CN103820398A - Mink enteritis virus recombinant subunit vaccine and preparation method thereof - Google Patents
Mink enteritis virus recombinant subunit vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a mink enteritis virus recombinant subunit vaccine and a preparation method thereof. The strain that produces the vaccine is recombinant autographa California nuclear polyhedrosis virus (CGMCC No. 8051), the expression vector of the strain is expressed through secretion, comprises six histidine labels, and is prone to be used in protein detection and analysis; mink enteritis virus recombinant subunit vaccine produced by the strain does not contain any viral genome and thus does not have any animal safety hazard or potential infectivity; and moreover the vaccine has a strong immunity specificity, can activate the immunized animals to generate enough immunity antibodies, and generates a long-term protection. An MEV virus-like particle is produced according to the preparation method, the titer of virus liquid generated by a bio-reactor with a volume of 1000 L can reach a hemagglutination titer of 1:4096 to 1:32768; the expression quantity of granule protein is 65 mg/L, and reach liter of virus liquid semi-finished product can produce 3000 to 8000 doses of standard mink vaccine. The recombinant subunit vaccine is a novel vaccine having a prominently better effect than that of conventional inactivated tissue vaccines.
Description
Technical field
The present invention relates to a kind of mink enteritis virus recombinant subunit vaccine and preparation method thereof.This vaccine is a kind of recombinant subunit vaccine that insect baculovirus expression system is produced of applying, and has the vaccine of anti-mink viral enteritis effect, belongs to veterinary biologics and biological technical field.
Background technology
Mink viral enteritis (Mink Viral Enteritis, MVE) be by mink enteritis virus (Mink Enteritis Virus, MEV) one that causes is acute, height contagious infection disease, and this disease, take diarrhoea as principal character, has higher M & M.
MEV is under the jurisdiction of Parvoviridae (Parvoviridae) in classification, and parvovirus belongs to (Parvovirus).MEV infects after mink, can cause intestinal mucosa inflammation, the necrosis of mink, then cause the violent diarrhoea of mink, the morbidity urgency of this disease, the high (Yan Xijun of lethality rate, bavin is beautiful, Wu Wei, etc. mink enteritis parvovirus isolation identification. herding and animal doctor .2007,39 (3): 52-53.).MEV can infect the mink of each age group, extremely strong to the infectivity of young ermine.1949, Schofield reported this disease the earliest in Canada.Wills in nineteen fifty-two isolation identification this cause of disease, and be this viral nomenclature.In the world, other Yang Diao state breaks out this disease successively subsequently, the states such as the U.S., Denmark, France, Britain, Japan in succession have the report that this disease breaks out (Liao Guoan. mink viral enteritis. Chinese livestock and poultry transmissible disease .1990,2:53-56.).This disease is one of viral infectious that universally acknowledged harm mink animal husbandry is larger, provisions ermine industry has been brought huge financial loss (Yan Xijun, Zhang Hailing, bavin is beautiful, Deng. clone and the sequential analysis of mink enteritis parvovirus structural protein VP2 gene. special product research .2008,47 (4): 1-6.).The same with other virus diseases, the control of mink viral enteritis is also mainly to put prevention first.Mainly inactivated vaccine for the vaccine of mink viral enteritis at present.
The conventional vaccine of control MEV is mainly inactivated vaccine at present.Conventional is MEV aluminium glue formalin-inactivated seedling.Get cat kidney primary cell (CRFK) or feline kidney cells (F81) is used tryptic digestion.Nutrient solution is the milk-protein containing 10% bovine serum.The advantage of this vaccine is that security is better, and side effect is less, but the MEV virus that the formaldehyde using because of its deactivation has after certain destruction and deactivation antigen protein can not be at mink proliferation in vivo, and therefore duration of immunity is short, needs repeatedly immunity; Often select virulent strain for the immune effect having reached, therefore need strict deactivation operation, if inactivating efficacy is undesirable, have loose malicious risk; Inactivated vaccine can not be induced and be produced local I gA, mucosal immunity defence capability a little less than.
Genetic engineering subunit vaccine is to utilize genetic engineering technique; structure is connected with the expression vector of protective antigen encoding gene; in cell or thalline, express target protein; vaccine (the J A Lopez de Turisod making through purification processing antigen protein; S E Corte; C Martinez, et al.Recombinant Vaccine for Canine Parvovirus in Dogs.Journal of Virology.1992,66 (5): 2748-2753.).
A major criterion of desirable MEV vaccine is exactly that cost is low.The recombinant VP 2 that uses baculovirus expression system to obtain high yield at insect cell line provides a kind of new selection for MEV vaccine.As everyone knows, applying biological reactor large scale culturing insect cell line is both difficult and expensive, only has the correlation technique of a solution difficult problem, as culture medium cost, high-density high expression level, Bioreactor scaleup technology, can make the commercialization of this vaccine become possibility.
MEV particle is the axisymmetric icosahedral structure of virus of diameter 20～24nm, and without cyst membrane, particle contains sub-thread minus-strand dna.The genome of MEV is approximately 5kb, and molecular weight is about 1.5～2.0 × 10
6kD(T Kariatsumari, M Horiuchl, E Hama, et al.Construction and nucleotide sequence analysis of an infections DNA clone of the autonomous pavovirus, mink enteritis virus.Journal of General Virology, 1991,72:867-875.).In its genome, have two main open reading frames, they are coding structure albumen (VP1 and VP2) and Nonstructural Protein (NSl and NS2) respectively.MEV is without cyst membrane, and or not containing carbohydrate and lipid, sturdy construction is not tight.MEV is to temperature-insensitive, and 65 ℃ are incubated 30min, and virus still can infect mink.
Virus-like particle (VLPs) is the half-finished functional ingredient of subunit vaccine, and the one-piece construction of its imitation virus particle, but containing being infectious genetic material.In fact, many virus-like particles lack the genome of DNA or RNA viruses completely, only have and deactivation, conception that the contained viral capsid proteins of attenuated virus vaccine is close.Under normal circumstances, the virus particle structure of only imitating compared with the virus-like particle of low dosage, as antigen immune host, is just enough to cause the similar aversion response producing with viral vaccine.Except they can excite the cell-mediated immune response of B, be also proved to be the antigenic substance of very effective stimulation CD4 proliferative response and cytotoxic T lymphocyte (CTL) response.
