CN102517331A - 2 type subunit vaccine for porcine circovirus as well as preparation method and application thereof - Google Patents
2 type subunit vaccine for porcine circovirus as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a 2 type subunit vaccine for a porcine circovirus as well as a preparation method and application thereof. A recombinant bacilliform virus contains double promoters (a polyhedrin promoter and a P10 promoter), a coding gene of a Cap protein with double copying can be expressed, and the expression efficiency of the protein is obviously enhanced; moreover, the Cap protein expressed by an inserted foreign gene does not contain an excess sequence, virus-like particles (VLPs) can be effectively formed, and the immunogenicity of an expressed protein is enhanced; furthermore, a produced antigen has high content; and according to the 2 type subunit vaccine for the porcine circovirus, which is disclosed by the invention, the productivity ratio and the quality of a viral protein of the 2 type subunit vaccine for the porcine circovirus are obviously enhanced, and a prepared vaccine composition has the advantages of stable and persistent immune effect, high safety and the like.
Description
Technical field
The present invention relates to the veterinary biologics technical field, be specifically related to a kind of porcine circovirus 2 type subunit vaccine and preparation method thereof and its application.
Background technology
Porcine circovirus 2 type (Porcine circovirus type 2; PCV2) be the main pathogen of pmws (PMWS); This disease is a characteristic with immunosuppression and the depletion of weanling pig multisystem, and sick pig mainly shows retarded growth, anaemia, expiratory dyspnea, diarrhoea, weak, clinical symptom such as weightening finish is slow, lymphadenectasis.From 1991 broke out PMWS in Canadian swinery since, PMWS caused great financial loss for whole world pig industry.PCV2 not only can cause the depletion of weanling pig multisystem, and the immunologic function of infected pigs is suffered damage, and causes Abwehrkraft des Koepers to descend, and causes secondary infection, increases the weight of the state of an illness.In addition, PCV2 is also relevant with illnesss such as sow breeding difficulty, hyperplasia property necrotizing pneumonia, the scorching nephrotic syndrome of pigskin and PRDCs.In recent years, China swinery also has PMWS popular, and the harm that is caused is very serious.
Vaccine immunity is the important means of control PCV2 infection and relative disease thereof.At present, the vaccine of domestic production is the totivirus inactivated vaccine, discloses a kind of totivirus inactivated vaccine like document CN101240264A; But PCV2 is a little less than cell in vitro internal breeding ability, and the cultivation difficulty is bigger, and the final titre of virus is low; The virus antigen content that provides is limited; Preparation high quality P CV2 vaccine needs concentrating virus antigen, and this will directly cause production of vaccine with high costs, can not satisfy the actual requirement of animal vaccine super quality and competitive price.Document CN101180406A, CN101884787A and CN101358182A all disclose the Cap albumen with recombinant baculovirus expression PCV2ORF2 genes encoding; And use it for preparation PCV2 vaccine; Wherein, Described recombinant baculovirus all only contains a promotor, and does not optimize its manufacturing condition, causes the viral protein productive rate and the quality of producing acquisition all lower; And expressed goal gene has the modification of other unnecessary sequence (like 6 * His label, secreted signal peptide etc.); The foreign protein that is unfavorable for expressing form virus-like particle (virus-like particles, VLPs), the defective that these recombinant baculovirus constructing technology strategies exist the target protein immunogenicity expressed partly to lose.In addition, because factor affecting such as the adjuvant of this subunit vaccine and immunologic stimulants, the immune effect in its practical is not good.PCV2 subunit vaccine of therefore, be badly in need of that a kind of productive rate of development is higher, immunogenicity and immune effect are better and preparation method thereof and its prepared vaccine composition.
Summary of the invention
The object of the present invention is to provide a kind of recombinant baculovirus transfer vector, said carrier is the PCV2Cap protein coding gene ORF2 that respectively inserts a copy respectively in the P10 of pFastBacDual transfer vector promotor and Ppolh promotor (polyhedrin promotor) afterwards.
In an optimal technical scheme of the present invention, described PCV2Cap protein coding gene ORF2 be complete, without the PCV2b ORF2 that modifies.
In an optimal technical scheme of the present invention, the nucleotide sequence of described PCV2Cap protein coding gene ORF2 is shown in SEQ ID NO:1.
In an optimal technical scheme of the present invention, said PCV2Cap protein coding gene ORF2 inserts in the pFastBac Dual transfer vector through BamH I/Hind III and Kpn I/Xho I double digestion respectively.
In an optimal technical scheme of the present invention, said recombinant baculovirus transfer vector is pFastBac Dual-2ORF2.
