CN103920146A - Porcine circovirus II type-porcine pseudorabies double-combination vaccine, and preparation methods and application thereof - Google Patents
Porcine circovirus II type-porcine pseudorabies double-combination vaccine, and preparation methods and application thereof Download PDFInfo
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Abstract
The invention relates to a porcine circovirus II type-porcine pseudorabies double-combination vaccine which is characterized by including at least one porcine circovirus II type antigen and at least one porcine pseudorabies virus antigen. The invention also relates to two preparation methods of the double-combination vaccine. One method comprises the steps: inactivating the porcine circovirus II type to obtain a porcine circovirus II type inactivated vaccine; and mixing the porcine pseudorabies virus antigen with a freeze-drying protective agent to obtain a porcine pseudorabies freeze-drying living vaccine, and then dissolving the porcine pseudorabies freeze-drying living vaccine with the inactivated vaccine as a diluent to obtain the porcine circovirus II type-porcine pseudorabies double-combination vaccine composition. The other method comprises the steps: mixing the porcine circovirus II type antigen with the porcine pseudorabies virus antigen in proportion, and then mixing with the freeze-drying protective agent to obtain the double-combination freeze-drying vaccine. With use of the double-combination vaccine, the cold chain operating cost can be greatly reduced; and the combination vaccine has a synergistic role in the immune effect on the porcine pseudorabies virus antigen, and does not affect the immune effect of the porcine circovirus antigen.
Description
Technical field
The invention belongs to technical field of vaccines, be specifically related to a kind of pig gyrate virus II type and porcine pseudorabies bigeminy vaccine and its preparation method and application.
Background technology
Pig circular ring virus (Porcine circovirus, PCV) is the animal virus of a kind of minimum of finding so far.Existing known PCV has two serotypes, i.e. PCV1 and PCV2.PCV1 is the virus of non-pathogenic, and PCV2 is pathogenic virus, form contrast with the PCV1 conventionally without virulence, the PCV2 of pig infects relevant with various diseases syndrome, these disease syndromes are called pig circular ring virus 2 toxicity disease jointly, are also called as pig circular ring virus relevant disease.
The pig in age in pig circular ring virus 2 toxicity affect 5-22 week, Clinical symptoms is to become thin, palor, growthing lag, respiratory distress, diarrhoea, jaundice and jaundice.In some affected pigs, can obviously there is Symptomatic combination, and also have some affected pigs only to there are one or both above-mentioned symptoms.The mortality rate of pig that infects PCV2 approaches 50%, in necropsy, and microscope visible and the visible multiple tissues of naked eyes and organ injury, wherein lymphatic organ is modal damage location.The amount of having observed PCV2 nucleic acid or antigen is strongly relevant to microscopic lymph degree of injury.In addition, also found that the amount of blood amplifying nucleic acid or antigen is relevant with the order of severity of clinical symptoms, had higher than 10 the pig that suffers from pig circular ring virus 2 toxicity disease
6the viral level of genome equivalent number/milliliter.
Porcine pseudorabies (Pseudorabies, PR) be the infectious disease that many animals is suffered from altogether, cause the infectious disease of domestic animal and wild animal acute fatal, taking heating, very itch and encephalomyelitis as cardinal symptom, pig is its natural susceptible host, mainly causes in-pig miscarriage, stillborn fetus and mummy tire, and Infection in Piglets is mainly nervous symptoms, Adult Pig is taking inapparent infection as main, and this disease has become the Etiological of kind of pig breeding dysfunction syndrome.Recent study discovery, has synergism when PCV2 and PRV mixed infection, and piglet aggravation after causing weaning, loses even more serious.
Compared with the clinical obvious disease performance of infecting with PCV2, subclinical PCV2 infects and is considered to be present in those and has infected PCV2 but in animal without clinical symptoms.Generally speaking, between these forms that PCV2 infects, have dependency, because subclinical infection can be transformed into pig circular ring virus 2 toxicity disease, and convalescent animal may keep long-lasting infection state.
Summary of the invention
The applicant infects in the test of pig circular ring virus 2 toxicity metabasis repeating subclinical PCV2, finds that unexpectedly PRV (Pseudorabies virus) can cause the generation of this transformation.
Therefore, the object of the invention is to pig Pigs Inoculated porcine circovirus vaccine, splice grafting boar pseudorabies virus vaccine in parallel, can be understood as to pig inoculation bigeminy vaccine or use pig circular ring virus vaccine and PRV (Pseudorabies virus) vaccine simultaneously, combine with pig gyrate virus II type and pseudorabies disease vaccine not only facilitate, multiple-effect, low cost, compared with single vaccine, can also reduce vaccination number of times, avoid can not obtaining omnidistance immunity because leaking to plant, can greatly reduce the expense of cold chain running.Especially, the applicant is surprised to find that, combines and porcine pseudorabies antigen immune effect had to synergism with vaccine, and do not affect the immune effect of pig circular ring virus antigen.
The invention provides a kind of pig gyrate virus II type and porcine pseudorabies bigeminy vaccine, it comprises at least one pig gyrate virus II type antigen and at least one PRV (Pseudorabies virus) antigen.