Baculovirus is the DNA virus that has cyst membrane, and specific infection insect utilizes efficient polyhedron or P10 promotor, is widely used in relying on insect cell expression albumen.The advantage of baculovirus expression system comprises high level expression, be easy to express recombinant protein, can express the foreign gene of large fragment, correct posttranslational modification, and (the T A Kost such as good biological safety, J P Condreay, D L Jarvis.Baculovirus as versatile vectors for protein expression in insect and mammalian cells.Nature biotechnology.2005,23:567-575).But, the still polyhedrin expressed far below wild-type virus of the expression level of foreign protein under normal circumstances.By insect cell genome being carried out to the effort such as the improvement on molecular level, the expression amount of foreign gene will be conducive to significantly improve.
Two kinds of structural protein: VPl of MEV main code and VP2, VP2 is positioned at the C end of VPl, and N terminal amino acid sequence and the VPl PROTEIN C terminal amino acid sequence of VP2 albumen are overlapped, and the two ends at same codon (Parrish CR, 1999).VP1 albumen comprises 727 amino acid, wherein contains 584 amino acid whose VP2 albumen and 143 amino acid whose distinct zones.
The tertiary structure of the capsid protein of parvovirus is similar, and its virus particle surface is assembled by 60 structural protein subunits, and wherein VPl accounts for 5～6; VP2 accounts for 54～55, is the major structural protein that forms capsid protein.Confirm after deliberation, on parvovirus, have three epitope district: 1.Loopl and Loop3 near field at least; Two-fold sink and three folding projection area between; 3. close on three folding central sections.These epitope districts can induce body to produce neutralizing antibody, and the amino acid in region changes the antibody that makes parvovirus can escape the generation of body preimmunization.
Summary of the invention
The object of the present invention is to provide a kind of new mink enteritis virus vaccine, for the production of safety, efficient mink viral enteritis (MVE) vaccine.Provide a kind of and can substitute traditional selection approach of organizing vaccine product, can avoid organizing the many unfavorable aspect of vaccine product.This novel vaccine contains restructuring MEV capsid protein, and does not contain MEV viral genome composition, thus the risk of having avoided viral diffusion and genome to discharge; First be to build a strain can express the structure one strain recombinant virus of MEV capsid protein as the production strain of this subunit vaccine for this reason.
Technological line is:
The technical scheme the present invention relates to comprises:
1. a kind of recombinant baculovirus involved in the present invention, its transfer vector comprises: the SnaB I restriction enzyme site after pVL1393 transfer vector Poly A inserts HS4 sequence, inserts melittin signal peptide before multiple clone site; In multiple clone site, insert the MEV structural protein VP2 gene of a copy; C at foreign gene holds the sequence label that contains 6 Histidines, this recombinant baculovirus called after restructuring clover silver powder californica nuclear polyhedrosis virus rBac-HBM-HS4-VP2.
2. restructuring clover silver powder californica nuclear polyhedrosis virus CGMCC No.8051 involved in the present invention can infected insect cell be, and efficient secretory expression recombinant protein VP2, above-mentioned recombinant VP 2 protein is equal to mink enteritis virus VP2 protein and can be further self-assembled into mink enteritis virus capsid protein in function.
3. above-described mink enteritis virus capsid protein is higher than the expression amount of conventional commercialization pVL1393 serial carrier in expressed in insect cells amount, and be secreted into outside born of the same parents, stable existence is in the supernatant liquor of expressing, and this culture supernatant does not need purified can use.
4. this restructuring mink enteritis virus capsid protein is the same with natural mink enteritis virus with on immunogenicity in antigenicity, and is a kind of albumen that can induce mink to produce the immunne response infecting for mink enteritis virus.
5. involved in the present invention for preventing the recombinant subunit vaccine of mink viral enteritis to comprise above-described restructuring mink enteritis virus capsid protein and expressing supernatant liquor.
6., when the recombinant subunit vaccine of prevention mink enteritis disease of the present invention is intramuscular injection formulation, contain adjuvant.
7. the large-scale producing method of a kind of mink enteritis virus subunit vaccine of the present invention, comprises the steps:
(1) utilize the full suspension culture Sf9 cell of triangle shaking flask serum-free to prepare seed cell;
(2) utilize triangle shaking flask serum-free entirely to suspend and prepare the restructuring clover silver powder californica nuclear polyhedrosis virus CGMCC No.8051 that expresses RHDV subunit protein VP60;
(3) seed cell is directly inoculated into the full suspension culture Sf9 cell of stirring type bioreactor, the cultivation scale of bio-reactor is amplified to 1 ton of cell tank, and by feeding culture technique, cell density is cultured to 2.0 × 10
6cells/ml～4.0 × 10
6cells/ml;
(4), take infection multiplicity (multiplicity of infection, MOI) as 0.5～5.0 virus inoculation CGMCC No.8051, cultivate 72～140h;
(5) gather in the crops above-mentioned nutrient solution supernatant, through deactivation, add aluminium hydroxide gel mix after make vaccine.
8. above-described preparation method, be that whole culturing process comprise cell cryopreservation and recovery, be unicellular full suspension, serum-free, protein free culture environment, also use the full suspension culture Sf9 cell of mechanical agitation type bio-reactor, and amplification culture scale reaches 1 ton of cell tank.
The present invention describes in detail
One, the structure of recombinant virus and characteristic thereof
1. the acquisition of mink enteritis virus (MEV) structural protein VP2 gene
GENBANK announce from the isolated strain MEV virus VP 2 protein sequence http://www.ncbi.nlm.nih.gov/nuccore/340764648 of Jilin Province, China.In addition, the VP2 protein sequence (sequence 1) of the mink enteritis virus production of vaccine strain RC1 strain that our company is had checks order, after both comparisons, find that there is 99% homology, take the latter's gene order as template, the synthetic VP2 gene upstream and downstream primer of design (synthetic by the raw work in Shanghai) VP2-F(sequence 2)/VP2-R(sequence 3).Then the method amplification VP2 gene (see figure 1) that uses RT-PCR, enters (pMD19-VP60) in pMD19-T carrier by this gene clone, is kept in E.coli with the form of plasmid.