Another object of the present invention is to provide the preparation method of a kind of porcine circovirus 2 type subunit vaccine production, comprise the steps: with strain
(1) obtains recombinant baculovirus transfer vector pFastBac Dual-2ORF2 of the present invention;
(2) homologous recombination produces recombinant baculovirus DNA;
(3) packing produces the recombinant baculovirus of expressing the PCV2 capsid protein.
In an optimal technical scheme of the present invention, described homologous recombination is that the described recombinant baculovirus transfer vector of step (1) is transformed among the competent escherichia coli cell DH10Bac that into contains shuttle vectors Bacmid, produces recombinant baculovirus DNA.
In an optimal technical scheme of the present invention, described packing is that the recombinant baculovirus DNA that step (2) produces is infected the sf9 cell, packs out recombinant baculovirus.
Another object of the present invention is to provide a kind of recombinant baculovirus, the preparation method with strain makes by porcine circovirus 2 type subunit vaccine of the present invention production.
In an optimal technical scheme of the present invention, said recombinant baculovirus is rBac-2ORF2.
Another object of the present invention is to provide a kind of preparation method of porcine circovirus 2 type subunit vaccine, comprise the steps:
(1) obtains the recombinant baculovirus that the present invention prepares;
(2) cultivate host cell, the recombinant baculovirus of inoculation step (1);
(3) inactivation of viruses;
(4) separation and purification reorganization PCV2Cap albumen;
(5) with purified recombinant PCV2Cap protein Preparation subunit vaccine.
In an optimal technical scheme of the present invention; Described step (2) comprises utilizes bio-reactor serum free medium suspension culture sf9 cell as host cell; By after infection multiplicity (MOI) is the described recombinant baculovirus of amount inoculation step (1) of 0.001-10; Continue to cultivate, PCV2Cap albumen is efficiently expressed in the sf9 cell.
In an optimal technical scheme of the present invention, the culture parameters of said bio-reactor is set at: pH 6.0-6.5, temperature 25-27 ℃; Dissolved oxygen 30-80%; Stirring velocity 100-180rpm, the culture parameters of preferred said bio-reactor is set at pH 6.2,27 ℃ of temperature; Dissolved oxygen 50%, stirring velocity 100-180rpm.
In an optimal technical scheme of the present invention, said step (2) comprises cell through the 5L-50L volume of culture step by step after the amplification culture, in the 50L bioreactor culture and inoculate said recombinant baculovirus; Perhaps, with cell through the 5L-50L-500L volume of culture step by step after the amplification culture, in the 500L bioreactor culture and inoculate said recombinant baculovirus.
In an optimal technical scheme of the present invention, said step (2) adopts any or its combination of batch formula cultural method, batch feeding cultural method, semicontinuous perfusion culture method or continous pouring culture.
In an optimal technical scheme of the present invention, said step (2) adopts the batch feeding cultural method.
In an optimal technical scheme of the present invention, said step (3) comprises the described recombinant baculovirus of adding divinyl imines (BEI) inactivator deactivation in cell culture fluid, and optional, finishes the back in deactivation and uses in the Sulfothiorine and excessive BEI.
In an optimal technical scheme of the present invention, said step (4) comprises the cell culture after the deactivation of collection step (3), removes by filter cell debris through centrifugal or tubular fibre, obtains containing the proteic cells and supernatant of PCV2Cap.
In an optimal technical scheme of the present invention, said step (5) comprises carries out quantitatively step (4) purified recombinant PCV2Cap albumen, adds adjuvant, the preparation vaccine.
In an optimal technical scheme of the present invention, described subunit vaccine comprises the purified recombinant PCV2Cap albumen of 8 μ g/ml and the aluminium hydroxide gel adjuvant of 1mg/ml.
In an optimal technical scheme of the present invention, described subunit vaccine also comprises an amount of sanitas.
In an optimal technical scheme of the present invention, described subunit vaccine also comprises immunostimulating complex.
In an optimal technical scheme of the present invention, the staple of said immunostimulating complex is the mixture of Quil A saponin(e, SUV and phosphatide, and wherein, Quil A saponin(e: SUV: the mass ratio of phosphatide is 1: 1: 1.
In an optimal technical scheme of the present invention, described subunit vaccine also comprises an amount of adjuvant.
In an optimal technical scheme of the present invention, said adjuvant is selected from any or its combination of water-based adjuvant, oil adjuvant, nanometer adjuvant or slowly-releasing adjuvant.
In an optimal technical scheme of the present invention; Every described subunit vaccine of part comprises: the PCV2Cap albumen that is not less than 8 μ g/ml; The aluminium hydroxide gel adjuvant of 1mg/ml; Consist of the immunostimulating complex of 50 μ g/ml Quil A saponin(es, 50 μ g/ml SUV and 50 μ g/ml phosphatide, by the vaccine final volume, content is not higher than 0.01% Thiomersalate.