Pig gyrate virus II type antigen of the present invention contains at least one PCV2 antigen, and the immunne response that opposing PCV2 infects can be induced, stimulates or be strengthened to described antigen inoculation pig.Preferably, the embedded virus that described PCV2 antigen is PCV2 totivirus, contain PCV2 immunogenicity aminoacid sequence (as ORF2 albumen), any other at least containing polypeptide or subunit or other compositions of PCV2 immunogenicity aminoacid sequence.PCV2 antigen can also comprise following any antigen: as Cimmeria company (Merial)
pfizer (Pfizer)
pCV2; The porcine circovirus type II inactivated vaccine (LG strain) of Harbin Wei Ke biotechnology development company; The porcine circovirus type II inactivated vaccine (DBN-SX07 strain) of Fuzhou Dabeinong Bioisystech Co., Ltd and genetic engineering subunit vaccine are (as the Ingelvac of Boehringer Ingelheim company (Boehringer-Ingelheim)
).
Preferably, the PCV2 totivirus antigen that in the present invention, pig gyrate virus II type antigen is deactivation, wherein preferably uses PCV2SH strain, and preserving number is CGMCC No.23890, is disclosed in patent documentation CN101240264A.Between the different strains of PCV2, nucleotide sequence homology is higher; all more than 90%, therefore PCV2SH strain (GenBank accession number AY686763), PCV2LG strain (GenBank accession number HM038034), PCV2DBN-SX07-2(GenBank accession number HM641752) antigen composition or vaccine combination all within protection domain of the present invention.
Preferably, in the present invention, pig gyrate virus II type antigen is polypeptide or subunit or other compositions that contains PCV2 immunogenicity aminoacid sequence, and more preferably, pig gyrate virus II type antigen is PCV2 subunit antigen, most preferably, pig gyrate virus II type antigen is Porcine circovirus type 2 ORF2 protein.Wherein, Porcine circovirus type 2 ORF2 protein of the present invention, the albumen of preferred sequence table SEQNO1.DNA sequential coding or the albumen of sequence table SEQ NO2. aminoacid sequence or with its homology more than 75%, preferably more than 80%, the more preferably albumen of more than 90% aminoacid sequence, for reaching the protein sequence of the object of the invention.The present invention has no particular limits Porcine circovirus type 2 ORF2 protein, as long as it has the immunogenicity retaining PCV2, any Porcine circovirus type 2 ORF2 protein all can be used as the source of PCV2ORF2DNA described herein and/or polypeptide.
Preferably, the PRV (Pseudorabies virus) antigen described in bigeminy vaccine of the present invention is pseudorabies live virus, improvement/chimeric pseudorabies live virus, a kind of or several combination arbitrarily of PRV (Pseudorabies virus) gene delection virus or weak viral disease poison.Preferably, described PRV (Pseudorabies virus) antigen is pseudorabies virus gene delection SA215 strain (CCTCC, deposit number V200002), Ea strain (CGMCC, deposit number N0.0907), WKQ-001 strain (CCTCC, preserving number V200511) or pseudorabies virus low virulent strain Bartha strain, Bartha-K61 strain, Bucharest strain, BUK strain, H strain (GCMCC, preserving number is No.5013) and C strain (CCTCC, deposit number V201114) a kind of or several combination arbitrarily.
Preferably, PCV2 viral level described in bigeminy vaccine of the present invention is 10
3～10
7tCID
50/ head part; More preferably, described PCV2 viral level is 10
4～10
6tCID
50/ head part.The content of described Porcine circovirus type 2 ORF2 protein biology is 0.5～50 μ g/ head part, more preferably 5～30 μ g/ head parts, more preferably 10～20 μ g/ head parts.Described PRV (Pseudorabies virus) antigenic content is 10
5～10
7tCID
50/ head part, is preferably 10
6tCID
50/ head part.Preferably, described PRV (Pseudorabies virus) antigen is PRV (Pseudorabies virus) gene-deleted strain (SA215 strain), and content is 10
5～10
7tCID
50/ head part, is preferably 10
6tCID
50/ head part.
Optimal way of the present invention relates to the adjuvant of bigeminy vaccine, is preferably one or more the compositions of IMS1313N VG, IMS2215VG, Gel01, carbomer, propolis, ISA206, ISA15A.
More preferably, in pig gyrate virus II type of the present invention and porcine pseudorabies bigeminy vaccine, also comprise freeze drying protectant.Preferably, freeze drying protectant of the present invention, can be one or more in lactose, skim milk, gelatin, trehalose, bovine serum albumin, polyvinylpyrrolidone, sorbitol, sodium glutamate.
Preferably, the lactose of the preferred 12wt%-30wt% of freeze drying protectant of the present invention, the gelatin of 1wt%-5wt%, the more preferably 1:1(v/v of 2wt% gelatin and 15wt% lactose) mixture.