2. build the transfer vector that contains insulator cis-acting elements and melittin signal peptide
Insulator is section of DNA sequence, can be by target gene and its controlling element isolation around, express (J A Wallace to guarantee it, G Felsenfeld, We gather together:insulators and genome organization.Current Opinion in Genetics and Development.2007 (17) 400 – 407).Two clasp Y insulation Y are determined: strengthen-blocking-up insulator and partition isolator.Strengthen-blocking-up insulator can be blocked end enhancement and prevent the genetic expression of unsuitable activation between enhanser and promotor.Blocking-up insulator can stop heterochromatic expansion and protection transcriptional activity region.Many at yeast, fruit bat, the insulator in the mankind and other eukaryotic gene group is out identified.They are positioned near region gene conventionally, and conventionally comprise the responsive site of DNA enzyme-I.Chicken β-myosin insulator (HS4) is about 1.2kb, is the vertebrate isolator of modal research.It has enhancing-blocking-up insulator and cuts off insulator dual-use function, can also block effect (the S Yao of silencer simultaneously, C S Osborne, R R Bharadwaj, P Pasceri, T Sukonnik, D Pannell, F Recillas-Targa, A G West, J Ellis.Retrovirus silencer blocking by the cHS4 insulator is CTCF independent.Nucleic Acid Research.2003 (31) 5317 – 5323)
The previous work discovery in our laboratory, HS4 insulator is placed in the downstream of the destination gene expression box of rhabdovirus expression vector, can significantly improve the expression amount of rabbit hemorrhagic fever virus VP2.Further, we attempt HS4 insulator to be placed in the downstream of MEV major structural protein (VP2) expression cassette, find that the expression amount of VP2 has significantly and improves at 72h, 96h, and wherein the expression amount of 72h is approximately 3 times of control group.Result hint VP2 protein expression is subject to the impact of heterochromatin effect and reduces its expression level, and HS4 can reduce the level of this impact.
The experiment in our early stage is found, Sf9 cell expressing VP2 albumen, after assembling assembly virus-like particle, can not be secreted in born of the same parents' external environment substantially, that is to say in the situation that there is no signal peptide, the virus-like particle that intracellular expression forms lacks a set of mechanism of secretion.Only have by the cell virus suspension of results is carried out to multigelation, can make a large amount of virus-like particle in born of the same parents discharge.So we consider to promote by increase signal peptide on VP2 the secretion of virus-like particle.
The signal peptide of most mammalian genes often can not be identified by insect cell, if by the full gene cloning that comprises signal peptide to recombinant baculovirus, usually can not realize secretion type expression.Melittin signal (Honeybee melittin signal peptide) can mediate foreign protein and be present in culture supernatant by the form of secreting, expressing, and can make the expression amount of foreign protein increase by 500.So we improve the secretion ratio of virus-like particle by the method for the upstream interpolation bee venom signal peptide in polyhedron promotor.
By insulator and melittin signal are inserted in recombinant virus structure common carrier pVL1393, to facilitate foreign gene to be incorporated into fast in carrier, be connected with two kinds of effect original papers, specifically see Fig. 3.
Concrete steps are as follows:
(1) vector construction that contains melittin signal peptide:
Melittin signal peptide sequence is according to reference (Van der Geld Y M, Smo ok M L, H uit ema M G, et al.Expr essio n o f recombinant pro teinase 3, the autoant-I gen in Wegener ' s g ranulomato sis, in insect cells.J Immuno l Methods, 2002,264 (1/2): 195-205).
DNA fragmentation (the sequence 4 that synthetic contains melittin signal peptide sequence (HBM), this DNA fragmentation comprises melittin signal peptide, 6 histidine-tagged (6 × His Tag), multiple clone site etc. are completed by Shanghai Sheng Gong biotechnology company limited), synthetic DNA fragmentation has been cloned in pMD19-T carrier (precious biotechnology (Shanghai) Co., Ltd.) (pMD19-HBM), be kept in E.coli with the form of plasmid, then this fragment of pcr amplification, use BamH I, Pst I double digestion pVL1393 carrier and PCR product, then the PCR product of double digestion is connected with carrier, the synthetic DNA fragmentation that contains melittin signal peptide has just been replaced the corresponding one section of sequence on pVL1393.
(2) insert HS4 insulator gene order at the carrier that contains melittin signal peptide
Announce the HS4 insulator sequence (sequence 5 of sequence (the http://www.ncbi.nlm.nih.gov/nuccore/U78775.2 number of logging in: U78775.2) synthetic total length according to GeneBank, by Shanghai, Sheng Gong biotechnology company limited completes) DNA fragmentation, see Fig. 2.Synthetic DNA fragmentation has been cloned in pMD19-T carrier (precious biotechnology (Shanghai) Co., Ltd.) (pMD19-HBM), be kept in E.coli with the form of plasmid, by this fragment of pcr amplification, the method then it being connected by single endonuclease digestion is inserted into the above-mentioned pVL-HBM that contains melittin signal peptide building and is positioned at the SnaB I restriction enzyme site in expression casette downstream.New carrier called after pVL-HBM-HS4.
3. build the baculovirus expression plasmid that contains VP2 gene and prepare recombinant insect cell baculovirus
Insect cell expression plasmid pVL-HBM-HS4 is after 37 ℃ of digested overnight hydrolysis of restriction enzyme EcoRI/Kpn I, with gel electrophoresis and Filter column extracting and purifying.Under the effect of T4 DNA ligase, be connected and spend the night in 16 ℃ with the VP2 gene fragment that uses EcoR I/Kpn I enzyme to cut the recovery of pMD19-VP2 plasmid, then adopt heat-shocked method that connection product is transduceed into E.coliDH5 α competent cell.
Concrete operations are as follows: 50 μ lDH5 α competent cells are moved in a little plastic centrifuge tube, add the ligation liquid of 5 μ l, after mixing, tubule is placed in to 30min on ice, proceed to heat-shocked 90s in 42 ℃ of water-baths, put back to rapidly on ice, after 2min, add 950 μ lLB substratum, cultivate 1h for 37 ℃.Get 1mL bacterium liquid and be condensed into 100 μ l to coat LB solid medium (containing 100 μ g/ml Ampicillin Trihydrates) upper, cultivate 16h for 37 ℃, use VP2-F(sequence 2)/VP2-R(sequence 3) primer carries out bacterium colony PCR, identifies the positive colony growing.
