CN1861195A - Fowl vaccine immunopotentiator of chicken interleuken-18, and its application - Google Patents
Fowl vaccine immunopotentiator of chicken interleuken-18, and its application Download PDFInfo
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- CN1861195A CN1861195A CN 200610050236 CN200610050236A CN1861195A CN 1861195 A CN1861195 A CN 1861195A CN 200610050236 CN200610050236 CN 200610050236 CN 200610050236 A CN200610050236 A CN 200610050236A CN 1861195 A CN1861195 A CN 1861195A
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Abstract
A chicken's interleukin 18 as the immunopotentiator of fowls' vaccine is the aqueous solution of the eukaryon expression plasmid, which is a recombinant carrier composed of the p CI as the eukaryon expression carrier and interleukin 18 genes. It has high bioactivity and low cost.
Description
Technical field
The invention belongs to biotechnology, relate to a kind of immunostimulant of bird vaccine, relate in particular to chicken interleukin-2 18 bird vaccine immunostimulant and application thereof.
Background technology
(interleukin 18 for interleukin-18, IL-18) be from the toxic shock mouse liver extracting solution that propionibacterium acnes (Propioibacteriumacnes) and lipopolysaccharide Combined Treatment are crossed, to separate first nineteen ninety-fives such as Okamura and clone a kind of novel cytokine of coming out, because this factor has intensive IFN-γ and induces ability, therefore at first its called after IFN-γ is induced the factor (IFN-γ inducingfactor, IGIF).Research subsequently finds that further IL-18 not only can induce IFN-γ, and has a various biological function, as promote the Th1 cell proliferation, induce the generation of Th1 type cytokines such as GM-CSF, IL-2 and TNF-α, and the lymphocytic differentiation and proliferation of promotion T, the various biological functions such as cytotoxicity of the cytotoxicity of the Th1 type cell of enhancing Fas mediation and NK cell, CTL cell are so IL-18 is a kind of multiple-effect cytokine.Usbio in 1996 etc. are with the Mus IGIF cDNA of people IGIF that has been probing pin clone, and subsequently, some other mammal is also successfully cloned in succession as the IL-18 of rat, pig, horse, cattle, sheep, Canis familiaris L..But relevant in this respect is that the birds IL-18 research of representative lags behind people and mammal far away with the chicken, and reason has two: know little about it to the chicken cell factor and regulator gene structure (1), and do not find chicken CD4 so far
+The Th1 of Txibao and the hypotype of Th2 exist; (2) differing greatly of chicken IL-18 and mammal IL-18, and IL-18 has very strong species specificity, makes the research that the many technology used in mammalian cell and method all can't be effectively applied to birds IL-18.
Along with developing rapidly of scale intensification aviculture, China has become maximum in the world egg and Carnis Gallus domesticus manufacturing country at present.In the evolution of aviculture, whether the successful control of infectious disease has become the key factor of restriction aviculture development.Simultaneously some zoonosis such as bird flu when bringing about great losses to aviculture also serious harm human beings'health, the generation of prevention and control fowl infection promotes that for the propagation of cutting off zoonosis human health is significant.Vaccination is the main means of preventing fowl infection to take place at present, but this method can not be controlled the generation of some infectious disease fully, and the intensive farm immuning failure happens occasionally, and causes enormous economic loss to aviculture.Using immunostimulant is to improve immune effect of vaccine, the pathogenetic effective way of control infection, but present widely used immunostimulant is a synthetic, costs an arm and a leg, and has limited the scope of its use, therefore, it is imperative to develop a kind of bird immunity reinforcing agent of cheapness.
Summary of the invention
Technical problem to be solved by this invention provides a kind of chicken interleukin-2 18 bird vaccine immunostimulants, it is the aqueous solution of chicken interleukin-2 18 eukaryon expression plasmids (pCI-IL-18), its concentration is 1-20 μ g/ μ l, this eukaryon expression plasmid is as carrier for expression of eukaryon with pCI, the recombinant vector that contains interleukin-18 (IL-18) gene of SEQ No 1 coded sequence, the preservation of this recombinant vector number is CGMCCNo.1599, date saved is on January 25th, 2006,591 nucleotide of this sequence total length, type is a nucleotide, topological structure is a line style, and chain is a strand, and total length is the coding region.
Be cloned into chicken IL-18 cDNA first until the talents such as Kirsten Schenider in 2000, the research for chicken IL-18 at present relatively lags behind.The present invention studies the molecular biology of chicken IL-18, successfully clone chicken IL-18 gene, made up the eukaryon expression plasmid of this gene, not only help to understand infectious disease and comprise the pathogenesis of immunosuppressive disease and the immunologic mechanism of vaccine, and novel gene engineered vaccine and novel fowl are all had important theoretical and using value with development of immunostimulant etc.