Another object of the present invention is to provide the preparation method of above-mentioned bigeminy vaccine, the present invention realizes by two kinds of technical schemes, and scheme one is that pig gyrate virus II type is carried out to deactivation, makes pig gyrate virus II type inactivated vaccine; PRV (Pseudorabies virus) antigen is mixed with freeze drying protectant, make pseudorabies freeze-dried live vaccine, then dissolve described pseudorabies freeze-dried live vaccine using described inactivated vaccine as diluent and be prepared into pig annulus II type, pseudorabies bigeminy vaccine compositions.Scheme two is that pig gyrate virus II type antigen and PRV (Pseudorabies virus) antigen are mixed in proportion, and is then mixed with into bigeminy freeze dried vaccine with freeze drying protectant.
Another object of the present invention is to provide a kind of described bigeminy vaccine in preparation prevention and/or control the application in pig gyrate virus II type disease and pseudorabies medicine, particularly, described bigeminy vaccine is being applied in the medicine of preparation prevention and/or control PCV2 clinical onset.
The beneficial effect that technical solution of the present invention is brought at least comprises:
1) contain two or more antigen, can prevent the combined vaccine of two or more disease to become the feature of vaccine research of new generation with its convenience, multiple-effect, low cost.Compared with single vaccine, combined vaccine can reduce the inoculation times of vaccine, avoids can not obtaining omnidistance immunity because leaking to plant; In addition, vaccine is scarcely heat-resisting, it is produced, transports, stores and even whole use procedure all needs to carry out at a lower temperature, i.e. so-called " cold chain ", this cold chain all linked with one another running, expense is high, make vaccine cost high, and use combined vaccine can reduce the expense that cold chain operates greatly, therefore there is significant superiority.
2) bigeminy vaccine that the prevention that prepared by pig gyrate virus II type of the present invention and PRV (Pseudorabies virus) antigen composition and use said composition and treatment pig gyrate virus II type and PRV (Pseudorabies virus) infect, not only can not produce mutual immune interference or the impact of two kinds of antigen compositions, and after the unexpected antigen mixing of finding pig gyrate virus II type and PRV (Pseudorabies virus), when protective immune response that the antigen composition of pig gyrate virus II type and PRV (Pseudorabies virus) can not only produce, also unexpected discovery pig gyrate virus II type and PRV (Pseudorabies virus) antigen have the effect of mutual enhancing immune effect, and prove as subsequent embodiment of the present invention, particularly exist wherein in a kind of situation of antigen, another kind of antigen amount reduces by half and still can maintain the immune effect of this antigen, and this exceeds those of ordinary skill in the art's expectation.
3) solved the difficulty of prior art, understanding prejudice when having changed people pig gyrate virus II type and PRV (Pseudorabies virus) being infected simultaneously, first pig gyrate virus II type and PRV (Pseudorabies virus) antigen are combined to use according to suitable ratio, and before the present invention, never people thinks that two kinds of diseases can be prevented and then these two kinds of vaccines can be combined use simultaneously;
4) bigeminy vaccine that the prevention that prepared by pig gyrate virus II type of the present invention and PRV (Pseudorabies virus) antigen composition and use said composition and treatment pig gyrate virus II type and PRV (Pseudorabies virus) infect, in the application of prevention and treatment pig gyrate virus II type disease and pig gyrate virus II type subclinical infection, can effectively prevent the transformation of pig gyrate virus II type subclinical infection to pig gyrate virus II type clinical infection, contain spreading of the state of an illness.
5) in addition, the bigeminy vaccine of the present invention's prevention and treatment pig gyrate virus II type relevant disease and PRV (Pseudorabies virus) relevant disease, preparation method is simple, the content of tiring of vaccine is high, immunity is convenient and swift, with repeatedly immunity of the prior art, at least needing to make a call to 2 pins or 3 pins could prevent and treat vaccine and the immunization method thereof of above two kinds of diseases and compare, the present invention only immunity just can prevent pig gyrate virus II type and two kinds of pathogen infections of PRV (Pseudorabies virus) for 1 time, reduce immune cost, save immune programme for children, reliable more economically.
Detailed description of the invention
For making the present invention easier to understand, describe the present invention in detail below in conjunction with embodiment, these embodiment only play illustrative effect, are not limited to range of application of the present invention, in the following example, NM specific experiment method, carries out according to normal experiment method conventionally.
" every part " of the present invention or "/head part " refers to that every pig uses amount of vaccine at every turn.Be not specifically noted part, described " every part " or "/head part " is 2ml in embodiments of the present invention.Vaccine adjuvant of the present invention includes but not limited to: Gel01(France SEPPIC), aluminium hydroxide gel, carbomer (Carbomer) (trade name Carbopol), propolis, ISA206(France SEPPIC) or ISA760VG(France SEPPIC), preferred water dissolubility vaccine adjuvant, for example Gel01(France SEPPIC).
The example of freeze drying protectant of the present invention, includes but not limited to: aqueous gelatin solution, lactose aqueous solution and composition thereof, preferably 2wt% aqueous gelatin solution mixes the mixture of gained with 1:1 (v/v) ratio with 15wt% lactose aqueous solution.