Concrete operations: single colony inoculation that picking grows in 2mlLB nutrient solution (containing 100 μ g/ml Ampicillin Trihydrates), 37 ℃, rotating speed 250r/min, 2h is cultivated in concussion, then get 1 μ l bacterium liquid as template PCR, then agarose gel electrophoresis, identifies positive colony.Then send order-checking.Preserve the correct clone of order-checking, obtain transfer vector pVL-HBM-HS4-VP2, see Fig. 3.By pVL-HBM-HS4-VP2 carrier and BD BaculoGold Linearized Baculovirus DNA(U.S. TRANSLAB product) coinfection Sf9 cell acquisition recombinant baculovirus.
In the tissue culture ware of 6cm, inoculate 2 × 10
6individual Sf9 cell, cell degree of converging is 50%～70%.After cell attachment, (approximately 15min) removes substratum from culture dish, then adds the transfection buffer A of 1mL.The pVL-HBM-HS4-VP2 plasmid of the BD BaculoGold Linearized Baculovirus DNA of 0.5 μ g and 2 μ g is mixed and places 5min in aseptic EP pipe, and then add 1mL transfection reagent B.Remix is even.Then the transfection reagent B/DNA mixture of drawing 1mL being added drop-wise in tissue culture ware drop by drop.Then culture dish is hatched to 4h at 27 ℃.After 4h, remove transfection composite, use cell culture medium to wash three times, then add 3mL fresh culture to cultivate 4～5 days at 27 ℃.Use sealed membrane that culture dish is sealed, in incubator, put into hygenic towelette simultaneously.After 4 days, gather in the crops supernatant, obtain recombinant virus.Then test and obtain mono-clonal baculovirus by plaque.
Concrete operations are as follows:
1) in the tissue culture ware of 6mm, inoculate 2～2.5 × 10
6individual Sf9 cell, places 5min under room temperature.Gradient dilution (10
-4～10
-7) the cotransfection virus supernatant of results.In each culture dish, add the virus of 1mL dilution.At room temperature place 1h, every 15min that crosses rocks, allows virus fully infect.
2) use sterilized water to configure 2% lower concentration agarose simultaneously, use microwave heating to 60 ℃ fully to melt.Then put into 42 ℃ of water-baths and be incubated, then add isopyknic Grace substratum (GIBCO, 2 times concentrated), mix.
3) siphon away supernatant in culture dish, use careful 1% agarose at cell surface covering 4mL of rifle head, siphon away all bubbles simultaneously.After about 10～15min, agarose solidifies.
4) then culture dish 27 ℃ of cultivations in moistening environment just can be seen to plaque for 7 days.On culture dish, draw a some mark plaque, then use a rifle choicest peek plaque, put into 700 μ lSF-SFM substratum (Wo Mei Bioisystech Co., Ltd of Suzhou City) and rock and spend the night with 4 ℃.Obtain viral suspension (P0 generation) ,-80 ℃ of freezing preservations are as seed bank.
To gather in the crops recombinant baculovirus called after restructuring clover silver powder californica nuclear polyhedrosis virus rBac-HBM-HS4-VP2, this virus was 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 08 02nd, 2013, deposit number is: CGMCC No.8051.
4. the mensuration of recombinant baculovirus titre
Adopt plaque counting process, prepare 20ml cell suspension, adjusting cell density is 5 × 10
5cells/ml, every hole paving 2ml cell in 6 orifice plates.After the liquefaction of low melting point agar, low melting point agar, insect substratum (Wo Mei Bioisystech Co., Ltd of Suzhou City product) are put into super clean bench at once.Draw 13ml4% low melting point agar and add and be equipped with in 39ml substratum (1.3 times that concentration is conventional medium) reagent bottle, gentleness mixes.After being ready to, be put in 40 ℃ of water-baths.With insect substratum by 7 concentration gradients (10 of viral dilution
-1～10
-7), method: get 0.5ml virus liquid and put into 4.5ml substratum (with the aseptic centrifuge tube of 10ml), dilution is 10
-1, the rest may be inferred for all the other.P1 surveys 10 for virus
-4, 10
-5, 10
-wthree titres, P2 surveys 10 for virus
-5, 10
-6, 10
-7, P3 surveys 10 for virus
-6, 10
-7, 10
-8three titres, 4 holes of each titre.After cell attachment, the substratum in 6 orifice plates is removed, added 1ml viral dilution liquid at once.In 27 ℃ of incubators, adsorb after 1h, by virus liquid sucking-off, the plate culture medium (low melting-point agarose substratum) that is placed on 40 ℃ of water-baths is put into super clean bench, the plate culture medium of 2ml is put at once.Orifice plate is wrapped with sealed membrane, and be put in valve bag, in 27 ℃ of incubators, cultivate.After transfection 7th～10 days time, the neutral red solution of preparation 1mg/ml (preparing with sterile purified water).Every hole adds the neutral red solution of the 1mg/ml of 0.5ml, incubated at room 1～2h.The approximate transparent point of observing recombinant virus formation is plaque.Counting statistics is observed viral plaque forming unit (plaque forming units, PFU).Utilize formula " titre=plaque number × extension rate × (1/ inoculation volume ml/ hole) ", calculate virus titer, optimization range is that on 6 orifice plates, 3～20 spots are calculated in each hole.
5. recombinant baculovirus infects detection and the evaluation of Sf9 cells produce MEV virus-like particle
(1) recombinant baculovirus infects Sf9 cells produce MEV virus-like particle
200ml Sf9 cell suspension is incubated in 1L screw socket triangle shaking flask, and cell culture medium is Wo Mei Bioisystech Co., Ltd of serum free medium SF-SFM(Suzhou City), shaking speed is 110r/min, homo(io)thermism is in 27 ℃.When cell density reaches 2 × 10
6when cells/ml, with baculovirus, rBac-HBM-HS4-VP2 inoculates with MOI=0.5.Cultivate after 96h, collect supernatant, 4 ℃ of centrifugal 10min of 3000r/min, collect supernatant liquor ,-20 ℃ of freezing preservations.Because virus-like particle in supernatant has protein characteristic, can be detected by sds gel electrophoresis (see figure 4) and Western blot detection (see figure 5).
