CN102352347A - Porcin circovirus type 2 (PCV2) recombinant baculovirus construction method and subunit vaccine preparation method thereof - Google Patents
Porcin circovirus type 2 (PCV2) recombinant baculovirus construction method and subunit vaccine preparation method thereof Download PDFInfo
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Abstract
The invention relates to a porcin circovirus type 2 (PCV2) recombinant autographa californica multicapsid nucleopolyhedrovirus construction method and a subunit vaccine preparation method thereof. The novel recombinant lucerne fork vein noctuid nuclear polyhedrosis virus construction method comprises the steps that: the main surface antigen which expresses the PCV2 is used to prepare the subunit vaccine with better immunity. Specifically, in the carrier expression system of the PCV2 recombinant autographa californica multicapsid nucleopolyhedrovirus, a hepatitis E virus open reading frame (ORF2) gene of the PCV2 is directionally inserted, so the gene efficiently expresses in insect cells and has good immunogenicity, the prepared corresponding subunit vaccine can stimulate pigs to generate the protective immunoreaction against the attack of the strong virus of the PCV2.
Description
Technical field
The present invention relates to a kind of PCV2 recombination to construct and subunit vaccine preparation method thereof, be meant that specifically a kind of PCV2 reorganization Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus makes up and the subunit vaccine preparation method, belongs to biological technical field veterinary biologics industry.
Background technology
(Porcine circovirus PCV) belongs to PCV-II section (Circoviridae) PCV-II and belongs to (Circovirus) pig circular ring virus, and no cyst membrane is the animal virus of the present minimum of finding.Difference according to pathogenic, antigenicity and nucleotide sequence can be divided into PCV-1 and PCV-2 with PCV.PCV-1 the unknown still has pathogenic; PCV-2 has pathogenic, can cause pig performance various clinical symptom.PCV-1 genome total length 1759bp, PCV-2 total length 1768bp or 1767bp, the two sequence homology is less than 80%.Verified at present, PVC has two main reading frames, ORF1 and ORF2.ORF1 is maximum reading frame, transcribes relevant with duplicating of virus.The Cap albumen of ORF2 coding virus can react with the antiserum(antisera) of PCV-2, is main immunogen.
Pig circular ring virus has become the swine disease virus of global wide-scale distribution at present, and it has, and popular scope is wide, each growth phase pig infects characteristics such as serious, that polyinfection is outstanding.PCV2 starts from 2000 at the popular of China; Positive rate that should virus in China also is the trend that rises year by year; The paroxism appearred one time in 2001-2002 in China South China; Serious in the harm of many provinces at present; Caused tremendous loss for China's pig industry, serious threat the development of China's pig industry.
The main kind of PCV-II vaccine of research and development has in the world at present: PCV2 totivirus inactivated vaccine CIRCOVAC; PCV1-PCV2 embedded virus inactivated vaccine SuvaxynPCV2; Baculovirus expression polypeptide PCV2 recombinant vaccine CircoFLEX and PorcilisPCV, wherein SuvaxynPCV2 is first vaccine that has obtained complete license licensed licenser licence in the U.S..
Compare with other expression systems, baculovirus expression system has following superiority: capsid and the genome of baculovirus are big, can hold 10kb left and right sides foreign DNA and not influence and duplicate, and also can carry out the expression of a plurality of exogenous segment simultaneously; Use the late protein promotor; Even the exogenous genes products pair cell is toxic; Do not influence yet expression level (Han Qinggong, Cui Yanhong, Liu Baoguo. the application of insect baculovirus expression system in avian influenza virus research. Jilin agricultural sciences .2010,35 (1): 38-41,44.); Baculovirus has tangible host's boundary, to the vertebrates no pathogenicity, can not in vertebrate cells, duplicate expression and more can not integrate, and therefore numerous biologies is had very high security; Polyhedrin and p10 gene are nonessential fragments, and promotor is all very strong; The seat that foreign gene inserts polyhedron gene causes the latter's disappearance or inactivation, no polyhedrin formation, thus causing recombinant virus inactivation very easily under physical environment, can not cause to people's harm with to the pollution of environment; A series of post-treatment of translating that insect cell can be accomplished foreign protein as eukaryotic cell are modified, and the physics and chemistry of expression product and biological characteristics and natural product similarity are very high; Its virus output in insect cell is high, therefore can efficiently express the swivel base gene protein.In addition, the serum-free culture technology of insect cell is very ripe, and the downstream processing of expression product is convenient.
Utilize traditional porcine kidney cell culture technique production PCV-II inactivated vaccine, virus titer is low, and antigenic content is not enough, and biological safety is poor.Utilize baculovirus expression system that PCV2 Cap albumen is efficiently expressed, can obtain more high-load antigen protein, and downstream processing technology is ripe more, and monitoring of PCV-II popular and prevention and control are all had huge using value.
Summary of the invention
In order to solve and suppress the wide-scale distribution of pig circular ring virus; The purpose of this invention is to provide that a kind of more effective prevention porcine circovirus 2 type infects porcine circovirus 2 type Cap protein subunit vaccine and its structure and the mode of production, also can prepare PCV2 specificity quick diagnosis articles for use.