Eukaryon expression plasmid of the present invention with pCI as expression vector, except the early promoter/enhancer that has CMV, also contain the intron sequences that derives from beta globin gene, the existence of this sequence is the expression efficiency of enhancing gene obviously, than strong 10-40 of vector expression ability such as this intron sequences of nothing doubly.
The invention discloses a kind of preparation method of chicken interleukin-2 18 immunostimulants, step is: the 1) clone of the extracting of inducing mode, RNA of chicken interleukin-2 18 and cDNA total length thereof; 2) comprise the structure of the expression plasmid of chicken interleukin-2 18 full-length genes; 3) reorganization chicken interleukin-2 18 proteic acquisition modes; 4) preparation of reorganization chicken interleukin-2 18 expression plasmids and protein immunization reinforcing agent.
Bird vaccine immunostimulant provided by the invention can be applicable to comprise vaccines such as chicken infectivity bursa of Fabricius virus dna vaccination, infectious bronchitis virus dna vaccination, Avian pneumo-encephalitis virus dna vaccination, iltv dna vaccine, bird flu virus dna vaccination, chicken anemia factor dna vaccination, chicken coccidiosis dna vaccination.
The immunostimulant of bird vaccine provided by the invention can also be for containing above-mentioned eukaryon expression plasmid expressed chicken interleukin-2-18 albumen in eukaryotic cell or animal body, and the shared weight ratio of chicken interleukin-2 18 albumen is 1-20%.
The immunostimulant of this bird vaccine can be applicable to attenuated vaccine, recombinant vaccine and dna vaccinations such as chicken infectivity bursa of Fabricius virus, infectious bronchitis virus, Avian pneumo-encephalitis virus, infectious laryngotracheitis virus, bird flu virus, chicken anemia factor, chicken coccidiosis.
Chicken leukocyte eukaryon expression plasmid pCI-IL-18 provided by the invention and reorganization chicken interleukin-2 18 albumen show as the advantage of the immunostimulant of bird vaccine: reorganization and interleukin-18 albumen that (1) eukaryotic expression system is produced, not only have biologic activity, and than other artificial adjuvant cheapness; (2) can strengthen the cellullar immunologic response of vaccine-induced body and humoral immunoresponse(HI) Ah, and prolong immunity and persistent period, also can lower the using dosage of vaccine, thereby can reduce cost; (3) chicken interleukin-2 18 has natural resistant effect, can strengthen the resistance of birds to infectious disease.(4) the invention provides the method that a kind of chicken interleukin-2 18 induces, the preparation method of chicken interleukin-2 18 as immunostimulant is provided simultaneously, this method prepares simple, with low cost, easy to use, easy to utilize.
Description of drawings
Fig. 1: the present invention uses RT-PCR to amplify the histogram of chicken interleukin-2-18 gene of 591bp.
Fig. 2: the enzyme action of chicken interleukin-2 provided by the invention-18 (IL-18) recombiant plasmid T-IL-18 is identified figure.
Fig. 3: the design of graphics of chicken interleukin-2 provided by the invention-18 (IL-18) recombiant plasmid T-IL-18.
Fig. 4: the present invention uses RT-PCR to amplify the histogram of chicken interleukin-2-18 coding gene.
Fig. 5: chicken interleukin-2 provided by the invention-18 (IL-18) gene eucaryon expression plasmid pCI-IL-18 enzyme action is identified figure.
Fig. 6: the design of graphics of chicken interleukin-2 provided by the invention-18 (IL-18) gene eucaryon expression plasmid pCI-IL-18.
Fig. 7: the design of graphics of chicken interleukin-2 provided by the invention-18 (IL-18) gene prokaryotic plasmid pGEX-4T-2-IL-18.
Fig. 8: the enzyme action of chicken interleukin-2 provided by the invention-18 (IL-18) gene prokaryotic plasmid pGEX-4T-2-IL-18 is identified figure.
Fig. 9: SDS-PAGE that chicken interleukin-2 provided by the invention-18 (IL-18) is expressed in the Vero cell and Western blot analysis chart.
Figure 10: SDS-PAGE that chicken interleukin-2 provided by the invention-18 (IL-18) is expressed in prokaryotic cell and Western blot analysis chart.
Figure 11: the present invention adopts mtt assay to the active detection figure of reorganization chicken interleukin-2-18 (IL-18).
Figure 12: chicken interleukin-2 provided by the invention-18 (IL-18) albumen strengthens the immunologic function datagram of chicken body.
Figure 13: chicken interleukin-2 provided by the invention-18 (IL-18) eukaryon expression plasmid strengthens the immunologic function datagram of chicken body.
The specific embodiment
The present invention is further described with accompanying drawing in conjunction with the embodiments.