(2) expression of gel protein electrophoresis detection VP2 albumen
Get the cell suspension of above-mentioned cultivation recombinant baculovirus rBac-HBM-HS4-VP2,4 ℃ of centrifugal (9000r/min) 5min, collect supernatant liquor, carry out sds gel electrophoresis.It is constant voltage 100V that deposition condition is set, and the time is 2h.After end, gel is placed in 0.1%R type coomassie brilliant blue staining liquid, and level is waved dyeing 1h, then with destainer decolouring, and takes pictures, and sees Fig. 6.
(3) Western blot detects the expression of analyzing VP2 albumen
Get the cell suspension of above-mentioned cultivation recombinant baculovirus rBac-HBM-HS4-VP2,4 ℃ of centrifugal (9000r/min) 5min, collect supernatant liquor, getting the 10 μ l supernatant liquors SDS sample-loading buffer concentrated with 2 times of 10 μ l mixes, process after 5min for 99 ℃, sample is added in 12% the point sample hole of sds polyacrylamide gel electrophoresis gel, carry out electrophoresis.When transferring film, constant current 300mA, shifts 1.5h at 4 ℃, takes out nitrocellulose filter and is put in 5% skim-milk and seals 3h.While hatching, primary antibodie adopts the rabbit source antibody (1: 500) of anti-6 Histidines, the goat anti-rabbit antibodies (1: 1000) of the horseradish peroxidase-labeled of two anti-employings.
(4) the HA titre of MEV virus-like particle detects
By continuous 18 hole marks of 96 hole blood-coagulation-boards, every hole adds 25 μ l physiological saline, add again the cell conditioned medium of the recombinant baculovirus rBac-HBM-HS4-VP2 of 25 μ l to first hole, sample is carried out successively to the doubling dilution of 1:2, the blank in 6～8 holes is set, only adds physiological saline.Add the pig red blood corpuscle of 25 μ l1% to each hole, mix latter 37 ℃ and place after 40min and check and record blood clotting result, see Fig. 7.
(5) purifying of MEV virus-like particle
Adopt sucrose gradient centrifugation, the viral suspension (about 150ml) of results point is filled in 50ml centrifuge tube, by high speed freezing centrifuge centrifugal (500g) 10min, supernatant discarded.With the about 10ml of density gradient centrifugation lysate, by resuspended the cell precipitation in 4 centrifuge tubes, then (1mL is for 10 for Shanghai Sheng Gong biotechnology company limited, Protease Inhibitor Cocktall to add proteinase inhibitor
9and PMSF proteinase inhibitor (phenylmethylsulfonyl fluoride, 100X, final concentration 0.1mM) cells).Cracked suspension is put on ice, with the ultrasonic 10min of ultrasonic cell disruptor, under microscope, seen ultrasonic effect.After ultrasonic, mixed solution divided and be filled in 2mlEP pipe, with high speed freezing centrifuge, 12000r/min, 4 ℃ of centrifugal 15min, proceed to supernatant in new EP pipe, more centrifugal 15min.Supernatant after centrifugal is transferred in new EP pipe, and 6 EP pipes of each sample,, go to sucrose gradient centrifugation by 1.5ml/.Rotary head can be placed 6 centrifuge tubes, 6 centrifuge tubes is filled it up with to 40% sucrose (Sigma Sucrose, BCBH5304V), and the appropriate sucrose of amount sucking-off per sample, is slowly added to sample above sucrose.Stainless steel sleeve pipe numbering is installed.Centrifugal speed: 200000g(39900r/min), centrifugal 2h, 4 ℃ of temperature.Centrifugal good sample is taken out, by the 400 resuspended sample precipitations of μ lTAE solution (detection keeps sample).In 2 centrifuge tubes, add successively 30%, 45%, 60%(W/W) sucrose, the time standby minute hand head adding up adds from bottom.In two ultracentrifugation pipes, respectively add the lysate of 200 μ l containing viral sample.150000g, 4 ℃ of centrifugal 2h.After centrifugal, finding has a bright band in 60% sucrose layer, is target protein, by albumen sucking-off.
(6) Electronic Speculum film making detects
After fixing processing of MEV virus-like particle sample after a small amount of purifying is concentrated, be placed on the coated grid of freshly prepd zero load plastic cement/carbon, after rinsing gently several times with several distilled water, add that 2% Tungstophosphoric acid, sodium salt solution carries out negative staining.Observed under electron microscope the (see figure 8) of making film.
Two, produce kind of preparation and a preservation that poison is criticized
Recombinant baculovirus preparation technology mainly comprises restructuring pVL1393 plasmid (being kept in Host Strains) and homologous recombination success and tests the recombinant baculovirus (first-generation poison) of purifying by plaque.The former adds glycerine to be kept in-80 ℃, and producing with planting a poison is in first-generation poison, to add 3% foetal calf serum packing to be kept in-80 ℃, and planting malicious long-term experiment chamber preservation is to be kept in-80 ℃ by extracting recombinant virus genomes.The malicious P1(first-generation of kind of batch normally packing being preserved by virus is produced in preparation) virus infection Sf9 cell prepares the P2(s-generation) virus, the like prepare the P3(third generation) virus.
Produce kind of the preparation that poison is criticized and preserve concrete grammar as follows:
1. test by plaque successfully viral the homologous recombination of results, screen several plaques and prepare pure recombinant virus.This is P0 virus.
2. then by P0 virus infection Sf9 cell, gather in the crops P1 virus.
3. after then P1 virus being added to 20% serum, carry out packing according to every cell cryopreservation tube 1ml, be then put in-80 ℃ of Refrigerator stores.The malicious storehouse of the inferior kind as production use.The P1 kind that increases successively poison obtains P2, P3 virus, and P3 virus is as producing by virus.
The kind poison of preparing according to the method one batch can be for the use of a production cycle, and plants toxicity matter homogeneous.Before kind poison batch is finished, do not need again to prepare kind of a poison.