Indication PCV2 of the present invention reorganization Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus is: Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus (the Autographa californica multicapsid nucleopolyhedrovirus that PCV2 ORF2 nucleotide sequence (SEQ ID No.3) with containing of obtaining behind the molecular biology method after codon optimized and aminoacid sequence (SEQ ID No.4) can efficiently express PCV2 surface antigen (Cap albumen); AcMNPV), the present invention is designated hereinafter simply as PCV2 recombinant baculovirus or recombinant baculovirus.
The present invention mainly implements through following technical scheme:
1. make up the PCV2 recombinant baculovirus that a strain contains SEQ ID No.3 nucleotide sequence and SEQ ID No.4 aminoacid sequence, can efficiently express PCV2 surface antigen (Cap albumen).
2. the construction process of PCV2 recombinant baculovirus of the present invention is:
(1) to design primer according to ORF2 gene order in the document, amplification PCV2HZ strain DNA is a template, is primer with Seq-U (SEQ ID No.1.) and Seq-D (SEQ ID No.2), carries out the segmental pcr amplification of PCV2-ORF2 with TaKaRa rTaq enzyme; Response procedures is: get into circulation behind the 95 sex change 5min, and 94 ℃ of sex change 60s, 56 ℃ of annealing 45s, 72 ℃ are extended 30s, and after 30 circulations, 72 ℃ are fully extended 10min.After getting 5 μ L amplified productions and adding 1 μ L, 6 * Loading Buffer mixing, electrophoresis in 1% sepharose, analysing amplified product.ORF2 gene clone amplified production DNA, consistent through the PCR detected result with expected result, be about 760bp.
(2) with amplified production through being connected to behind the double digestion on the Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus shuttle vectors pPAK/mod, be converted into again in the intestinal bacteria DH10Bac competent cell, extract highly purified plasmid pPAK/CY;
(3) with the Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus genomic dna cotransfection insect cell of pPAK/CY plasmid with disappearance ORF1629 gene, the baculovirus that screening obtains recombinating;
(4), and infect the Sf9-F cell according to plaque cloning process purified virus; (this strain has been delivered No. 3 China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center preservations in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 09 26th, 2011, deposit number is: CGMCC No.5267) to go out the positive recombinant baculovirus Bac/CY strain of high expression level amount through seroimmunity fluorescent method evaluation and screening.
3. subunit vaccine preparation: adopt suspended cell culture technic to cultivate insect cell Sf9-F; Culture environment is 27 ℃ of 120r/min; Cultivate the baculovirus Bac/CY that will insert reorganization when its cell of about 48h is in logarithmic phase; Cultivate through 72h again; The results cells infected, virus titer can reach 10
8.0TCID
50More than/the ml, multigelation 3 times is got supernatant with PBS centrifuge washing method for several times, and above-mentioned recombinant protein is purified and added adjuvant and can produce the ORF2 subunit vaccine to PCV2 through Over emulsfication.
Description of drawings
Fig. 1 pcr amplification product electrophorogram
Fig. 2 shuttle vectors synoptic diagram
Fig. 3 shuttle plasmid synoptic diagram
The serum NAT experiment of Fig. 4 immune mouse
Fig. 5 cell growth curve
Fig. 6 antigen presentation content comparison diagram
Embodiment
To combine specific embodiment to come aid illustration the present invention below, persons skilled in the art can better be understood the present invention in view of the above, but following embodiment does not constitute any restriction to the present invention only as technical show-how.
The present invention clone also optimize PCV2 HZ strain (inventor by the morbidity pig that Zhejiang Province infects 2 type PCV-II separate, identify obtain) the ORF2 sequence, and with its recombinate to shuttle vectors pPAK/mod (available from Clonetech Laboratories, Inc.) on.(available from Clonetech Laboratories, Inc.) (available from Takara Bio, Inc.) enzyme is cut, and makes its disappearance ORF1629 gene with restriction enzyme Bsu36I with Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus genome BacPAK6.The BacPAK6 baculovirus genome cotransfection insect cell Sf9-F strain of extracting pPAK/mod plasmid and above-mentioned disappearance ORF1629 gene is (available from Cambivac; Ltd.); Obtain a strain Bac/CY strain recombinant baculovirus, utilize recombinant baculovirus, infected insect cell Sf9-F; After cultivating 48-72h; Efficiently express porcine circovirus 2 type Cap albumen, the viral protein mixture is through deactivation, purifying; After the emulsification, process subunit vaccine.
One, the PCV2 recombinant baculovirus makes up
1. the clone of porcine circovirus 2 type Zhejiang strain ORF2 gene
In accordance with Invitrogen?
manual extraction of porcine circovirus type 2 HZ strain DNA as template to Seq-U (serial 1:5 '-AGTGCTCGGAGGATCCATGACGTATCCAGGGAGGC-3') and Seq-D (serial 2:5 '-GAGCAGATCTTTAGGTGTTAAGTGGGGGGTCTTTAAG-3') as primers with TaKaRarTaq enzyme PCV2-ORF2 fragments PCR amplification reactions were: 95 for 5min after entering the loop, 94 ℃ denaturation 60s, 56 ℃ annealing 45s, 72 ℃ extension 30s, 30 cycles, 72 ℃ fully extended 10min.After getting 5 μ L amplified productions and adding 1 μ L, 6 * Loading Buffer mixing, electrophoresis in 1% sepharose, analysing amplified product.ORF2 gene clone amplified production DNA, consistent through the PCR detected result with expected result, be about 760bp (having comprised primer length), sequencing fragment is the result show, this fragment is the PCV2-ORF2 fragment.