Another object of the present invention is to provide a kind of porcine circovirus 2 type subunit vaccine, utilize recombinant baculovirus according to the invention to prepare.
Another object of the present invention is to provide a kind of porcine circovirus 2 type subunit vaccine, prepare by preparation method of the present invention.
In an optimal technical scheme of the present invention; Every described subunit vaccine of part comprises: the PCV2Cap albumen that is not less than 8 μ g/ml; The aluminium hydroxide gel adjuvant of 1mg/ml consists of the immunostimulating complex of 50 μ g/ml Quil A saponin(es, 50 μ g/ml SUV and 50 μ g/ml phosphatide.
In an optimal technical scheme of the present invention; The composition of every said vaccine composition of part comprises: every described subunit vaccine of part comprises: the PCV2 Cap albumen that is not less than 8 μ g/ml; The aluminium hydroxide gel adjuvant of 1mg/ml; Consist of the immunostimulating complex of 50 μ g/ml Quil A saponin(es, 50 μ g/ml SUV and 50 μ g/ml phosphatide, and by the vaccine final volume, content is not higher than 0.01% Thiomersalate.
Another object of the present invention is to provide the application of recombinant baculovirus transfer vector of the present invention in preparation PCV2 recombinant C ap albumen or PCV2 subunit vaccine.
Another object of the present invention is to provide the application of recombinant baculovirus of the present invention in preparation PCV2 recombinant C ap albumen or PCV2 subunit vaccine.
In an optimal technical scheme of the present invention, described PCV2 recombinant C ap albumen or PCV2 subunit vaccine are used for prevention and are infected the pmws that causes by porcine circovirus 2 type (PCV-2).
For clear statement protection scope of the present invention, the present invention defines following term as follows:
Its staple of immunostimulating complex of the present invention is the mixture of Quil A saponin(e, SUV and phosphatide, and wherein, Quil A saponin(e: SUV: the mass ratio of phosphatide is 1: 1: 1; This mixture can form a kind of lipid vesicle with greater activity; Be again a kind of brand-new antigen presentation system, body is had immuno-potentiation, have the dual-use function of adjuvant and antigen presentation; Can produce the effectiveness of " comprehensively " immunne response, and strengthen specific antibody for a long time and reply.
" every part " of the present invention is meant the dosage of the each immunity of every pig.
Except as otherwise noted; When per-cent of the present invention is the per-cent between liquid and the liquid volume per-cent; When per-cent is the per-cent between liquid and the solid volume/weight per-cent; Be weight/volume percent when per-cent is the per-cent between solid and the liquid, all the other are weight/weight percent.
Compared with prior art, the present invention has following advantage:
1, through verification experimental verification, the present invention adopts the proteic gene order of coding Cap of Chinese popular PCV2b hypotype can be used to prevent Chinese popular pig circular ring virus 2 viral disease pointedly;
2, recombinant baculovirus according to the invention contains double-promoter (polyhedrin promotor and P10 promotor), can express the Cap protein coding gene of two copies, has significantly improved proteic expression efficiency; And the expressed Cap albumen of the foreign gene of insertion does not contain unnecessary sequence, can effectively form virus-like particle (VLPs), has improved the immunogenicity of expressing protein, and the antigenic content of producing is high;
3, preparation method of the present invention utilizes bio-reactor, extensive serum-free suspension culture sf9 insect cell; And use it for preparation porcine circovirus 2 type subunit vaccine; Significantly improved the viral protein productive rate and the quality of porcine circovirus 2 type subunit vaccine, prepared vaccine composition has advantages such as immune effect is stable, lasting, security height;
4, the porcine circovirus 2 type subunit vaccine of the present invention's preparation has good security and immune efficacy, can resist the circovurus type 2 infection to pig good immune protection is provided;
5, the preparation method of applying biological reactor drum porcine circovirus 2 type subunit vaccine provided by the invention has that occupation area of equipment is little, industrial scale is big, production efficiency is high, and can realize advantages such as production automation control and suitability for industrialized production.
Description of drawings
Fig. 1 recombinant baculovirus transfer vector of the present invention makes up schema.
Fig. 2 recombinant baculovirus of the present invention makes up schema.
Fig. 3 list copy, two copy reorganization bacmid PCR identify figure.
Fig. 4 PCV2 subunit vaccine of the present invention preparation technology schema.
The Cap albumen that Fig. 5 the present invention expresses forms virus-like particle (VLPs) Electronic Speculum picture.
Embodiment
Below will combine embodiment to specify the present invention, embodiments of the invention only are used to technical scheme of the present invention is described, and non-limiting essence of the present invention.