Present embodiment has been described the preparation method of chicken interleukin-2 provided by the invention-18 (IL-18) gene, may further comprise the steps:
(1) the fresh spleen of the aseptic separation 14 age in days Zhejiang area kind Xiaoshan chickling of the preparation of chicken spleen lymphocyte suspension grinds the back with tissue grinder and adds no Ca
2+, Mg
2+Hank ' s liquid, the centrifugal 20min washed twice of 1300g is again with RPMI1640 culture fluid washed twice.Behind trypan blue (1%Trypan blue) dyeing viable count, cell dilution is become certain density single cell suspension with RPMI1640 (containing 10% calf serum, 1% penicillin and 1% streptomycin) culture fluid.
(2) the synthetic chicken IL-18cDNA of RT-PCR cultivates spleen lymphocyte by the external evoked IL-18 optimum condition of screening, through final concentration respectively the LPS stimulation 5h of 10 μ g/ml, 10h, 24h, 48h extracts cell, and Trizol test kit one-step method is extracted the total RNA of lymphocyte.Design a pair of primer:
P1(5’-ATGAGCTGTGAAGAGATCG-3’)
P2(5’-TCATAGGTTGTGCCTTTCA-3’)
Primer is synthetic by Shanghai biological engineering company limited.The extractive total RNA of different incubation times uses superscript respectively
TMPreamplification System reverse transcription test kit (can available from Gibco company) carries out reverse transcription.Reverse transcription product carries out PCR by Expand High Fidelity PCR System (can available from Gibco company), and the cycling condition through optimizing is: 94 ℃ of pre-degeneration 2min, 94 ℃ of degeneration 45s, 52 ℃ of annealing 1min, 72 ℃ are extended 45s, and 72 ℃ are extended 10min, totally 30 circulations.The PCR product is identified with 1% agarose gel electrophoresis.
The result shows: the chicken spleen lymphocyte is after LPS stimulates different time, extracted total RNA is carried out RT-PCR, the result shows at LPS to be stimulated about 10 hours, can amplify about 600bp specific fragment (referring to accompanying drawing 1, the chicken IL-18 gene band of 1 expression 600bp size in the accompanying drawing 1; 2 expression DNA MarkerDL2000), after the PCR product reclaims, directly clone in pGEM-T easy Vector (can available from Promega company), with recombiant plasmid called after T-IL-18 (referring to accompanying drawing 2,1 expression EcoR I single endonuclease digestion recombiant plasmid T-IL-18 result in the accompanying drawing 2,2 show Not I single endonuclease digestion recombiant plasmid T-IL-18 result, 3 expression PCR qualification results, 4 expression DNA Marker), carry out single endonuclease digestion respectively with EcoR I and Not I, as seen size is respectively the band about 3.0kb and 600bp, and PCR identifies the band (referring to 3 swimming lanes in the accompanying drawing 2) that can amplify about 600bp.
(3) clone of chicken IL-18 gene and sequence obtain low melting point glue and reclaim the purpose fragment, utilize T-A clone strategy to be directly connected in pGEM-T easy Vector (referring to accompanying drawing 3), connect product Transformed E .coli Dh5 α competent cell, be incubated at the LB flat board that contains Amp, IPTG and X-gal (all can available from Gibco company), the picking white colony carries out that PCR identifies and the enzyme action evaluation.Sequence Identification is undertaken by Shanghai Bo Ya biotech company.
Sequencing is the result show: clone's Xiaoshan chicken IL-18cDNA total length is totally 591 nucleotide, 196 aminoacid of encoding, and its aminoacid sequence is referring to SEQ No 1.
Present embodiment has been described the preparation method of chicken interleukin-2 18 (IL-18) gene eucaryon expression plasmid pCI-IL-18, and its step comprises:
(1) amplification of chicken IL-18cDNA is at the two ends of above-mentioned chicken IL-18 gene redesign primer (total length is 591bp altogether, 196 aminoacid of encoding)
P3(5’-CG
GAATTCATGAGCTGTGAAGAGATCG-3’)
P4(5’-CC
GTCGACTCATAGGTTGTGCCTTTCA-3’)
The primer two ends are introduced restriction enzyme site EcoR I (P3 line place) and SalI (P4 line place) respectively, and primer is synthetic in Shanghai Bo Ya biotech company.With T-IL-18 is masterplate, and P3 and P4 are primer, by the cDNA:94 ℃ of pre-degeneration 2min of following PCR cyclic program amplification chicken IL-18, and 94 ℃ of degeneration 45s, 55 ℃ of annealing 1min, 72 ℃ are extended 45s, and 72 ℃ are extended 10min, totally 30 circulations.The PCR product is identified with 1% agarose gel electrophoresis.