The PCV2-ORF2 fragment that reclaims after identifying is connected with pGEM-T easy carrier (available from Promega Corporation), and explanation is operated with reference to Promega pGEM-T easy support agent box.Get above-mentioned connection product, under aseptic condition, join in the competent cell, ice bath 30min behind the mixing.Put into 42 ℃ of water-bath heat shock 90s rapidly, after taking out immediately, ice bath 2～3min makes it cooling.Mixture after the heat shock is added 800 μ L be preheated in 37 ℃ the LB nutrient solution (Amp-), 37 ℃, shaking culture 1.5h in the 170r/min shaking table.With the centrifugal 2min of culture 5000r/min room temperature, discard 800 μ L supernatants.Remaining nutrient solution of mixing and cell precipitation are evenly coated on the Amp+ flat board.Be inverted plate and transform bacterium colony in 37 ℃ of incubator overnight incubation pickings.
From the positive bacterium colony of the dull and stereotyped picking recombinant conversion of LB, be inoculated in 3mL and contain in the LB nutrient solution of 100 μ g/mLAmp, 200r/min, 37 ℃ of shaking culture 12h～16h (or spending the night).Asepticly pipette above-mentioned bacterium liquid 1.5mL and place the sterilization centrifuge tube, carry out plasmid with reference to Qiagen Plasmid mini kit test kit and extract.
2. with restriction enzyme Xho I and BamH I PCV2 ORF2 gene is carried out double digestion, 1% agarose gel electrophoresis inspection observations.Reclaim the PCV2 ORF2 fragment after enzyme is cut.
3. the structure of recombinant shuttle plasmid pPAK/mod
(Clonetech Laboratories, Inc.) with restriction enzyme Xho I and BamH I double digestion, the separation of 1% agarose gel electrophoresis is also reclaimed the pPAK8 plasmid after enzyme is cut with the pPAK8 plasmid.Segmental purity and concentration are reclaimed in the ultraviolet spectrophotometer analysis.PCV2 ORF2 fragment after pPAK8 plasmid after enzyme is cut and enzyme are cut adds in the 10 μ L linked systems with 1: 3 mol ratio, and 16 ℃ of connections are spent the night.Transform DH10Bac competent cell (the Clonetech Laboratories of 100 μ L with whole connection products; Inc.); The suspicious conversion bacterium colony of picking; Carry out plasmid with reference to Qiagen Plasmid mini kit test kit and extract, Xho I and BamH I double digestion identify whether contain recombinant plasmid pPAK/CY.
The positive bacterium colony of picking recombinant conversion is inoculated in 6mL and contains in the antibiotic nutrient solution, 200rpm, 37 ℃ of shaking culture 12h～16h (or spending the night).Asepticly pipette above-mentioned bacterium liquid 1.5mL and divide and be installed in the sterilization centrifuge tube, carry out plasmid with reference to Qiagen Plasmid mini kit test kit and extract.Obtain highly purified pPAK plasmid.Plasmid carries out sequencing simultaneously, further confirms to contain the PCV2-ORF2 fragment.
4. recombinant plasmid pPAK/CY and baculovirus ORF1629 missing gene group DNA cotransfection SF9-F cell
The present invention adopts the BacPAK baculovirus expression system to select the baculovirus of a special structure; Behind this viral genome BacPAK6 process Bsu36 I single endonuclease digestion; Obtained lacking the baculovirus genomic dna of ORF1629 gene; Have only when with the pPAK/CY plasmid on the common transfection success of the ORF1629 gene that carries, just can produce recombinant virus particle.
Get grow to logarithmic phase the Sf9-F cell with 3x10
4Cell/cm
2The density inoculum density, the inoculation diameter be the culture dish of 35mm, obtain the Sf9-F monolayer cell.Get 2 Eppendorf tubes; The baculovirus genome proportional mixing that recombinant plasmid pPAK/CY, positive control plasmid, sterilized water, the ORF1629 that purifying is extracted lacks; Negative control and positive control are set; Substitute pPAK/mod as positive control with the pBacPAK8-GUS plasmid, negative control does not contain pPAK/mod plasmid and baculovirus genome.In the system that mixes, add 4 microlitre Bacfectin transfection reagent (Clonetech Laboratories; Inc.) after; Abundant mixing; Dropwise add on the Sf9-F monolayer cell; After 27 degree are hatched 5 hours, inhale and remove supernatant liquid, add the 1.5mLBacPAK perfect medium; Add 50 microlitre X-gluc substrates in the substratum of the upper strata of heliotropism contrast simultaneously, 27 degree were cultivated about 5 days.Positive control should transfer blueness to, proves that transfection is normal.Inspection Sf9-F cytopathy situation, after pathology appearred in the Sf9-F of transfection cell, the Sf9-F cell culture supernatant of results pPAK/CY transfection promptly contained recombinant baculovirus in this supernatant.