Embodiment 1The structure of recombinant baculovirus
Use Bac-to-Bac system constructing recombinant baculovirus, the gene order of announcing according to GENBANK (the NCBI number of landing EU340257.1) designs following primer:
P1：TCTGGATCCATGACGTATCCAAGGAGGCG
P2：GCGAAGCTTTAAGGGTTAAGTGGGGGGTC
P3：TCTCTCGAGATGACGTATCCAAGGAGGCG
P4：GCGGGTACCTAAGGGTTAAGTGGGGGGTC
With PCV2b strain virus ORF2 sequence is template (the PCV2b ORF2 sequence that this template basis has been announced: the number of landing EU340257.1 is synthetic to be obtained), P1, P2 amplification PCV2 ORF2 gene; And this gene clone gone in the pMD-19T carrier, obtain recombinant vectors pMD-19T-ORF2-1, be template with PCV2b strain virus ORF2 sequence; P3; P4 amplification PCV2 ORF2 gene, and this gene clone gone in the pMD-19T carrier, recombinant vectors pMD-19T-ORF2-2 obtained.
Through BamH I and Hind III double digestion pMD-19T-ORF2-1, the ORF2 gene clone is gone among the transfer vector pFastBac Dual carrier construction pFastBac Dual-ORF2; Through Kpn I and Xho I double digestion pMD-19T-ORF2-2; The ORF2 gene clone is gone among the transfer vector pFastBac Dual-ORF2; Structure comprises the recombinant transfer vector pFastBac Dual-2ORF2 of two copy ORF2 genes; With this recombinant transfer vector transformed into escherichia coli DH10Bac, obtain to insert the recombinant plasmid bacmid-2ORF2 (PUC M13F/R primer identifies that bacmid result sees Fig. 3 A) of two copy ORF2 genes, this recombinant plasmid bacmid-2ORF2 is transfected among the insect cell Sf9; Obtain recombinant baculovirus rBac-2ORF2 (ORF2 sequence such as SEQ ID NO:1 are said), rBac-2ORF2 is subsequent use as kind of poison for the amplification recombinant baculovirus.With recombinant transfer vector pFastBac Dual-ORF2 transformed into escherichia coli DH10Bac; Obtain to insert the recombinant plasmid bacmid-ORF2 of single copy ORF2 gene; This recombinant plasmid bacmid-ORF2 is transfected among the insect cell Sf9; Obtain recombinant baculovirus rBac-ORF2 (ORF2 sequence such as SEQ ID NO:1 are said), amplification recombinant baculovirus rBac-ORF2 subsequent use (PUC M13F/R primer identifies that bacmid result sees Fig. 3 B).
Embodiment 2The bio-reactor serum-free suspension culture and the proteic expression of Cap of insect cell are quantitative
Shook in the bottle sterile culture Sf9 insect cell 3-4 days at 1000ml, treat that concentration is long to 3-5 * 10
6Cells/ml, vigor seeded cells in the bio-reactor of 5L greater than 95% o'clock, and inoculum density is 3-8 * 10
5Cells/ml.When cell concn reaches 3-5 * 10
6During cells/ml, seed cells in the 50L bio-reactor, treat that it is 3-5 * 10 that cell grows to concentration
6Cells/ml is inoculated in the 500L bio-reactor, treats that cell concn reaches 2-8 * 10
6During cells/ml, inoculation recombinant virus rBac-2ORF2 or rBac-ORF2, infection multiplicity (MOI) is 0.001-10, the reactor drum culture condition is pH 6.0-6.5, temperature 25-27 ℃, dissolved oxygen 30-80%, stirring velocity 100-180rpm.Consider the optimum condition of cell cultures, preferred pH 6.2,27 ℃ of cell cultures phase temperature settings, dissolved oxygen 50%, stirring velocity 100-180rpm.After infecting, continue to cultivate after 5-9 days, adding final concentration is the BEI of 5mmol/L, behind 37 ℃ of effect 24h, adds 1mol/L Na
2S
2O
3To final concentration 5mmol/L termination deactivation.Through the centrifugal or filtering method harvested cell of tubular fibre culture supernatant, put 2-8 ℃ and preserve vaccinogen liquid.
