-N125/Cap expression circovirus is observed
Whether albumen completes self assembly into virus-like particle VLPs, finds Fim-1ura3 Δ-pUKD-N125/ after shaking flask culture 92h
There are the virion of size about 17-20nm or so (such as Fig. 5) by Cap.
Embodiment 4, kluyveromyces marxianus genetic engineering bacterium Fim-1ura3 Δ-pUKD-N125/Cap ferment and take orally
The preparation of vaccine
Kluyveromyces marxianus genetic engineering bacterium Fim-1ura3 Δ-pUKD-N125/Cap in 5 liters of fermentor into
Row high density fermentation is added using synthetic media (containing ingredients such as glucose, ammonium sulfate, magnesium sulfate, potassium dihydrogen phosphates) by stream
Fed-batch fermentation, for cell density OD600 up to 271 (such as Fig. 6), biomass reaches 102g/L (dry cell weight), passes through electrophoresis after the 48h that ferments
The yield of detection and gray scale scanning, Porcine circovirus type 2 Cap reaches 2g/L (such as Fig. 7).
Fermentation collected thallus after 48 hours, was washed with deionized 3 times, after being resuspended with phosphate buffer PBS, using high pressure
The oral virus of oral Porcine Circovirus of protective agent sucrose or the dry system of Direct spraying is added in the method smudge cells of homogeneous
Sample particle vaccines.
Embodiment 5 takes orally the circovirus vaccine immune mouse of kluyveromyces marxianus preparation
SPF grades of Balb/c mouse of 6 week old Healthy female are selected, it is random to be grouped, every group each 7.First group is blank group, is fed
Food PBS (PBS) is not cooked immune；Second group is control group, is injected import company circovirus type II vaccine (0.3mL/ is only), the
Three groups are immune group: feeding Porcine Circovirus provided by the invention takes orally virus-like oral vaccine (KM is oral), and every
100 μ g Cap protein of mouse feeding dosage/time, after head exempts from 1 week, 4 (feeding times: 0 of feeding are repeated by identical approach and dosage
It, 7 days, 14 days, 21 days, 28 days).
Mouse orbit rear vein beard takes blood before feeding, and the blood taken is put into 1h in 37 degree of incubators, then 4 DEG C,
3000rpm is centrifuged 4min, is drawn in serum to new EP with the pipettor of 10 μ l, puts -20 DEG C of refrigerators and saves.The excrement of acquisition claims
Ten times of TBST (sodium azide containing 0.01%) is added in weight, and after vortex oscillation mixes, 14000rpm is centrifuged 10min, takes
Clearly in new EP pipe, marks, put -20 DEG C of refrigerators.
The detection of the content of anti-PCV cap IgG antibody uses Wuhan Ke Qian biological products Co., Ltd pig in mice serum
Circovirus type II ELISA antibody assay kit, detecting step are as follows:
1) antigen coated microplate is taken, every hole is added TBST 200ul, outwells after static 3min, pat dry on blotting paper, washs
Twice.
2) the diluted serum to be checked of 1 ︰ 100, static 45min in 37 degree of incubators is added.
3) sample in hole is discarded, 200ul TBST is added after being patted dry with blotting paper, outwells, pats dry after static 3min, altogether
Meter washing 5 times.
4) the diluted sheep anti-mouse igg ELIAS secondary antibody of 1 ︰ 33000,37 degree of standing 30min are added in every hole.
5) liquid in plate is discarded, is cleaned, method is the same as 3)
6) the substrate solution A and B of 100ul mixed in equal amounts is added, is protected from light colour developing 10min at room temperature.
7) sulfuric acid reaction terminate liquid 50ul is added in every hole, detects the light absorption value under 450nm with microplate reader.
Mouse feeding oral circovirus type II vaccine prepared by the present invention generates antibody for 2 weeks in serum, mice serum produces
Raw anti-PCV Cap IgG antibody (such as Fig. 8).After 6 weeks immune, the PCV Cap IgG antibody in mice serum reaches peak value, average
Titre reaches 10000 or more, and (purifying PCV Cap protein) is vaccinated with import, and antibody level is suitable, but the present invention more premature labor
Raw antibody.In addition, 20 weeks or more can be continued by taking orally the antibody that circovirus epidemic disease II type vaccine provided by the invention generates, and resist
Body titre is maintained at 10000 or more, illustrates that oral circovirus prepared by the present invention has good immune effect.
The detection of anti-PCV Cap IgA antibody content uses sheep anti mouse IgA α chain-HRP (English using ELIAS secondary antibody in excrement
Abcam company, state, article No. ab97235), detection of the operating method with PCV Cap IgG antibody anti-in serum.Referring to Fig. 9, mouse
Oral circovirus vaccine of the present invention (KM is oral) is fed after 28 and 35 days, the IgA antibody in excrement is significantly higher than blank control group
(PBS), illustrate that circovirus oral vaccine prepared by the present invention can significantly improve the mucosal immunity effect of mouse, generate PCV
Cap IgA antibody.