5. the purifying of recombinant virus and evaluation
It is first for recombinant baculovirus to take a morsel, and behind 10 times of doubling dilutions, inoculation individual layer Sf9-F cell after 27 degree are hatched 1 hour, siphons away viral liquid, adds the BacPAK perfect medium that contains 1% agar, puts 27 degree incubators, humidification cultivation 4～6 days.By a day observation of cell pathology situation, behind the single plaque of picking, inoculate new Sf9-F cell, treat that the viable cell ratio is reduced to below 20% to gather in the crops, obtain several baculovirus mono-clonals.Virus liquid is measured with the plaque method and is tired, and presses plaque forming unit and calculates.
Get round cover glass several, put in 24 orifice plates, add 100 μ l poly-lysines and encapsulated 1 hour, PBS flushing 3 times adds Sf9-F cell 3x10 in 24 orifice plates
4Individual/cm
2After the overnight incubation; With the baculovirus of infection multiplicity MOI 10.0 inoculations through the clone; 27 degree were cultivated 4 days, and acetone fixed is anti-as one with the PCV2 positive serum; Immunofluorescence technique detects antigen presentation; The positive strain of screening high expression level amount, and called after recombinant baculovirus (Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus Autographa californica multicapsid nucleopolyhedrovirus, AcMNPV) Bac/CY (CGMCCNo.5267).
6. the expression of the specific recombinant proteins of recombinant baculovirus strain and analysis
Antigen capture ELISA quantitative method is analyzed the antigen presentation amount:
With the method purifying PCV2 HZ strain of CsCl density gradient centrifugation, after freund's adjuvant mixes, 2 of immune 4-5 rabbit in age in week.Respectively 28,42, carried out two in 56 days to exempt from, three exempt from and four exempt from.Preceding twice immunity Freund's complete adjuvant, the twice immunity Freund's incomplete adjuvant in back.Finally carried out the serum collection at the 63rd day.
With Protein A gel chromatography column to rabbit anti--PCV2 IgG carries out purifying, the Bradford method is measured antibody purified content.
Utilize coating buffer to rabbit anti--after PCV2 IgG carries out 1: 10000 times of dilution, encapsulate in microtiter plate, 100 μ L/ holes, 4 ℃ of sealings are cultivated and are spent the night.Then should flat board three times with the phosphoric acid buffer PBS washing that contains 1% polysorbas20.Then seal 37 ℃ of sealing function 60min with the 1%BSA confining liquid.Continuing should flat board three times with the phosphoric acid buffer PBS washing that contains 1% polysorbas20.Next step determined antigen sample that will dilute 200 times or 500 times in advance joins in dull and stereotyped first each hole of row, doubling dilution successively then, 37 ℃ of sealing function 60min.With washing lotion washing three times.The proteic monoclonal antibody of anti-PCV2 Cap (available from VMRD, Inc.) carries out joining in each hole after 1: the 500 times of dilution 100 μ L/ holes, 37 ℃ of sealing function 60min with diluent.With washing lotion washing three times.Goat anti-rabbit igg two with the diluent that contains 1%BSA dilution in 1: 10000 alkali phosphatase enzyme mark is anti-, and every hole adds 100 μ L, 37 ℃ of sealing function 60min.With washing lotion washing three times.Add 100 μ L substrates colour developings, 37 ℃ of sealing function 30min then add the NaOH termination reaction of 100 μ L 3M with every hole.Carry out reading through microplate reader then, confirm the antigen diluent terminal point,, calculate antigenic content as benchmark.
The Sf9-F insect cell shakes a bottle suspension culture for 27 ℃ with 120r/min, when concentration reaches 1～2 * 10
6/ mL and viable cell ratio are higher than at 95% o'clock, insert the baculovirus of reorganization with the ratio of infection multiplicity (M.O.I.) 1.0.Through cultivation in 3-4 days, 10000g centrifugal 1min isolated cell fragment and supernatant detected the antigen presentation amount with above-mentioned antigen capture ELISA method.Recombinant virus involved in the present invention is inoculated the insect cell of a small amount of suspension culture, and the target protein expression amount can reach about 150 μ g/ml.
7. recombinant baculovirus Bac/CY immunogenic response research on mouse.
Inoculation recombinant baculovirus Bac/CY cultivates 72h on the Sf9-F insect cell, with its results also-20 ℃/37 ℃ of freeze thawing three times, centrifugal 10 minutes of 3000g gets supernatant.Detect antigenic content with antigen capture ELISA.
8 adult Kunming mouses of picked at random, peritoneal injection 16 a μ g/ 0.1ml; Alternative is got 8 adult mices and is done the contrast experiment at random simultaneously, takes peritoneal injection physiological saline equally.Immunity for the first time after 14 days the equivalent booster immunization once, the 28th day eyeball blood sampling separation of serum.
With mixing 37 ℃ of incubations are inoculated the PK15 cell after 1 hour cell plate with the PCV2 virus of serum and 200 TCID50 in the doubling dilution, add cell growth medium then and cultivate 72h.Remove viral liquid, with 1: 1 ethanol and acetone fixed cell plate 30 minutes; Clean cell plate 3 times with PBS, add 5% skimmed milk sealing treatment 1h; Clean with PBS then, adds the anti-effect 1.5 hours of the anti-PCV2 virus of dilution in 1: 400, clean 3 times with PBS then, added the two anti-lucifuges processing that contain FITC of diluting at 1: 200 1 hour.And then with PBS cleaning 3 times.Under immunofluorescence microscopy, observe at last, judge the NAT of mice serum.