Cap albumen contained in the vaccine antigen of preparation is through the ELISA detection by quantitative.How anti-with the anti-PCV2-Cap albumen of capture antibody rabbit that encapsulates damping fluid dilution purifying to suitable concn, every hole 100 μ l, 4 ℃ are spent the night, PBST washing three times, 1%BSA sealing 1 hour.Add the antigen standard substance (the Cap albumen of the formation virus-like particle VLPs through CsCl density gradient centrifugation purifying) and gradient dilution sample to be checked of different concns, hatched 1 hour for 37 ℃, PBST gives a baby a bath on the third day after its birth inferior.Every hole adds detects antibody-proteic monoclonal antibody of anti-PCV2Cap, hatches 1 hour for 37 ℃, and PBST gives a baby a bath on the third day after its birth inferior.Every hole adds the sheep anti-mouse igg of two anti--HRP marks, hatches 1 hour for 37 ℃, and PBST gives a baby a bath on the third day after its birth inferior.TMB colour developing 10 minutes, 2M H
2SO
4Termination reaction.The ELIASA reading calculates the proteic amount of Cap in the sample to be checked through typical curve.
Use three kinds of different viruses of different MOI (0.01,0.1,1) inoculating two kinds, respectively at the target protein content in inoculation back sampling in the 7-11 days quantitative analysis culture supernatant, the result sees table 1.
The different infection multiplicity inoculating two kinds of table 1 viral protein expression amount (μ g/ml) is analyzed
The result shows by the Cap protein quantification; Use bio-reactor serum-free suspension culture to express Cap albumen; Under the righttest MOI (MOI=0.1); Its expression amount can reach 180 μ g/ml, and the same terms uses the recombinant baculovirus expression Cap albumen (only containing the polyhedrin promotor) of single copy goal gene down, and its expression amount is merely 70 μ g/ml.This shows that construction strategy according to the invention can significantly improve the proteic expression level of Cap.
Embodiment 3VLP particulate purifying and electron microscopic observation
Results are expressed cell culture, and with cell culture 10000g, 4 ℃ of centrifugal 30min remove cell debris; Get supernatant, with the centrifugal 3h of supernatant 31000rpm (Beckman SW70 rotor), will precipitate with a small amount of PBS resuspended, treat the deposition fully the dissolving after; Press 2.1g/4.5ml solution and add CsCl, after mixing, divide to install in the 5ml ultracentrifugation pipe, add PBS; Arrive apart from mouth of pipe 2-3mm place, after the accurate trim, 149000g, 10 ℃ of centrifugal 24h.After centrifugal, visible two faint yellow zona pellucidas.Purpose band (lower floor's band) sucking-off is collected, be the virus-like particle of purifying.
Get under the equal conditions, cultivate the rBac-ORF2 and the cell culture that rBac-2ORF2 expresses of equivalent, handle as stated above, the purpose band of sucking-off is diluted to equal volume, take a sample and carry out phospho-wolframic acid negative staining, electron microscopic observation.The result is as shown in Figure 5, and wherein, Fig. 5 A is an electron microscopic observation picture behind the rBac-ORF2 list copy expression sample purifying, and Fig. 5 B is an electron microscopic observation picture behind the two copy expression of the rBac-2ORF2 sample purifying.It is thus clear that rBac-2ORF2 can give expression to more virus-like particle, has good immunogenicity.
Embodiment 4The bio-reactor serum-free condition of suspension culture of insect cell is optimized
Present embodiment adopts batch formula cultural method and batch feeding cultural method that condition of suspension culture is optimized, and wherein, Cap protein expression quantivative approach is with embodiment 2.
During the formula of criticizing is cultivated, in the 10L bio-reactor, according to 5 * 10
5Cells/ml inoculation sf9 cell is when cell density reaches 2 * 10
6During cells/ml, inoculation recombinant virus rBac-2ORF2 cultivated 7 days, and the harvested cell culture is measured the proteic expression amount of Cap.
During batch feeding is cultivated, in the 10L bio-reactor, according to 5 * 10
5Cells/ml inoculation sf9 cell is when cell density reaches 8 * 10
6During cells/ml, inoculation recombinant virus rBac-2ORF2, the while is carried out feed supplement according to the amount of volume of culture every day 1/1000, cultivates 7 days, and the harvested cell culture supernatant is put 2-8 ℃ and is preserved vaccinogen liquid.Measure the proteic expression amount of Cap.
The Cap protein quantification is the result show: batch formula culture expression amount is 150 μ g/ml, and batch feeding culture expression amount reaches 221 μ g/ml.Thus it is clear that, use the batch feeding cultural method can significantly improve cell culture density and the proteic expression level of Cap.
Embodiment 5The vaccine adjuvant comparative studies
Prepare vaccine compositions according to following different adjuvants:
Adjuvant 1 vaccine: press 1% of vaccine antigen TV and add Nonidet P40 (NP-40), stirring at room 2h; Press protein concentration calculating antigen head umber (every part 8 μ g antigens) in the vaccine antigen; Add staple and be Quil A saponin(e, SUV and phosphatide immunostimulating complex (wherein; Quil A saponin(e: SUV: the mass ratio of phosphatide is 1: 1: 1; Every part 50 μ g promptly contain 50 μ g Quil A saponin(e+50 μ g SUV+50 μ g phosphatide in every part), stirring at room 2h; Add Thiomersalate to final concentration 0.01% (final volume of pressing vaccine calculates); To every part of 1ml, add the aluminium hydroxide gel adjuvant with PBS dilution vaccine, mix by every part 1mg; Quantitatively packing seals.