Embodiment
the preparation of embodiment 1 pig gyrate virus II type antigen
The preparation of 1 pig gyrate virus II type subunit antigen
(1) porcine circovirus 2 type ORF2 gene is introduced to melittin signal peptide gene sequence, proceed to pMD-18T carrier, the recombinant vector that acquisition comprises porcine circovirus 2 type ORF2 gene: first design primer Spe-ATG (5'-cgactagtatgacgtatccaaggaggcgt-3') and primer Hind-TAA (5'-gcaagctttattcattaagggttaagttggg-3'), the genomic DNA of the PCV2-SH strain taking preserving number as CGMCC No.2389 is as template, amplification PCV2-0RF2 gene, and the ORF2 gene clone of amplification is entered in pMD-18T carrier, obtain recombinant vector pT-ORF2.The about 730bp of PCR product size.PCR product is checked order, and from electrophoresis and sequencing result, recombinant vector pT-ORF2 is containing the SEQ ID N01 sequence in sequence table.Concrete sequence refers to patent CN101884787A.
Design 4 primers, taking pT-ORF2 as template, introduce melittin signal peptide core former times acid sequence and Restriction Enzyme SpeI and Hind III restriction enzyme site by merging PCR method at ORF2 gene order 5 ' end, primer is as follows:
HBM-F15'-tttcttacatctatgcgacgtatccaaggaggcgt-3'
HBM-R15'-ggcaacgttgactaagaatttcatactagtatcgtccac-3'
HBM-F25'-ttatggtcgtatacatttcttacatctatgcg-3'
HBM-R25'-aaacaagggcaacgttgactaagaatttc-3'
ORF2 sequence by SpeI and Hind III double digestion with melittin signal skin core former times acid sequence, and be cloned into transfer vector pFastBacTM1 (Invitrogen), form the carrier pFastBacP2 that comprises porcine circovirus 2 type restructuring ORF2 gene.With pcr amplification order-checking, from sequencing result, recombinant transfer vector pFastBacP2 is containing melittin signal skin core former times acid sequence, containing the SEQ ID N02 sequence in sequence table.Concrete sequence refers to patent CN101884787A.
(2) transfer vector that comprises porcine circovirus 2 type restructuring ORF2 gene is transformed in the competent escherichia coli cell that contains shuttle vector; the shuttle vector that acquisition comprises porcine circovirus 2 type restructuring ORF2 gene; again this shuttle vector is transfected in insect cell, obtains recombinant baculovirus vBac-P2; PFastBacP2 is transformed to the escherichia coli DH10Bac (purchased from Invitrogen company) that contains viral shuttle plasmid Bacmid, obtain recombiant plasmid Bacmid-P2; Through PCR screening, extract positive plasmid, prepare positive recombiant plasmid Bacmid-P2DNA.Adopt
rReagent transfection Sf9 cell (purchased from Invitrogen company), after pathological changes appears in cell, carries out recombinant virus plaque purification and virus amplification, then carries out PCR checking object restructuring ORF2 gene by universal primer M13F and M13R.Purification obtains recombinant baculovirus, called after vBac-P2.Recombinant baculovirus vBac-P2 is as kind of a poison in amplification, is tired after titration and is saved backup in 4 ° of C by plaque ethods.
(3) cultivation of recombinant baculovirus vBac-P2 infected insect cell and insect cell, by the insect cell High Five of vBac-P2 recombinate shape virus infection suspension culture
tM(purchased from Invitrogen company).Aseptic suspension culture High Five in the 500ml rolling bottle of the EXPRESS FIVE SFM culture medium that contains 200-350ml
tMcell, inoculating cell density is 0.3-0.6 × 10
6individual cell/mL works as cell density and reaches 1 × 10
6when individual cell/ml, inoculate each rolling bottle with recombinant virus vBac-P2, after being inoculated into recombinant baculovirus in each rolling bottle and thering is different infection multiplicity (M.O.1.) and be respectively 0.01,0.1,10 inoculation recombinant baculovirus, with 26-28 DEG C of cultivation, rotating speed is 100rpm, cultivates 4 days.
(4) porcine circovirus 2 type of recombinant baculovirus restructuring ORF2 gene expression porcine circovirus 2 type restructuring ORF2 albumen, results and Purification of Pig circovurus type 2 restructuring ORF2 albumen.Embody condition and authentication method with reference to patent CN101884787A.
(5) by culture through 10000g centrifugal 20 minutes, precipitation separation and supernatant, supernatant filters by the filter membrane of 1 μ m, then by ultrafiltration and concentration purification, ultrafilter membrane aperture is 10000 (molecular weight cut offs), sample solution after purification is through 7mM divinyl imines (BEl) deactivation recombination bacillary viral vector, and 36-48 hour, adds the sodium thiosulfate of equivalent to neutralize.Then carry out proteantigen assay by ultraviolet spectrophotometer, measurement result is 500 μ g/ml.4 DEG C of preservations.
The preparation of 2 pig gyrate virus II type totivirus inactivation antigens
The preparation of pig gyrate virus II type (SH strain) virus liquid: by covering with the PK15 cell (purchased from ATCC) of monolayer, remove cell culture fluid, PCV2 seed culture of viruses is pressed to 0.1～0.2TCID
50inoculum concentration be inoculated on PK15 cell, 37 DEG C absorption 30 minutes, add cell maintenance medium, put 37 DEG C of rotating and culturing.Observe every day 1～2 time, Growth of Cells is good, cultivates harvesting culture after 4～7 days for 36～37 DEG C, and after freeze thawing 2-3 time, measuring malicious valency result is 10
6.5tCID
50/ ml, is placed in antigen liquid-20 DEG C of following preservations.