Specific embodiments of the present invention are described in detail above, but it is merely an example, the present invention is simultaneously unlimited
It is formed on particular embodiments described above.To those skilled in the art, any couple of present invention carries out equivalent modifications and
Substitution is also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by equal transformation and
Modification, all should be contained within the scope of the invention.
Claims (12)
1. a kind of recombination kluyveromyces marxianus recombination engineering, which is characterized in that convert Marx's Crewe by recombinant vector
It ties up constructed by yeast nutrition deficient strain, wherein the recombinant vector will be by that will encode Porcine Circovirus capsid protein
Nucleotides inserted to kluyveromyces marxianus expression vector constructed by, wherein kluyveromyces marxianus expression carries
Body includes kluyveromyces marxianus inulinase promoter gene sequence, kluyveromyces marxianus inulinase terminator gene sequence
Column, the nucleotides sequence of the kluyveromyces marxianus expression vector are classified as SEQ ID No.6.
2. recombination kluyveromyces marxianus recombination engineering according to claim 1, which is characterized in that the pig annulus
Virus Type II capsid protein amino acid sequence is as shown in SEQ ID No.1 or 2.
3. recombination kluyveromyces marxianus recombination engineering according to claim 1, which is characterized in that the Marx
The auxotrophic strain of kluyveromyces is CGMCC No.10621.
4. a kind of preparation method of Porcine Circovirus virus-like particle, which is characterized in that step includes:
Kluyveromyces marxianus recombination engineering cultivation and fermentation is recombinated described in claim 1, is assembled into pig annulus in the cell
Virus Type II virus-like particle；
The bacterium of cultivation and fermentation is collected, or the bacterium of collection is crushed.
5. according to the method described in claim 4, it is characterized in that, further including constructing recombination Marx's Crewe dimension ferment as follows
The step of female recombination engineering:
By the nucleotides inserted for encoding Porcine Circovirus capsid protein to kluyveromyces marxianus expression vector, weight is constructed
The recombinant vector is converted kluyveromyces marxianus auxotrophic strain by group carrier, and building obtains the recombination horse
Gram this kluyveromyces recombination engineering.
6. according to the method described in claim 4, it is characterized in that, according to the codon preference of kluyveromyces marxianus,
Guaranteeing that encode Porcine Circovirus capsid protein sequence carries out codon optimization under the same conditions.
7. recombinating kluyveromyces marxianus recombination engineering described in a kind of claim 3 or by claim 4 the method system
The application of standby Porcine Circovirus virus-like particle, which is characterized in that be used to prepare prevention and/or treatment pig circular ring virus
The drug for the disease that II type virus causes.
8. application according to claim 7, which is characterized in that the application, which is selected from as oral vaccine, as feed, to be added
Add agent, any one or a few in drinking water additive.
9. application according to claim 7, which is characterized in that the disease that the Porcine Circovirus virus causes is selected from
Appointing in postweaning multisystemic wasting syndrome, pigskin inflammation nephrotic syndrome, piglet congenital tremors and sow breeding difficulty
Meaning is one or more of.
10. a kind of method for preparing Porcine Circovirus virus-like particle oral vaccine, which is characterized in that step includes:
To be obtained after rupture after collection thallus step in the thallus of cultivation and fermentation collected in claim 4 or claim 4
Virus-like particle prepare Porcine Circovirus virus sample particle vaccines as active constituent or as one of active constituent.
11. according to the method described in claim 10, it is characterized in that, direct broken wall, products therefrom is as mouth after collecting thallus
Take vaccine.
12. a kind of oral drugs for the disease that prevention and/or treatment Porcine Circovirus virus cause, which is characterized in that packet
Include the Porcine Circovirus virus-like particle prepared by claim 4 the method.
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| CN107661496A (en) * | 2017-10-25 | 2018-02-06 | 复旦大学 | A kind of pig parvoviral immune composition and preparation method and application |
| CN108273055A (en) * | 2018-01-03 | 2018-07-13 | 复旦大学 | Canine parvovirus vaccine composition and the preparation method and application thereof |
| CN108864259A (en) * | 2018-07-03 | 2018-11-23 | 复旦大学 | A kind of dissolving-out method of 2 porcine circovirus virus-like particle |
| CN112501186B (en) * | 2020-11-26 | 2023-04-07 | 浙江鼎持生物制品有限公司 | Porcine circovirus 2 d-type CAP protein and application thereof in preparation of subunit vaccine |
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| CN102827259A (en) * | 2012-08-30 | 2012-12-19 | 青岛康地恩药业股份有限公司 | Porcine circovirus type 2 (PCV2) Cap protein gene and application thereof |
| CN105063081A (en) * | 2015-09-07 | 2015-11-18 | 复旦大学 | Recombinant vector used in Kluyveromyces marxianus auxotrophic strain |
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| CN105658660A (en) * | 2013-10-02 | 2016-06-08 | 勃林格殷格翰动物保健公司 | Pcv2 orf2 protein variant and virus like particles composed thereof |
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