Every part of adjuvant 1 vaccine comprises: the PCV2 Cap albumen of 8 μ g/ml, the aluminium hydroxide gel adjuvant of 1mg/ml, consist of 50 μ g/ml Quil A saponin(es, 50 μ g/ml SUV and the immunostimulating complex of 50 μ g/ml phosphatide, certain density NP-40, and calculate by the vaccine final volume and not to be higher than 0.01% sanitas Thiomersalate.
Adjuvant 2 vaccines:, add Thiomersalate to final concentration 0.01% (final volume of pressing vaccine calculates) with PBS dilution vaccine to every part of 1ml (every part 8 μ g/ml antigens); Add the aluminium hydroxide gel adjuvant by every part 1mg/ml, mix; Quantitatively packing seals.
With 25 of the piglets of buying back, be divided into 3 groups, first group, second group is respectively 10, through intramuscular inoculation adjuvant 1 vaccine and adjuvant 2 vaccines.The 3rd group (5 piglets) is as blank (being non-immune group).Separation of serum was used for antibody test to every group of piglet blood sampling in the 7th day, the 14th day, the 21st day, the 28th day, the 60th day, the 90th day, the 120th day, the 150th day, the 180th day in before the immunity and immunity back, and the result sees table 2.
Antibody test result behind the different adjuvant immunities of table 2
Annotate: the result is negative in "-" expression antibody test.Greatest dilution with the positive serum of OD value is an antibody titer.
Can find out according to the antibody test result, compare, add adjuvant 1 vaccine of immunostimulating complex and the antibody of adjuvant 2 vaccines and produced fast with the blank group; In immunity back 28 days; Antibody titer can reach higher level, and the antibody extended period is longer, has significantly improved immune effect; And, added the antibody valence higher level of adjuvant 1 vaccine of immunostimulating complex.
Embodiment 6The preparation of porcine circovirus 2 type subunit vaccine
Use novel immunostimulating complex adjuvant and aluminium hydroxide gel adjuvant preparation vaccine, concrete preparation method is following:
Press 1% of vaccine antigen TV and add NP-40 (Nonidet P40), stirring at room 2h; Press protein concentration calculating antigen head umber (every part 8 μ g antigens) in the vaccine antigen, add immunostimulating complex (its staple is the mixture of Quil A saponin(e, SUV and phosphatide), stirring at room 2h; Add Thiomersalate to final concentration 0.01% (final volume of pressing vaccine calculates); To every part of 1ml, add the aluminium hydroxide gel adjuvant with PBS dilution vaccine, mix by every part 1mg; Quantitatively packing seals.
Every part of vaccine of above method configuration comprises: the PCV2Cap albumen of 8 μ g/ml, the aluminium hydroxide gel adjuvant of 1mg/ml, consist of 50 μ g/ml Quil A saponin(es, 50 μ g/ml SUV and the immunostimulating complex of 50 μ g/ml phosphatide, the NP-40 of vaccine antigen stoste TV 1%, and calculate by the vaccine final volume and not to be higher than 0.01% sanitas Thiomersalate.
Embodiment 7The porcine circovirus 2 type subunit vaccine is renderd a service experiment
Main pathogen and associated antibodies are carried out in the pig farm, Wuhan detect, select 15 of the negative pigs of cause of disease such as porcine circovirus 2 type, CSFV, porcine reproductive and respiratory syndrome virus, pig parvoviral, the negative pigs of porcine circovirus 2 type antibody of 21-25 age in days for use.
Qualified piglet is divided into 3 groups at random, and the 1st group (5 pigs) is immune group, gives the porcine circovirus 2 type subunit vaccine (8 μ g/ head part) by preparation among the embodiment 6; The 2nd group (5 pigs) is for attacking malicious control group; The 3rd group (5 pigs) is the blank group.Immunity was carried out PCV2 virus (7.01gTCID to the 1st, 2 group in back 28 days simultaneously
50/ ml) attack every pig musculi colli injection 3ml, collunarium 1ml, isolated rearing.Attack poison back the 4th, 7 day respectively in two oxters and two buttocks injection Freund's incomplete adjuvant emulsive keyhole hemocyanin (1mg/ml, every position 0.5ml), attack poison and cutd open in back 28 days and kill.The 3rd group as negative control group, and isolated rearing is separately attacked poison at the 1st, 2 group and cutd open in back 28 days and kill.All pigs before immunity, immunity back 7 days, 14 days, 21 days, 28 days and cuing open kill before blood sample collection, carry out PCV2 serum antibody analysis (seeing table 3) through the ELISA method.Attack poison and cutd open the whole porklings of inspection in back 28 days, meet two in following three, i.e. decidable morbidity (seeing table 4).