By doughnut for virus liquid, (0.5 μ m～2 μ m) filters post and filters, and removes cell debris, and after concentrated, measuring malicious valency result is 10
7.0tCID
50/ ml adds 37 DEG C of deactivation 24h of formalin of 0.1%～0.2% again, adds in 0.1%～0.2% nertralizer and the toxicity of formaldehyde.
the preparation of embodiment 2 PRV (Pseudorabies virus) antigens
By PRV (Pseudorabies virus) gene-deleted strain, (SA215 strain is TK
--/ gE
--/ gI
--three gene delections, be disclosed in Chinese patent CN101186902, this strain is preserved in CCTCC with deposit number V200002) do suitably dilution with viral dilution liquid (the DMEM culture medium of serum-free), be 0.1 to be inoculated in ST cell (purchased from CCTCC, numbering GDC0060) cultivation by infection multiplicity (M.O.I), 37 DEG C of absorption 30min, add containing 3%(v/v) the DMEM cell maintenance medium of calf serum, cultivate 2～4 freeze thawing 2～3 times for 37 DEG C, results virus, virus titer is 10
8.7tCID
50/ ml.
the preparation of embodiment 3 pig gyrate virus II type antigens and PRV (Pseudorabies virus) antigen bigeminy vaccine
1. porcine circovirus type II inactivated vaccine dilution porcine pseudorabies freeze-dried live vaccine
Pig gyrate virus II type totivirus inactivation antigen is mixed with 1:1 ratio with water quality adjuvant IMS1313N VG, with IKA agitator with 350rpm) stirring and evenly mixing 5min makes Emulsion.According to national standard, it is tested, all conform with the regulations.Concrete vaccine proportioning is carried out with reference to table 1, establishes altogether A～G7 kind, and wherein in A, B, C, D, E, F group, pig gyrate virus II type part adopts PCV2 totivirus inactivation antigen, and in every part (2ml), content is respectively 10
3.0tCID
50, 10
4.0tCID
50, 10
6.0tCID
50, 10
6.0tCID
50, 10
7.0tCID
50with 10
6.0tCID
50.
A, B, C, D, E, G group PRV (Pseudorabies virus) part are mixed with freeze drying protectant (2wt% aqueous gelatin solution is prepared with 1:1 (v/v) ratio with 15wt% lactose aqueous solution) PRV (Pseudorabies virus) antigen with 1:1 (v/v) ratio; stirring and evenly mixing 30～60min; aseptic subpackagedly carry out lyophilizing, make pseudorabies freeze-dried live vaccine according to content proportioning in table 1.
When use, will be in A, B, C, D, E group dissolve pseudorabies freeze-dried live vaccine using porcine circovirus type II inactivated vaccine as diluent, by each group of pseudorabies live vaccine that adds to respectively lyophilizing shown in table 1, dissolving mixes.Make altogether the vaccine combination of 5 kinds of different proportionings.F group is independent porcine circovirus type II inactivated vaccine, and G group is independent PRV (Pseudorabies virus) freeze-dried live vaccine.
Table 1 pig gyrate virus II type totivirus antigen and the concrete proportioning of pseudorabies antigen
| Vaccine formulation group | PCV2 totivirus antigenic content (every part) | PRV antigenic content (every part) |
| A | 10 3.0TCID 50 | 10 5.0TCID 50 |
| B | 10 4.0TCID 50 | 10 6.0TCID 50 |
| C | 10 6.0TCID 50 | 10 6.0TCID 50 |
| D | 10 6.0TCID 50 | 5×10 5.0TCID 50 |
| E | 10 7.0TCID 50 | 10 7.0TCID 50 |
| F | 10 6.0TCID 50 | / |
| G | / | 10 6.0TCID 50 |
2. the sick and weak malicious antigen of pig gyrate virus II type subunit antigen and pseudorabies is prepared bigeminy freeze-dried live vaccine
According to H, I, J, K, L, M, N group in table 2, pig gyrate virus II type subunit antigen and PRV (Pseudorabies virus) antigen are carried out to proportioning according to content shown in table 2; then mix with 1:1 ratio with freeze drying protectant (2wt% aqueous gelatin solution is prepared with 1:1 ratio with 15wt% lactose aqueous solution); stirring and evenly mixing 30～60min; aseptic subpackagedly carry out lyophilizing, make pig gyrate virus II type, pseudorabies bigeminy freeze-dried live vaccine.O group is mixed pig gyrate virus II type subunit antigen with freeze drying protectant (2wt% aqueous gelatin solution is prepared with 1:1 ratio with 15wt% lactose aqueous solution) according to content shown in table 2 with 1:1 ratio; stirring and evenly mixing 30～60min; aseptic subpackagedly carry out lyophilizing, make pig gyrate virus II type subunit freeze dried vaccine.