Three, production of vaccine
(1) recover the seed cell 1ml of frozen Sf9 cell in 100ml serum free medium, be placed in 500ml screw socket glass triangle shaking flask and cultivate, 27 ℃ of shaking table temperature, rotating speed 110r/min.After cultivating 48h, add 200ml serum free medium, diluted passage is in 3000ml screw socket glass triangle shaking flask.After cultivating 48h, add 400ml serum free medium.According to every 48h, within 1: 2, Dilution ratio is passaged to 3000ml seed cell, and cell density reaches 3.0 × 10
6cells/ml;
(2) above-mentioned 3000ml seed cell is inoculated into 30L stirring type bioreactor (working volume 20L).Cultivate after 72～96h, add nutritional concentrated solution by feeding culture, continue to cultivate 72h, cell density reaches 4.0～8.0 × 10
6cells/m;
(3) the 20L seed cell in above-mentioned 30L stirring type bioreactor is directly inoculated to the bio-reactor into 1000L, progressively add fresh culture to total volume of culture 750L, and by feeding culture technique, cell density is cultured to 2.0 × 10
6cells/ml～4.0 × 10
6cells/ml;
(4) utilize the full suspension culture preparation of triangle shaking flask serum-free to express the restructuring clover silver powder californica nuclear polyhedrosis virus CGMCC No.8051 of RHDV subunit protein VP60, it is inoculated in the cultured cells of above 1000L bio-reactor take infection multiplicity (multiplicity of infection, MOI) as 0.5～5.0;
(5) gather in the crops above-mentioned nutrient solution supernatant, through deactivation, add aluminium hydroxide gel mix after make vaccine.
Four, mink viral enteritis virus baculovirus vector inactivated vaccine detection method
1. proterties check: after leaving standstill, upper strata is clarified liq, and lower floor is white precipitate, is even suspension after jolting.
2. steriling test; Test by existing " Chinese veterinary pharmacopoeia " appendix, answer asepsis growth.
3. loading amount check: test by existing " Chinese veterinary pharmacopoeia " appendix, 100ml/ bottle should be no less than 100ml.
4. safety detection: by healthy enteritis VLP inactivated vaccine subcutaneous inoculation susceptible mink (anti-MEV-HI value≤1:4) 5,3ml/, Continuous Observation 10 days, all minks should be all good for and be lived.
5. effect detects: the optional one of following method.
Serology test: by healthy 2～12 monthly ages of enteritis VLP inactivated vaccine subcutaneous inoculation susceptible mink (anti-MEV-HI value≤1:4) 5,1ml/ only, arranges simultaneously and do not inoculate 5 of contrast minks.Immunity blood sampling in latter 14 days, separation of serum, measures respectively anti-MEV-HI value for antibody.Immunity mink HI antibody titer all should be not less than 1:32, and contrast mink HI antibody titer all should be higher than 1:4.
Immunization method: by healthy 2～12 monthly ages of enteritis VLP inactivated vaccine subcutaneous inoculation susceptible mink (anti-MEV-HI value≤1:4) 5,1ml/ only, arranges simultaneously and do not inoculate 5 of contrast minks.Latter 14 days of immunity, to all minks difference, oral mink enteritis virus MEV-RC1 strain virus liquid 15ml(contains 15 × 107.0TCID50), Continuous Observation 10 days.Immunity mink should be all good for and be lived, and control group mink should all fall ill.
6. the antiseptic mercurials determination of residual amount: measure by existing " Chinese veterinary pharmacopoeia " appendix respectively, antiseptic mercurials residual quantity is 0.01%.
The Microbial resources information the present invention relates to
The recombinant baculovirus the present invention relates to is to utilize contained carrier pVL1393 in commercial kit (purchased from BD company), the method of transforming by carrier, melittin signal peptide (HBM) and insulator (HS4) are inserted into the appropriate location in carrier, by mink enteritis virus capsid protein (VP2) gene, (this gene is made as template using the commercialization mink enteritis virus production of vaccine strain RC1 strain that our company was had again, this commercialization mink enteritis virus vaccine authentication code is: veterinary drug new word (2011) 150256020) be inserted into the multiple clone site of above-mentioned transformation carrier, then with BaculoGold Linearized baculovirus DNA(Baculovirus Gene group) cotransfection Sf9 cell, there is homologous recombination in pVL1393 plasmid and BaculoGold Linearized baculovirus DNA, goal gene is inserted into the viral genome that forms ring-type in Baculovirus Gene group, then be assembled into complete virion and be secreted into extracellular.There is not the viral genome of homologous recombination because wherein contain a lethality deletion mutantion, can not be assembled into intact virus and build the recombinant baculovirus genome Bacmi that contains said gene, thereby make homologous recombination rate more than 99%.Then test to be further purified by plaque and select recombinant baculovirus.This recombinant baculovirus is named as restructuring clover silver powder californica nuclear polyhedrosis virus rBac-HBM-HS4-VP2, this virus was 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 08 02nd, 2013, deposit number is: CGMCC No.8051.
Positive effect of the present invention
The present invention relates to a kind of mink enteritis virus recombinant subunit vaccine and preparation method thereof.The production strain that the present invention relates to vaccine is restructuring clover silver powder californica nuclear polyhedrosis virus CGMCC No.8051, and the expression vector that this strain system adopts is secreting, expressing, and contain 6 histidine-tagged, be easy to Protein Detection analysis and use; Produce mink enteritis virus recombinant subunit vaccine with it as producing strain, this vaccine is not containing viral genome, without animal safety harm and potential diffusive; Immunity is with strong points, can excite immune animal to produce enough immune antibodies, and produce permanent protection.Produce MEV virus-like particle according to technology provided by the invention, the virus liquid of gathering in the crops in 1000L bio-reactor is tired and is reached blood clotting valency 1: 4096～1: 32768, become the about 65mg/L of granule protein expression amount, every liter of virus liquid work in-process can be made 3000～8000 parts of standard mink vaccine; This recombinant subunit vaccine has added adjuvant, effectively raises immune effect and this vaccine safety of immunizing potency, effective of vaccine, and steady quality, controlled is the novel vaccine that vaccine is organized in a kind of deactivation that is obviously better than prior art production.