A body temperature symptom: piglet fervescence (>=40 ℃) should continue 3 days at least;
The B weight standard: the relative weight gain rate descends should be not less than 5.0%, and the average daily gain of attacking malicious piglet should be less than non-average daily gain of attacking malicious control group piglet.Pursue all test piglet body weight of a weighing respectively the same day in attacking poison, attacked malicious back 28 days all test piglet body weight of weighing once more; Wherein, the calculating of the relative weight gain rate is undertaken by following formula in " B weight standard ":
The C virus antigen detects: detect lymph node tissue with immunohistochemistry technique, detect PCV2 virus.
Table 3 antibody test result
Table 4 is respectively organized experimental animal morbidity result of determination
The result shows, back 21 days of immunity, most of piglet antibody changes sun, antibody all changes sun after 28 days, attack poison after, the immune group protection ratio can reach 100%, all experimental animals are morbidity all.
This shows that the subunit vaccine that method described in the present invention is produced can provide protection to animal, can effectively protect pig only to resist PCV2 and infect, and the PCV2 vaccine of the present invention of employing novel adjuvant can significantly improve the immune effect of vaccine.
Claims (22)
1. recombinant baculovirus transfer vector; Said carrier is the PCV2Cap protein coding gene ORF2 that respectively inserts a copy afterwards in the P10 promotor and the Ppolh promotor (polyhedrin promotor) of pFastBac Dual transfer vector respectively; Preferred described PCV2 Cap protein coding gene ORF2 be complete, without the PCV2b ORF2 that modifies, more preferably said PCV2 Cap protein coding gene ORF2 is respectively through in BamH I/Hind III and the Kpn I/Xho I double digestion insertion pFastBac Dual transfer vector.
2. recombinant baculovirus transfer vector as claimed in claim 1 is characterized in that, the nucleotide sequence of described PCV2Cap protein coding gene ORF2 is shown in SEQ ID NO:1.
3. like each described recombinant baculovirus transfer vector of claim 1-2, it is characterized in that said recombinant baculovirus transfer vector is pFastBac Dual-2ORF2.
4. porcine circovirus 2 type subunit vaccine production comprises the steps: with the preparation method of strain
(1) obtains like each described recombinant baculovirus transfer vector of claim 1-5;
(2) homologous recombination produces recombinant baculovirus DNA;
(3) packing produces the recombinant baculovirus of expressing the PCV2 capsid protein.
5. preparation method as claimed in claim 4; It is characterized in that; The described homologous recombination of step (2) is that the described recombinant baculovirus transfer vector of step (1) is transformed among the competent escherichia coli cell DH10Bac that into contains shuttle vectors Bacmid, produces recombinant baculovirus DNA.
6. like claim 4 or 5 described preparing methods, it is characterized in that the described packing of step (3) is the recombinant baculovirus DNA transfection sf9 cell that step (2) is produced, and packs out recombinant baculovirus.
7. a recombinant baculovirus is prepared by each described method of claim 4-6, and preferred said recombinant baculovirus is rBac-2ORF2.
8. the preparation method of a porcine circovirus 2 type subunit vaccine comprises the steps:
(1) obtains the described recombinant baculovirus of claim 9;
(2) cultivate host cell, the recombinant baculovirus of inoculation step (1);
(3) inactivation of viruses;
(4) separation and purification reorganization PCV2 Cap albumen;
(5) with purified recombinant PCV2 Cap protein Preparation subunit vaccine.
9. preparation method as claimed in claim 8; It is characterized in that; Described step (2) comprises utilizes bio-reactor serum free medium suspension culture sf9 cell as host cell; By after infection multiplicity (MOI) be the described recombinant baculovirus of amount inoculation step (1) of 0.001-10, continue cultivation, PCV2 Cap albumen is efficiently expressed in the sf9 cell.
10. preparation method as claimed in claim 9; It is characterized in that; The culture parameters of bio-reactor is set at: pH 6.0-6.5, temperature 25-27 ℃, dissolved oxygen 30-80%, stirring velocity 100-180rpm, and the culture parameters of preferred bio-reactor is set at pH 6.2,27 ℃ of temperature; Dissolved oxygen 50%, stirring velocity 100-180rpm.