Table 2 pig gyrate virus II type subunit antigen and the concrete proportioning of pseudorabies antigen
| Vaccine formulation group | PCV2 subunit antigen content (every part) | PRV antigenic content (every part) | |
| H | 0.5μg | 10 5.0TCID 50 | |
| I | 5μg | 10 6.0TCID 50 | |
| J | 10μg | 10 7.0TCID 50 | |
| K | 20μg | 10 6.0TCID 50 | |
| L | 20μg | 5×10 5.0TCID 50 | |
| M | 30μg | 10 7.0TCID 50 | |
| N | 50μg | 10 5.0TCID 50 | |
| O | 20μg | / |
the checking of embodiment 4A～O vaccine combination effectiveness
145 of sodium selenites choosing 14～21 age in days PCV2 antigens, antibody and PRV neutralizing antibody feminine gender, are divided into 29 groups, 5 every group at random.The 1st, 1 part vaccine A of 2 groups of pig musculi colli injections, the 3rd, 1 part vaccine B of 4 groups of pig musculi colli injections, the 5th, 1 part vaccine C of 6 groups of pig musculi colli injections, the 7th, 1 part vaccine D of 8 groups of pig musculi colli injections, the 9th, 1 part vaccine E of 10 groups of pig musculi colli injections, 1 part vaccine F of the 11st group of pig musculi colli injection, 1 part vaccine G of the 12nd group of pig musculi colli injection, the 13rd, 1 part vaccine H of 14 groups of pig musculi colli injections, the 15th, 1 part vaccine I of 16 groups of pig musculi colli injections, the 17th, 1 part vaccine J of 18 groups of pig musculi colli injections, the 19th, 1 part vaccine K of 20 groups of pig musculi colli injections, the 21st, 1 part vaccine L of 22 groups of pig musculi colli injections, the 23rd, 1 part vaccine M of 24 groups of pig musculi colli injections, the 25th, 1 part vaccine N of 26 groups of pig musculi colli injections, 1 part vaccine O of the 27th group of pig musculi colli injection.28th, 29 groups not immune, as negative control, isolated rearing.
Within latter 28 days, carry out piglet protest test in immunity, the 1st, 3,5,7,9,11,13,15,17,19,21,23,25,27 and 28 groups of every pigs are with 10
6.0tCID50/ml PCV2SH strain counteracting toxic substances, adopts nasal drip, every pig 2.0ml.2nd, 4,6,8,10,12,14,16,18,20,22,24,26 and 29 groups of every pigs are with 10
6.0tCID
50counteracting toxic substances is carried out in/ml PRVFa strain, adopts nasal drip, every pig 2.0ml.Observe two weeks.
As a result, after immune group 1-27 group pig counteracting toxic substances, observe and all do not find clinical symptoms.And all there is the symptoms such as fervescence, lethargy, loss of appetite, dyspnea, wherein 1 death in the 28th group of negative control pig.All dead after the 29th group of 5 pig counteracting toxic substances.Result of the test shows, in embodiment 3, the vaccine of the different antigenic contents of preparation is all effective, the attack that can resist PCV2 and PRV.
embodiment 5 pig gyrate virus II type subunit antigen, PRV (Pseudorabies virus) bigeminy Seedling compositions and single Seedling immune animal aftereffect
the test of power evaluation comparison
1. vaccine immunity: choose surface healthy, without clinical symptoms, blood-serum P CV2 polymerase chain reaction (PCR) positive, PCV2 neutralizing antibody is less than 1:4, PRV antigen and neutralizing antibody feminine gender, 45 of 4-6 piglets in age in week in subclinical infection, be divided at random 5 groups, 10 every group of immune group, 5 of matched groups.First group of immune swine circovirus type II inactivated whole virus list Seedling F, (amount that is every single Seedling F of pig immunity is 10 to 1 part
6.0tCID
50); Second group of single Seedling G of immune swine pseudorabies, 1 part (10
6.0tCID
50); Prepared by the 3rd group of immune swine circovirus type II inactivated whole virus, pseudorabies bigeminy vaccine C(embodiment 3), 1 part; The 4th group of immune swine circovirus type II inactivated whole virus, pseudorabies bigeminy vaccine D(pseudorabies antigenic content reduce by half), 1 part; The 5th group not immune, as blank.
2. antibody detection: take a blood sample weekly after immunity, after separation of serum, adopt neutralization test method to detect respectively pig circular ring virus antibody and pseudorabies antibody.Concrete testing result is as follows:
Table 2 is antibody dynamic change after pig circular ring virus vaccine F immunity piglet, table 3 is antibody dynamic change after pseudorabies live vaccine G immunity piglet, table 4 is antibody dynamic change after pig gyrate virus II type inactivated whole virus, pseudorabies bigeminy vaccine C immunity piglet, table 5 is antibody dynamic change after pig gyrate virus II type inactivated whole virus, pseudorabies bigeminy vaccine D immunity piglet, and table 6 is the dynamic change of blank group Serum Antibody.Known according to Antibody Results, after two kinds of bigeminy Seedlings immunity piglets, pig circular ring virus antibody horizontal and single Seedling immune antibody level all remain basically stable, and that the more single Seedling immune antibody of pseudorabies antibody level all promotes is faster, and antibody horizontal is relatively higher.When the piglet of bigeminy vaccine immunity in subclinical infection PCV2, in vaccine, pseudorabies antigenic content reduces by half still higher than the antibody horizontal of the single Seedling of immune swine pseudorabies only.As can be seen here, in this research, two kinds of bigeminy vaccine compositionss of preparation have synergistic function to pseudorabies antigen immune effect, and do not affect the immune effect of pig circular ring virus antigen.