Accompanying drawing explanation:
The agarose gel electrophoresis of the VP2 gene of Fig. 1 synthetic after pcr amplification, in figure 1: blank; 2:VP2 gene PCR amplified production; M:DL2000DNA molecular weight standard.
The agarose gel electrophoresis of the HS4 insulator sequence DNA fragment of Fig. 2 synthetic after pcr amplification.
Fig. 3 recombinant transfer vector plasmid schematic diagram.
Fig. 4 baculovirus is infected insect cell (Sf9) and expresses the sds gel electrophoresis figure of MEV VP2 protein expression, in figure 1: cultivate 72h supernatant after connecing poison; 2: after connecing poison, cultivate 96h supernatant; M:Marker; 3: do not connect poison and cultivate 72h supernatant (negative control 1).
Fig. 5 baculovirus is infected insect cell (Sf9) and expresses the Western blot of MEV VP2 protein expression and analyze, in figure 1: cultivate 72h cell suspension after connecing poison; 2: after connecing poison, cultivate 72h cell precipitation; M:Marker; 3: after connecing poison, cultivate 72h supernatant.
Fig. 6 gel method is surveyed the titre of recombinant baculovirus
Fig. 7 recombinant baculovirus is at the hemagglutinative titer of the MEV of insect cell inner expression virus-like particle
Virus-like particle electromicroscopic photograph after Fig. 8 purifying
Embodiment:
Can further clearly understand the present invention by the following example providing.But following examples of implementation are not limitation of the invention.
---batch preparation of recombinant baculovirus kind poison
Preparation rBac-HBM-HS4-VP2P1 kind poison storehouse (200,1ml/ props up): the P0 of purifying, for recombinant virus, is infected to logarithmic phase Sf9 cell 250mL, connect malicious MOI=0.1～0.01, after infecting, 96h results supernatant, obtains P1 for kind of a poison, and concrete operations are with embodiment 3.The P1 of collection is got to appropriate sample detection virus titer for kind of a poison, and all the other points install to 1.8ml cell cryopreservation tube, and every pipe adds 1mlP1 virus and 0.11ml foetal calf serum (Invitrogen company product).The above-mentioned point of cryopreservation tube installing is put in to Ultralow Temperature Freezer-80 ℃ preservation.
---produce the preparation with seed lot seed culture of viruses
To after the Sf9 cell recovery of liquid nitrogen cryopreservation, be inoculated in 250ml shaking flask, put on 27 ℃ of constant-temperature tables and cultivate, rotating speed is 110r/min.Treat that cell density reaches 3.0 × 10
6when cells/ml is above, be amplified in 1L shaking flask according to 1:2 ratio, be finally amplified to 3L shaking flask.3L shaking flask cell reaches 2.0 × 10
6when cells/ml, by MOI=0.05～0.1 inoculation rBac-HBM-HS4-VP2 recombinant virus, 27 ℃ of suspension culture 96h left and right, results nutrient solution, sampling and measuring HA tires, quantitative separating, freezing preservation, is production seed culture of viruses and criticizes.Dated harvest date, Virus passages etc.
---vaccine virus liquid preparation (take by 1311001 batches, 1311002 batches, 1311003 batches vaccines of technical solution of the present invention trial production as example)
(1) recover the seed cell 1ml of frozen Sf9 cell in 100ml serum free medium, be placed in 500ml screw socket glass triangle shaking flask and cultivate, 27 ℃ of shaking table temperature, rotating speed 110r/min.After cultivating 48h, add 200ml serum free medium, diluted passage is in 3000ml screw socket glass triangle shaking flask.After cultivating 48h, add 400ml serum free medium.According to every 48h, within 1: 2, Dilution ratio is passaged to 3000ml seed cell, and cell density reaches 3.0 × 10
6cells/ml;
(2) above-mentioned 3000ml seed cell is inoculated into 30L stirring type bioreactor (working volume 20L) culture condition and is set as: inoculum density: 1.5 × 10
6cells/ml, temperature: 27 ℃, pH:6.2, rotating speed: 60r/min, DO:45%.Cultivate after 72～96h, add nutritional concentrated solution by feeding culture, continue to cultivate 72h, cell density reaches 4.0～8.0 × 10
6cells/m;
(3) the 20L seed cell in above-mentioned 30L stirring type bioreactor is directly inoculated to the bio-reactor into 1000L, progressively add fresh culture to total volume of culture 750L, and by feeding culture technique, cell density is cultured to 2.0 × 10
6cells/ml～4.0 × 10
6cells/ml;
(4) utilize the full suspension culture preparation of triangle shaking flask serum-free to express the restructuring clover silver powder californica nuclear polyhedrosis virus CGMCC No.8051 of RHDV subunit protein VP60, by it with infection multiplicity (multiplicity of infection, MOI) be 0.5～5.0 to inoculate in the cultured cells of above 1000L bio-reactor, cultivate 72～140h; Sampling and measuring HA tires, and swine erythrocyte agglutination titer is 1:32765.
Embodiment 4
---deactivation and join seedling (take by 1311001 batches, 1311002 batches, 1311003 batches vaccines of technical solution of the present invention trial production as example)
The virus liquid that above-described embodiment 3 is cultivated is put in deactivation container, and under whipped state, adding concentration is that the BEI of 1mol/L is 0.005mol/L to final concentration, and limit edged stirs, and after mixing, keeps 37 ℃, deactivation 40～48h.When deactivation stops, in inactivation of viruses liquid, add immediately the 2mol/L Sulfothiorine (Na of filtration sterilization
2s
2o
3) solution, making its final concentration is 0.005mol/L, stirs.Virus liquid after deactivation and aluminium hydroxide gel are joined seedling by the volume ratio of 9: 1, make aluminium hydroxide gel content in vaccine represent to be no more than 3.9mg/ml with aluminum oxide, add 1% Thiomersalate solution, making its final concentration in vaccine is 0.01% again, after fully stirring, carries out packing.
Embodiment 5
---vaccine test (take by 1311001 batches, 1311002 batches, 1311003 batches vaccines of technical solution of the present invention trial production as example)
1. after proterties check leaves standstill, upper strata is clarified liq, and lower floor is white precipitate, is even suspension after jolting.
2. steriling test is tested by existing " Chinese veterinary pharmacopoeia " appendix, all asepsis growth.