11., it is characterized in that step (2) comprises cell through the 5L-50L volume of culture step by step after the amplification culture like each described preparation method of claim 8-10, in the 50L bioreactor culture and inoculate said recombinant baculovirus; Perhaps, after the cultivation that cell is amplified through the 5L-50L-500L volume of culture step by step, in the 500L bioreactor culture and inoculate said recombinant baculovirus.
12. like each described preparation method of claim 8-11; It is characterized in that; Said step (2) adopts any or its combination of batch formula cultural method, batch feeding cultural method, semicontinuous perfusion culture method or continous pouring culture to cultivate, and is preferably the batch feeding cultural method and cultivates.
13., it is characterized in that said step (3) comprises the described recombinant baculovirus of adding BEI inactivator deactivation in cell culture fluid like each described preparation method of claim 8-12, and finish the back in deactivation and use in the Sulfothiorine and excessive BEI.
14. like each described preparation method of claim 8-13; It is characterized in that; Said step (4) comprises the cell culture after the deactivation of collection step (3), removes by filter cell debris through centrifugal or tubular fibre, obtains containing the proteic cells and supernatant of PCV2Cap.
15., it is characterized in that said step (5) comprises carries out quantitatively step (4) purified recombinant PCV2Cap albumen like each described preparation method of claim 8-14, add adjuvant, the preparation vaccine.
16. like each described preparation method of claim 8-15; It is characterized in that; Described subunit vaccine comprises the purified recombinant PCV2 Cap albumen of 8 μ g/ml, the aluminium hydroxide gel adjuvant of 1mg/ml, and preferred described subunit vaccine also comprises an amount of sanitas.
17. like each described preparation method of claim 8-16; It is characterized in that; Described subunit vaccine also comprises immunostimulating complex; Preferred said immunostimulating complex is the mixture of Quil A saponin(e, SUV and phosphatide, and wherein, Quil A saponin(e: SUV: the mass ratio of phosphatide is 1: 1: 1; Preferred described subunit vaccine also comprises an amount of adjuvant, and more preferably said adjuvant is selected from any or its combination of water-based adjuvant, oil adjuvant, nanometer adjuvant or slowly-releasing adjuvant.
18. like each described preparation method of claim 8-17; It is characterized in that every described subunit vaccine of part comprises: the immunostimulating complex of the PCV2 Cap albumen of 8 μ g/ml, the aluminium hydroxide gel adjuvant of 1mg/ml, the Quil A saponin(e that consists of 50 μ g/ml, 50 μ g/ml SUV and 50 μ g/ml phosphatide; The described subunit vaccine of preferred every part comprises: the immunostimulating complex of the PCV2 Cap albumen of 8 μ g/ml, the aluminium hydroxide gel adjuvant of 1mg/ml, the Quil A saponin(e that consists of 50 μ g/ml, 50 μ g/ml SUV and 50 μ g/ml phosphatide, and calculate by the vaccine final volume and not to be higher than 0.01% Thiomersalate.
19. a porcine circovirus 2 type subunit vaccine utilizes the said recombinant baculovirus of claim 7 to make, and perhaps prepares like each described method of claim 8-18.
20. porcine circovirus 2 type subunit vaccine as claimed in claim 19; It is characterized in that; The composition of every said vaccine composition of part comprises, the immunostimulating complex of the PCV2 Cap albumen of 8 μ g/ml, the aluminium hydroxide gel adjuvant of 1mg/ml, the Quil A saponin(e that consists of 50 μ g/ml, 50 μ g/ml SUV and 50 μ g/ml phosphatide; The composition of preferred every said vaccine composition of part comprises: the immunostimulating complex of the PCV2 Cap albumen of 8 μ g/ml, the aluminium hydroxide gel adjuvant of lmg/ml, the Quil A saponin(e that consists of 50 μ g/ml, 50 μ g/ml SUV and 50 μ g/ml phosphatide, and calculate by the vaccine final volume and not to be higher than 0.01% Thiomersalate.
21. each described recombinant baculovirus transfer vector of claim 1-3 or the said recombinant baculovirus of claim 7 application in preparation PCV2 recombinant C ap albumen or PCV2 subunit vaccine.
22. application as claimed in claim 21, described PCV2 recombinant C ap albumen or PCV2 subunit vaccine are used to prevent to infect the pmws that causes by porcine circovirus 2 type.
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| CN 201210270504 CN102925486B (en) | 2011-12-26 | 2012-08-01 | Porcine circovirus type 2 subunit vaccine, and preparation method and application thereof |
| CN201210356552.7A CN103033622B (en) | 2011-12-26 | 2012-09-21 | PCV2 (porcine circovirus 2) ELISA (enzyme-linked immuno sorbent assay) antigen detection kit as well as preparation method and applications thereof |
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