Antibody dynamic change after table 2 pig circular ring virus vaccine F immunity piglet
Antibody dynamic change after table 3 pseudorabies vaccine G immunity PCV2 infection piglet
Antibody dynamic change after table 4 pig gyrate virus II type inactivated whole virus and pseudorabies bigeminy vaccine C immunity piglet
Table 5 pig gyrate virus II type inactivated whole virus, pseudorabies bigeminy vaccine D(pseudorabies antigen reduce by half) immune PCV2 infects antibody dynamic change after piglet
Table 6 blank group pig gyrate virus II type, pseudorabies antibody dynamic change
effect after embodiment 6 pig gyrate virus II type subunit antigen, PRV (Pseudorabies virus) bigeminy Seedling compositions and single Seedling immune animal
evaluation comparison test
1. vaccine immunity: choose surface healthy, without clinical symptoms, blood-serum P CV2 polymerase chain reaction (PCR) positive, PCV2 neutralizing antibody is less than 1:4, PRV antigen and neutralizing antibody feminine gender, 45 of 4-6 piglets in age in week in subclinical infection, be divided at random 5 groups, 10 every group of immune group, 5 of matched groups.First group of immune swine circovirus type II subunit list Seedling O, 1 part; Second group of single Seedling G of immune swine pseudorabies, 1 part; Prepared by the 3rd group of immune swine circovirus type II subunit, pseudorabies bigeminy vaccine K(embodiment 3), 1 part; The 4th group of immune swine circovirus type II subunit, pseudorabies bigeminy vaccine L(pseudorabies antigenic content reduce by half), 1 part; The 5th group not immune, as blank.
2. antibody detection: take a blood sample weekly after immunity, after separation of serum, adopt neutralization test method to detect respectively pig circular ring virus antibody and pseudorabies antibody.Concrete testing result is as follows:
Table 7 is antibody dynamic change after pig circular ring virus subunit vaccine O immunity piglet, table 8 is antibody dynamic change after pseudorabies live vaccine G immunity piglet, table 9 is antibody dynamic change after pig gyrate virus II type subunit, pseudorabies bigeminy vaccine K immunity piglet, table 10 is antibody dynamic change after pig gyrate virus II type subunit, pseudorabies bigeminy vaccine L immunity piglet, and table 11 is the dynamic change of blank group Serum Antibody.Known according to Antibody Results, after two kinds of bigeminy Seedlings immunity piglets, pig circular ring virus antibody horizontal and single Seedling immune antibody level all remain basically stable, and that the more single Seedling immune antibody of pseudorabies antibody level all promotes is faster, and antibody horizontal is relatively higher.When the piglet of bigeminy vaccine immunity in subclinical infection PCV2, in vaccine, pseudorabies antigenic content reduces by half still higher than the antibody horizontal of the single Seedling of immune swine pseudorabies only.As can be seen here, in this research, two kinds of bigeminy vaccine compositionss of preparation have synergistic function to pseudorabies antigen immune effect, and do not affect the immune effect of pig circular ring virus antigen.The phenomenon that this and embodiment 5 obtain is basically identical.
Antibody dynamic change after table 7 pig circular ring virus subunit vaccine O immunity PCV2 infection piglet
Antibody dynamic change after table 8 pseudorabies vaccine G immunity PCV2 infection piglet
Antibody dynamic change after table 9 pig gyrate virus II type subunit, pseudorabies bigeminy vaccine K immunity PCV2 infection piglet
Table 10 pig gyrate virus II type subunit, pseudorabies bigeminy vaccine L(pseudorabies antigen reduce by half) immune PCV2 infects antibody dynamic change after piglet
Table 11 blank group pig gyrate virus II type, pseudorabies antibody dynamic change
embodiment 7 pig gyrate virus II types, PRV (Pseudorabies virus) are to the pathogenic synergism of pig
Choose all 40 of negative 21～28 age in days piglets of health, pig gyrate virus II type and PRV (Pseudorabies virus) antigen-antibody, be divided into 4 groups, 10 every group, isolated rearing respectively.It is 1.0 × 10 that first group of every piglet shot total amount through collunarium
2tCID
50pCV2 virus liquid, observe its clinical condition.It is 1.0 × 10 that second group of every piglet shot total amount through collunarium
1tCID
50pRV(Fa strain) virus liquid, observe its clinical condition.It is 1.0 × 10 that the 3rd group of every piglet first shot total amount through collunarium
2tCID
50pCV2 virus liquid, after 14 days, shooting total amount again through collunarium is 1.0 × 10
1tCID
50pRV(Fa strain) virus liquid, observe its clinical condition.The 4th group as blank, not counteracting toxic substances.