3. safety detection is got 20 of the healthy susceptible minks of 49 ages in days, is divided at random 4 groups, and 5/group, do not inoculate for first group, in contrast, rear 3 groups of single doses (4ml/ is only) are inoculated respectively the mink enteritis virus recombinant subunit vaccine vaccine of different batches, last.Each group isolated rearing 14 days, records its spirit, diet, ight soil, body temperature situation and inoculation position and whether occurs swelling, necrosis and systemic adverse reactions.14 days time, cut open and kill all minks and carry out pathology detection, latter 14 days immune mink spirit of record immunity, body temperature, diet, ight soil situation; Whether inoculation position there is the reaction such as swelling, necrosis; Immunity is cutd open all minks to kill for latter 14 days, observes and record, and the mink viral enteritis pathological change that whether exists mink enteritis virus to cause, the results are shown in Table 1.
Table 1 mink enteritis virus recombinant subunit vaccine proof test result
4. effect detects:
(1) Serology test
By healthy 2～12 monthly ages of enteritis VLP inactivated vaccine subcutaneous inoculation susceptible mink (anti-MEV-HI value≤1:4) 5,1ml/ only, arranges simultaneously and does not inoculate 5 of contrast minks.Immunity blood sampling in latter 14 days, separation of serum, measures respectively anti-MEV value for antibody.The results are shown in Table 2.
Anti-MEV antibody test result after the immunity of table 2 mink enteritis virus recombinant subunit vaccine
(2) Immunization method
By healthy 2～12 monthly ages of enteritis VLP inactivated vaccine subcutaneous inoculation susceptible mink (anti-MEV-HI value≤1:4) 5,1ml/ only, arranges simultaneously and does not inoculate 5 of contrast minks.Latter 14 days of immunity, to all minks difference, oral mink enteritis virus MEV-RC1 strain virus liquid 15ml(contains 1.5 × 10
8.0tCID
50), Continuous Observation 10 days, the results are shown in Table 3.
After the immunity of table 3 mink enteritis virus recombinant subunit vaccine, attack poison protection result
Claims (9)
1. a shaft-like recombinant virus, is characterized in that the transfer vector of described recombinant baculovirus comprises: the SnaB I restriction enzyme site after pVL1393 transfer vector PolyA inserts HS4 sequence, inserts melittin signal peptide before multiple clone site; In multiple clone site, insert the MEV structural protein VP2 gene of a copy; C at foreign gene holds the sequence label that contains 6 Histidines, this recombinant baculovirus called after restructuring clover silver powder californica nuclear polyhedrosis virus rBac-HBM-HS4-VP2 has 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 08 02nd, 2013, deposit number is: CGMCC No.8051.
2. recombinant baculovirus according to claim 1, it is characterized in that this restructuring clover silver powder californica nuclear polyhedrosis virus CGMCC No.8051 can infected insect cell be, and efficient secretory expression recombinant protein VP2, above-mentioned recombinant VP 2 protein is equal to mink enteritis virus VP2 protein and can be further self-assembled into mink enteritis virus capsid protein in function.
3. mink enteritis virus capsid protein as claimed in claim 2, it is characterized in that in expressed in insect cells amount higher than the expression amount of conventional commercialization pVL1393 serial carrier, and be secreted into outside born of the same parents, stable existence is in the supernatant liquor of expressing, and this culture supernatant does not need purified can use.
4. restructuring mink enteritis virus capsid protein as claimed in claim 2, it is characterized in that this restructuring mink enteritis virus capsid protein is the same with natural mink enteritis virus with on immunogenicity in antigenicity, and be a kind of albumen that can induce mink to produce the immunne response infecting for mink enteritis virus.
5. for preventing a recombinant subunit vaccine for mink viral enteritis, it is characterized in that: it has comprised restructuring mink enteritis virus capsid protein claimed in claim 2 and expression supernatant liquor claimed in claim 3.
6. the recombinant subunit vaccine of prevention mink enteritis disease according to claim 5, is characterized in that: when vaccine is intramuscular injection formulation, contain adjuvant.
7. a large-scale producing method for mink enteritis virus subunit vaccine, comprises the steps:
(1) utilize the full suspension culture Sf9 cell of triangle shaking flask serum-free to prepare seed cell;
(2) utilize triangle shaking flask serum-free entirely to suspend and prepare the restructuring clover silver powder californica nuclear polyhedrosis virus CGMCC No.8051 that expresses RHDV subunit protein VP60;
(3) seed cell is directly inoculated into the full suspension culture Sf9 cell of stirring type bioreactor, the cultivation scale of bio-reactor is amplified to 1 ton of cell tank, and by feeding culture technique, cell density is cultured to 2.0 × 10
6cells/ml～4.0 × 10
6cells/ml;
(4), take infection multiplicity as 0.5～5.0 virus inoculation CGMCC No.8051, cultivate 72～140h;
(5) gather in the crops above-mentioned nutrient solution supernatant, through deactivation, add aluminium hydroxide gel mix after make vaccine.
8. preparation method according to claim 7, is characterized in that whole culturing process, comprises cell cryopreservation and recovery, be unicellular full suspension, serum-free, protein free culture environment.
9. preparation method according to claim 7, it is characterized in that using the full suspension culture Sf9 cell of mechanical agitation type bio-reactor, and amplification culture scale reaches 1 ton of cell tank.
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| CN110412028A (en) * | 2018-04-27 | 2019-11-05 | 中国科学院动物研究所 | A kind of method of counting of insect PuGV |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110412028A (en) * | 2018-04-27 | 2019-11-05 | 中国科学院动物研究所 | A kind of method of counting of insect PuGV |
| CN110412028B (en) * | 2018-04-27 | 2020-10-16 | 中国科学院动物研究所 | Counting method of insect particle viruses |
| CN110368490A (en) * | 2019-07-22 | 2019-10-25 | 齐鲁动物保健品有限公司 | Raccoon dog parvovirus enteritis, canine distemper bivalent inactivated vaccine and preparation method thereof |
| CN110368490B (en) * | 2019-07-22 | 2022-08-02 | 齐鲁动物保健品有限公司 | Raccoon dog parvovirus enteritis and canine distemper bivalent inactivated vaccine and preparation method thereof |
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