Experimental result: like accompanying drawing 4, experimental result is found in twice immunity back mouse with serum that neutralization is tired and is up to 1: 128, minimum 1: 32.Illustrate that the PCV2 Cap albumen that gives expression in the viral liquid of results can produce very strong cellular immunization by inducing mouse, the PCV2 Cap albumen that the present invention processes has good immunogenicity.
Two, the recombinant baculovirus strain (Bac/CY) that makes up with the present invention, the PCV2 Cap protein product of inoculation insect cell expression is an antigen, the preparation subunit vaccine.
Get the Sf9-F cell that is cultured to logarithmic phase, blue dyeing of tongue phenol and counting cells number, cell quantity should be at 1-2
6Between/the ml, the viable cell ratio should be more than 98%.According to the cell counting result, inoculate required viral load.27 ℃ of 120r/min cultivate, and sampling in per 24 hours once.Antigen capture ELISA method detects the antigen presentation amount.
Research trial is optimized in the expression parameter of insect cell the preparation recombinant protein, with 5 infectious unit kind poison inoculation insect cells, has carried out the development of subunit vaccine as antigen with 5 batches of recombinant proteins of expressing.Recombinant protein culture and immunological adjuvant emulsification are prepared subunit vaccine.
Making method is following:
At first with after the recombinant virus culture results, freeze thawing is carried out cytoclasis three times, and 17000r/min is centrifugal in the HITACHI continuous flow centrifuge, goes out cell debris.Prepare the hypo solution of 1M bromine ethylamine hydrobromide (BEA) solution and 20%, subsequent use after the filtration sterilization.Supernatant liquor after centrifugal is carried out temperature equilibrium in 37 ℃, add final concentration 0.25% phenol red after, slowly in viral supernatant liquor, add BEA, behind the mixing with NaOH adjust pH to 7.5～8.0 of 2M; 37 ℃ of deactivation 18h use the HCl adjust pH to 6.5 of 2M afterwards again, add at last in the hypo solution and superfluous BEA, 37 ℃ hatch 2h after 4 ℃ of preservations subsequent use.
The Sf9-F cell that torrent reactor A P20C cultivates, inoculation recombinant baculovirus strain Bac/CY (CGMCC No.5267), a large amount is expressed PCV2 Cap albumen.
The growth of insect cell is limited by the supply of oxygen and the consumption of nutrition; Often can not reach higher cell density; With torrent formula best cultivation (seeing note), can when producing low-shearing power, make intrasystem dissolved oxygen homogeneous obtain higher cell culture density.After inoculating this cell, detected result shows that the target protein expression amount is higher.
The test operation program: with 5L SF900-II insect cell substratum inject AP20C system torrent bag (available from AmProtein-China, Inc.) in, 27 ℃ of 50r/min pH 7.1DO 60% runnings are spent the night, whether checking system aseptic.Inoculation culture to 7.5
7The Sf9-F cell suspension of/ml is 200ml altogether, 27 ℃ of all the other parameter constants of 80r/min.Whenever once, when treating that cell grows to 36/ml, with 10 infection multiplicities (MOI) inoculation CGMCC No.5267 strain virus seed suspension, culture parameters is constant at a distance from sampling in 6 hours.Whenever once at a distance from sampling in 12 hours, the centrifugal 1min of 10000g, isolated cell and supernatant, cell precipitation is resuspended with equal-volume PBS, antigen presentation amount in detecting supernatant and precipitating.
Test-results: inoculation Sf9-F cell is cultivated through 84 hours in the torrent bag, and cell count reaches 2.98
6Individual/ml (accompanying drawing 5).Inoculation recombinant baculovirus continued was cultivated 6 days, and the antigen presentation detected result shows: at the virus infection initial stage, antigen protein mainly concentrates in the cell precipitation, and antigenic content seldom in the supernatant; In the virus infection later stage, antigen mainly is distributed in the supernatant; The antigen presentation highest level appears at and connects back 120 hours of poison, and total antigenic content can reach 238.17 μ g/ml (accompanying drawing 6).
Below carry out emulsification:
At first carry out the preparation of oil phase and water: white oil and Si Ben-80 prepare according to 94% and 6% proportioning, mixing, and the horizontal high voltage of going forward side by side sterilization is treated when temperature is reduced to 20～30 ℃ subsequent use.Tween-80 is carried out autoclaving, treat when temperature is reduced to 20～30 ℃ subsequent use.Carry out mixing with being diluted to the antigen stock of 50 μ g/ml and the tween-80 of sterilization according to 95% and 5% ratio, and allow tween-80 be dissolved in fully in the antigen liquid, be stored to 2～8 ℃ subsequent use.
Carry out the emulsification packing in 1: 1 ratio after oil phase and water preparation are accomplished, the final content of antigen is about 25 μ g/ml.By " Chinese veterinary drug allusion quotation " for animals (the Chinese veterinary drug allusion quotation council. three Chinese agriculture press of in 2005 version of People's Republic of China's veterinary drug allusion quotation; 2006; The present invention is called for short " Chinese veterinary drug allusion quotation ") require to have carried out the check of work in-process and finished product vaccine, goods carry out clinical trial with being up to the standards.