Above-mentioned result of the test shows: the 4th group of blank piglet is all normal, has no any clinical symptoms.First group of piglet is through infecting separately PCV2, and mortality rate is 0, does not all show any obvious clinical symptoms, has after testing the existence of pig gyrate virus II type cause of disease.Second group of piglet is through infecting separately PRV(Fa strain), also there is not death, all do not show any obvious clinical symptoms, for the existence of pseudorabies cause of disease being detected yet.The 3rd group of piglet all do not show any obvious clinical symptoms in 2 weeks after infecting PCV2, had after testing the existence of pig gyrate virus II type cause of disease; After low content PRV infects, have 70%(7/10) there is the wherein 2-3 kind clinical symptoms such as palor, lassitude, dyspnea, cough in piglet, but the existence of pig gyrate virus II type cause of disease still can only be detected, still can't detect the existence of pseudorabies cause of disease.This success of the test copies the subclinical infection of pig gyrate virus II type, and confirms that PRV infection has important synergism in piglet is converted into clinical onset process by PCV2 subclinical infection.
embodiment 8 pig gyrate virus II types, pseudorabies bigeminy compositions are being prevented the application of PCV2 clinical onset
Choose surface healthy, without clinical symptoms, continuous 6 weeks blood-serum P CV2 polymerase chain reaction (PCR) positives, and in every milliliter of serum PCV2 content lower than 10
6individual genome copy, 30 of the 6-8 piglets in age in week in subclinical infection, are divided into 3 groups, 10 every group at random.First group with collunarium approach artificial challenge 1.0 × 10
1tCID
50pRV(Fa strain) virus liquid.Second group of first immune swine circovirus type II, PRV (Pseudorabies virus) bigeminy Seedling, after 21 days, then with collunarium approach artificial challenge 1.0 × 10
1tCID
50pRV (Pseudorabies virus).The 3rd group not immune, not counteracting toxic substances, isolated rearing.Choose in addition healthyly without clinical symptoms, all 10 of negative 6-8 piglets in age in week of pig gyrate virus II type, pseudorabies antigen, antibody, are designated as the 4th group, with collunarium approach artificial challenge 1.0 × 10
1tCID
50pRV(Fa strain) virus liquid, quarantine.
More than test, 70%(7/10 in first group) piglet there is the wherein 2-3 kind symptom such as palor, lethargy, cough, dyspnea, nervous symptoms, 10% death.PCR detects in 100% piglet serum PCV2 content all higher than 10
6individual genome copy, and PRV (Pseudorabies virus) detection is negative.2nd, all there is not any clinical onset symptom in 3 and 4 groups of piglets.Also all there is not any clinical onset symptom in piglet.Result of the test shows, the pig gyrate virus II type of this research preparation, the generation that PRV (Pseudorabies virus) bigeminy vaccine can effectively prevent PCV2 clinical onset, and porcine pseudorabies can be prevented or treat to the while.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any amendment of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. pig gyrate virus II type and a porcine pseudorabies bigeminy vaccine, is characterized in that, comprises at least one pig gyrate virus II type antigen and at least one PRV (Pseudorabies virus) antigen.
2. bigeminy vaccine according to claim 1, is characterized in that, described pig gyrate virus II type antigen is PCV2 totivirus and/or polypeptide or subunit containing PCV2 immunogenicity aminoacid sequence.
3. bigeminy vaccine according to claim 1 and 2, it is characterized in that, described PRV (Pseudorabies virus) antigen is pseudorabies live virus, improvement/chimeric pseudorabies live virus, a kind of or several combination arbitrarily of PRV (Pseudorabies virus) gene delection virus or weak viral disease poison.
4. bigeminy vaccine according to claim 1 and 2, is characterized in that, the content of described PRV (Pseudorabies virus) antigen is 10
5～10
7tCID
50/ head part, the content of described pig gyrate virus II type antigen is 10
3～10
7tCID
50/ head part.
5. according to the bigeminy vaccine described in any one in claim 1～4, it is characterized in that, also comprise vaccine adjuvant, described adjuvant is one or more the compositions of IMS1313N VG, IMS2215VG, Gel01, carbomer, propolis, ISA206 or ISA15A.
6. according to the bigeminy vaccine described in any one in claim 1～5, it is characterized in that, also comprise freeze drying protectant, described freeze drying protectant is the 1:1(v/v of 2wt% gelatin and 15wt% lactose) mixture.
7. a method of preparing pig gyrate virus II type and porcine pseudorabies bigeminy vaccine, is characterized in that, comprises the steps: pig gyrate virus II type to carry out deactivation, makes pig gyrate virus II type inactivated vaccine; PRV (Pseudorabies virus) antigen is mixed with freeze drying protectant, make pseudorabies freeze-dried live vaccine, then dissolve described pseudorabies freeze-dried live vaccine using described inactivated vaccine as diluent.
8. a method of preparing pig gyrate virus II type and porcine pseudorabies bigeminy vaccine, is characterized in that, comprises the steps: pig gyrate virus II type antigen and PRV (Pseudorabies virus) antigen to be mixed in proportion, and then mixes with freeze drying protectant and carries out lyophilizing.
9. the application of the bigeminy vaccine described in any one in preparation prevention and/or control pig gyrate virus II type disease and pseudorabies medicine in claim 1～6.
10. the application of the bigeminy vaccine described in any one in the medicine of preparation prevention and/or control PCV2 clinical onset in claim 1～6.
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