Test-results: with 5 of PCV2-Cap subunit vaccine 2 multiple doses (4mL/ head) the intramuscular inoculation 25 age in days piglets of manufacturing experimently, clinical observation was not seen no abnormal reaction in 28 days, proved that vaccine product is safe to this animal.The vaccine immunity potency test is to adopt 0.5mL, 1mL, three kinds of dosage immunity of 2mL, 25 age in days piglets, 5 every group, establishes 3 of not vaccination contrast pigs.Sun is all changeed in antibody test in 28 days behind all vaccine immunities; The strong virus attack test-results shows that 1mL and 2mL immune group all obtain 100% protection, and the 0.5mL group obtains 80% (4/5) protection ratio.
Three, the check of subunit vaccine
1. use the PCV2 Cap protein Preparation subunit vaccine of the inventive method expression and carry out safety examination.
With the piglet of 4 20 ages in days of said prepared subunit vaccine finished product inoculation, observed continuously 30 days, the spiritual appetite of piglet is normal during this period, and no abnormality seen changes, and all can detect porcine circovirus 2 type antibody through the ELISA method.4 piglets of strong malicious control group PCV2 all occurs and infect, and 3 death (being mortality ratio 75%) are arranged, and the check of corpse tissue is found the lymphoglandula oedema all takes place, the lungs oedema, and kidney turns to be yellow or pathology such as spotty necrosis is arranged.And 4 no any unusual clinical manifestations of control group.
Prepared inoculation of subunit vaccine finished product and nonvaccinated pregnant sow, the nest litter size is suitable basically, phenomenons such as stillborn foetus all do not occur, confirms that this subunit vaccine is safe.
2. use the PCV2 Cap protein Preparation subunit vaccine of the inventive method expression and carry out efficacy test.
Said prepared subunit vaccine finished product is inoculated the piglet of 4 20 ages in days, and after 14 days, carry out two and exempt from, exempt to carry out strong poison in back 21 days two and attack poison, attack the poison back and tangible clinical symptom all do not occur by a day observation, spiritual appetite is normal.Strong malicious control group, fervescence, appetite stimulator appear after attacking poison, become thin and the speed of growth slow.Cut open the inspection back and find that lymphoglandula, pulmonary edema are arranged, kidney has the point-like pathology.
With said prepared subunit vaccine finished product in the replacement gilt inoculation in preceding 15 days of breeding; Carry out strong poison at 42,60 days respectively then and attack poison; Tangible clinical symptom does not all appear in pregnant sow, and spiritual appetite is normal, and can detect high-caliber porcine circovirus 2 type serum neutralizing antibody.Phenomenons such as stillborn foetus do not appear in the childbirth pig when childbirth.And the contrast pregnant sow of the strong poison of inoculation, phenomenon such as part fetus occurs and heavily absorbs, and abdominal circumference reduces termination of pregnancy, gives birth to the weak tire of stillborn foetus and minority, and fervescence, appetite stimulator then appear in sow, become thin and the speed of growth is slow.These results show that said subunit vaccine can protect pig to avoid the attack of circovurus type 2 virulent strain.
3. the sundry item check is carried out the check of vaccine finished product by " Chinese veterinary drug allusion quotation " predetermined items.
Proterties: appearance milky white milk sap.
Formulation: be water-in-oil-type.Get a cleaning suction pipe, draw a small amount of vaccine and drip in the cold water surface, except that the 1st, all should indiffusion.
Stability: 37 ℃ of left and right sides condition held 21 days or get vaccine 10mL and be loaded in the centrifuge tube, with the centrifugal 15min of 3000r/min, not breakdown of emulsion.
Viscosity: undertaken by " Chinese veterinary drug allusion quotation " regulation, should be no more than 200cP.
Steriling test:, answer asepsis growth by the method check of " Chinese veterinary drug allusion quotation " regulation.
The formaldehyde and the antiseptic mercurials determination of residual amount: undertaken by the beastly officinal regulation of China, residual formaldehyde should be no more than 0.2% formaldehyde solution (containing 40% formaldehyde) amount; The phenol residual quantity should be no more than 0.5%; The antiseptic mercurials residual quantity should be no more than 0.01%.
Four, the recombinant baculovirus strain (Bac/CY) that makes up with the present invention; The PCV2 Cap protein product of inoculation insect cell expression is an antigen; Because this albumen has good immunogenicity, can process porcine circovirus 2 type virus detection kit (like the ELISA detection kit) by prior art for preparing and be used for sick the learning and virological investigation of non-medical diagnosis on disease purpose detection porcine circovirus 2 type viral prevalence.
The PCV2 Cap protein 12 0 μ g/ml and the emulsification of equivalent freund's adjuvant of recombinant baculovirus expression is complete, 2 of immune 5-6 rabbit in age in week.Carry out two 28,42 respectively and exempt from, three exempt from.The immune for the first time Freund's complete adjuvant of using, the twice immunity Freund's incomplete adjuvant in back.Finally carried out the serum collection at the 50th day.
With Protein A gel chromatography column to rabbit anti--PCV2 IgG carries out purifying, the Bradford method is measured antibody purified content.
Antibody purified encapsulates in microtiter plate after carrying out 1: 8000 times of dilution with coating buffer, 100 μ L/ holes, and 4 ℃ of sealings are cultivated and are spent the night.Then should flat board three times with the phosphoric acid buffer PBS washing that contains 1% polysorbas20.Then seal 37 ℃ of sealing function 60min with the 1%BSA confining liquid.Continuing should flat board three times with the phosphoric acid buffer PBS washing that contains 1% polysorbas20.With the dilution proportion determined antigen sample of the PBS that contains 1%BSA by 1: 40; The sample to be checked 100 μ L that dilution is good join in the sample well; Respectively add 100 μ L negative serums and positive serum in its adjacent both sides as contrast, the blank hole is set simultaneously, 37 ℃ of sealing function 60min.With washing lotion washing three times.The proteic monoclonal antibody of anti-PCV2 Cap (available from VMRD, Inc.) carries out joining in each hole after 1: the 500 times of dilution 100 μ L/ holes, 37 ℃ of sealing function 60min with diluent.With washing lotion washing three times.Goat anti-rabbit igg two with the diluent that contains 1%BSA dilution in 1: 10000 alkali phosphatase enzyme mark is anti-, and every hole adds 100 μ L, 37 ℃ of sealing function 60min.With washing lotion washing three times.Add 100 μ L and be diluted to the p-NPP substrate colour developing of working concentration, 37 ℃ of sealing function 30min then add the NaOH termination reaction of 100 μ L 3M with every hole.Read OD405 through microplate reader then, calculate the P/N value.Being judged to be the positive with P/N >=2.1, is suspicious between 2.1 and 1.5, is lower than 1.5 negative.When negative positive control serum is all set up, judge that detected result is effective.
Embodiment
Following examples are for further setting forth the present invention, but these embodiment are not construed as limiting the invention.
The preparation of subunit vaccine
Adopt the ordinary cells culture apparatus to cultivate insect cell Sf9-F with the suspended culture cell technology; Culture environment is 27 ℃ of 120r/min; Cultivate the baculovirus Bac/CY (CGMCCNo.5267 strain) that will insert reorganization when its cell of about 48h is in logarithmic phase; Cultivate through 72h again; The results cells infected, virus titer can reach 10
8.0TCID
50More than/the ml, multigelation 3 times is got supernatant with PBS centrifuge washing method for several times, above-mentioned recombinant protein purify add the conventional vaccine adjuvant and through Over emulsfication PCV2 recombinant baculovirus subunit vaccine.
The Sf9-F cell that torrent reactor A P20C cultivates, the preparation subunit vaccine
The Sf9-F cell inoculation to the torrent bag, was cultivated through 84 hours, and cell count reaches 2.98
6Individual/ml (accompanying drawing 5).Inoculation recombinant baculovirus CGMCC No.5267 strain virus seed suspension continued was cultivated 6 days, and the antigen presentation detected result shows: at the virus infection initial stage, antigen protein mainly concentrates in the cell precipitation, and antigenic content seldom in the supernatant; In the virus infection later stage, antigen mainly is distributed in the supernatant; The antigen presentation highest level appears at and connects back 120 hours of poison, and total antigenic content can reach 238.17 μ g/ml (accompanying drawing 6).Other processes are with embodiment 1.
Embodiment 3
The preparation of PCV2 recombinant baculovirus subunit vaccine
Can adopt rolling bottle culturing cell technology to cultivate insect cell Sf9-F, preparation PCV2 recombinant baculovirus subunit vaccine.Other processes are with embodiment 1.
Embodiment 4
Vaccine test
Proterties: appearance milky white milk sap.
Formulation: be water-in-oil-type.Get a cleaning suction pipe, draw a small amount of vaccine and drip in the cold water surface, except that the 1st, all indiffusion.
Stability: 37 ℃ of left and right sides condition held 21 days or get vaccine 10mL and be loaded in the centrifuge tube, with the centrifugal 15min of 3000r/min, not breakdown of emulsion.
Viscosity: undertaken by " Chinese veterinary drug allusion quotation " regulation, be no more than 200cP.
Steriling test: by the method check of " Chinese veterinary drug allusion quotation " regulation, all asepsis growth.
Safety verification: with 5 of PCV2-Cap subunit vaccine 2 multiple doses (4mL/ head) intramuscular inoculation 25 age in days piglets, clinical observation was not seen no abnormal reaction in 28 days, proved that vaccine product is safe to this animal.
Efficacy test: the vaccine immunity potency test is to adopt 5 of three kinds of dosage immunity of 2mL, 25 age in days piglets, establishes 3 of not vaccination contrast pigs.Behind all vaccine immunities antibody test in 28 days all change the sun (5/5), for immune swine (3/3) still negative; The strong virus attack test, three all collunarium inoculation PCV2-HZ strains after the vaccine immunity, every pig inoculation 10
6.5TCID
50The result shows that 1mL and 2mL immune group all obtain (5/5) 100% protection, and control group pig (3/3) is fervescence, appetite stimulator, spiritual depressed all, dissects and finds that oedema all appears in lung's lymphoglandula, and kidney has spotty necrosis.
The formaldehyde and the antiseptic mercurials determination of residual amount: undertaken by the beastly officinal regulation of China, residual formaldehyde is no more than 0.2% formaldehyde solution (containing 40% formaldehyde) amount; The phenol residual quantity is no more than 0.5%; The antiseptic mercurials residual quantity is no more than 0.01%.
Annotate: torrent formula bio-reactor is a kind of novel cell culture device, adopts efficient " torrent formula oxygen supply mechanism " (WO2007/142664 and WO2006/138143) to pass oxygen.Produce torrent through mechanical oscillation, wash away the oxygen molecule layer that is gathered in the culture bag internal surface repeatedly, oxygen molecule is dissolved in the nutrient solution rapidly, and constantly mix, fully transmit, each cell can both obtain competent oxygen, keeps the metabolism of cell normal growth.The bubble that this torrent formula oxygen supply mechanism has avoided traditional bubbling style oxygen supply to produce break pair cell damage that forms and the shearing force that strong mechanical stirring pair cell forms increase substantially cell culture density, and viral yield obviously increases.
Claims (7)
1. a PCV2 reorganization Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus is characterized in that this PCV2 reorganization Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus contains SEQ ID No.3 nucleotide sequence and SEQ ID No.4 aminoacid sequence, can efficiently express PCV2Cap albumen; This Bac/CY strain virus has been delivered No. 3 China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center preservations in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 09 26th, 2011, deposit number is: CGMCC No.5267.
2. a kind of according to claim 1 PCV2 reorganization Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus makes up, and the structure key step of the Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus that it is characterized in that recombinating comprises:
(1) to design primer according to ORF2 gene order in the document, amplification PCV2 HZ strain DNA is a template, is primer with Seq-U (SEQ ID No.1.) and Seq-D (SEQ ID No.2), carries out the segmental pcr amplification of PCV2-ORF2;
(2) amplified production is connected on the cloning vector pGEM-T identifies; Positive plasmid is converted in the intestinal bacteria DH10Bac competent cell through being connected to behind the double digestion on the Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus shuttle vectors pPAK/mod again, extracts highly purified plasmid pPAK/CY;
(3) with the Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus genomic dna cotransfection insect cell of pPAK/CY plasmid with disappearance ORF1629 gene, the Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus that screening obtains recombinating;
(4), and infect the Sf9-F cell according to plaque cloning process purified virus; Go out the positive Bac/CY strain (CGMCC No.5267) of high expression level amount through seroimmunity fluorescent method evaluation and screening.
3. the subunit vaccine that contains the described a kind of PCV2 reorganization Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus of claim 1, the main component that it is characterized in that this subunit vaccine are by the expressed porcine circovirus 2 type Cap albumen of Bac/CY strain PCV2 reorganization Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus (CGMCC No.5267).
4. the preparation method who prepares claim 3 PCV2 reorganization Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus subunit vaccine; It is characterized in that; Adopt the suspended culture cell technology to cultivate insect cell Sf9-F; Culture environment is 27 ℃ of 120r/min; Cultivate and to insert PCV2 reorganization Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus CGMCCNo.5267 when its cell of about 48h is in logarithmic phase; Cultivate through 72h again, the results cells infected, virus titer can reach 10
8.0TCID
50More than/the ml, multigelation 3 times is got supernatant with PBS centrifuge washing method for several times, above-mentioned recombinant protein purify add the conventional vaccine adjuvant and through Over emulsfication PCV2 reorganization Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus subunit vaccine.
5. a kind of PCV2 reorganization Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus subunit vaccine preparation method as claimed in claim 4; It is characterized in that; Can adopt suspended cell culture technic to cultivate insect cell Sf9-F to duplicate PCV2 reorganization Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus CGMCC No.5267, preparation PCV2 reorganization Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus subunit vaccine.
6. like a kind of PCV2 reorganization Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus subunit vaccine preparation method as described in the claim 4; It is characterized in that; Can adopt torrent formula bioreactor culture insect cell Sf9-F to duplicate PCV2 reorganization Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus CGMCC No.5267, cultivate insect cell to 1～4x10
6/ ml, concussion speed is 80r/min, the dissolved oxygen saturation ratio is 60%, cultivates 4～6 days and results preparation PCV2 reorganization Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus subunit vaccine.
7. the described a kind of PCV2 reorganization Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus of right 1 is characterized in that its PCV2 reorganization Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus CGMCC No.5267 expressed products can use preparation PCV2 antibody diagnosing reagent kit.
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| CN102839195A (en) * | 2012-07-18 | 2012-12-26 | 华南农业大学 | Method for expression of PCV 2 Cap protein by pFast Bac Dual baculovirus |
| CN103122352A (en) * | 2012-09-27 | 2013-05-29 | 华中农业大学 | Porcine circovirus II-type recombinant baculovirus as well as preparation method and application thereof |
| CN103122352B (en) * | 2012-09-27 | 2015-02-11 | 华中农业大学 | Porcine circovirus II-type recombinant baculovirus as well as preparation method and application thereof |
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| CN103173470A (en) * | 2013-03-11 | 2013-06-26 | 斯澳生物科技（苏州）有限公司 | Preparation of PCV2 ORF2 capsid protein virus-like particles derived from escherichia coli |
| CN104984335A (en) * | 2015-07-14 | 2015-10-21 | 浙江诺倍威生物技术有限公司 | Construction of PCV (Porcine Circovirus) double subtype ORF2 co-expression vector and vaccine preparation |
| CN106566814A (en) * | 2016-11-14 | 2017-04-19 | 中国动物卫生与流行病学中心 | Construction and application of porcine circovirus type-2 three-Cap gene linked and recombined baculovirus vaccine |
| CN108085293A (en) * | 2018-01-10 | 2018-05-29 | 杭州洪晟生物技术股份有限公司 | Culture medium for preparing pig annulus subunit vaccine for Hi5 cells and usage thereof |
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