(2) the structure low melting point glue of chicken interleukin-2 18 (IL-18) gene eucaryon expression plasmid pCI-IL-18 reclaims the chicken IL-18 gene of 591bp, behind EcoRI and the SalI double digestion, directly is cloned into EcoRI and the SalI multiple clone site place of pCI.
The result shows: the chicken IL-18 gene that will comprise 591bp is (referring to accompanying drawing 4, the purpose band of 1,2 expression 591bp in the accompanying drawing 4,3 expression DNA Marker) be cloned into pCI, be built into pCI-IL-18 eukaryon expression plasmid (referring to accompanying drawing 6), EcoR I and SalI double digestion can cut out the band about 4.0kb and 600bp, PCR can amplify the band (referring to accompanying drawing 5, the purpose fragment of 1 expression EcoRI and SalI double digestion in the accompanying drawing 5,2 expression DNA Marker) of 600bp.
(3) structure of chicken interleukin-2 18 (IL-18) gene prokaryotic plasmid pGEX-4T-2-IL-18
At the two ends of above-mentioned chicken IL-18 gene redesign primer (total length is 591bp altogether, 196 aminoacid of encoding)
P5(5’-CGC
GGATCCGCCTTTTGTAAGGATAAAAC-3’)
P6(5’-CCC
CTCGAGTCATAGGTTGTGCCTTCATT-3’)
The primer two ends are introduced BamHI (P5 line place) and XhoI (P6 line place) restriction enzyme site respectively, carry out PCR.
Primer is synthetic in Shanghai Bo Ya biotech company.With T-IL-18 is masterplate, and P5 and P6 are primer, by the cDNA:94 ℃ of pre-degeneration 2min of following PCR cyclic program amplification chicken IL-18, and 94 ℃ of degeneration 45s, 55 ℃ of annealing 1min, 72 ℃ are extended 45s, and 72 ℃ are extended 10min, totally 30 circulations.The PCR product is identified with 1% agarose gel electrophoresis.
Low melting point glue reclaims the chicken IL-18 gene of 591bp, behind BamHI and the XhoI double digestion, directly is cloned into BamHI and the XhoI multiple clone site place of pGEX-4T-2.
The result shows: the chicken IL-18 gene (referring to SEQ No 1) that will comprise 591bp is cloned into pGEX-4T-2, be built into pGEX-4T-2-IL-18 prokaryotic expression plasmid (seeing accompanying drawing 7), BamHI and XhoI double digestion can cut out the band about 4.0kb and 600bp, PCR can amplify the band of 600bp and (see accompanying drawing 8, the 1st, the pGEX-4T-2 empty plasmid, the 2 and 3pGEX-4T-2 recombiant plasmid BamHI/XhoI double digestion result, the 4th, PCR result, the 5th, DNA Marker).
Present embodiment has been described the proteic preparation method of reorganization chicken interleukin-2 18 (IL-18).May further comprise the steps:
(1) expression of chicken interleukin-2 18 (IL-18) gene eucaryon expression plasmid pCI-IL-18 in mammal Vero cell
The operating procedure of the expression of liposome-mediated chicken pCI-IL-18 in mammal Vero cell is as follows:
On 6 porocyte culture plates, inoculate 2 * 10
5Individual cell contains the culture medium of hyclone in 2ml; 37 ℃ of 5%CO
2The cell that incubator is cultivated 18-24h to 40-60% converges: prepare liquid in the Eppendorf pipe: Slolution A: 8 μ g DNA are diluted in the OPTI-MEM I ReducedSerum Medium culture medium that 100 μ l do not contain serum (can available from Gibco company); Solution B: dilute 10 μ l LipofectinReagent (can available from Life Technology company) and do not contain in the OPTI-MEM IReduced Serum Medium culture medium of serum to 100 μ l.In order to reach the transfection effect of best transfection, DNA can not surpass 20 μ g/ml and Lipofectin Reagent can not surpass 200 μ g/ml; With Slolution A and Solution B mixing gently, room temperature is placed 10-15min, and solution may become turbid, but can not influence transfection effect subsequently; The Vero cell that does not contain the OPTI-MEM I Reduced Serum Medium culture medium washing cultivation of serum with 2ml, add 0.8ml and do not contain the OPTI-MEM I Reduced Serum Medium culture medium of serum to DNA-Lipofectin mixing species, behind the mixing, mixture is layered on the Vero cell gently, can not added with antibiotic in whole process; 37 ℃ of 5%CO
2Incubator is cultivated 6h; The DNA-Lipofectin mixture that inclines adds the OPTI-MEM I Reduced Serum Medium culture medium that 2ml contains 5% hyclone and continues to cultivate 48-72h, detection of active.Expression product carries out the SDS-PAGE electrophoresis to be identified.
The result is referring to Fig. 9, after recombiant plasmid pCI-IL-18 is purified, behind transfection Vero cell 72h under the mediation of liposome, collecting cell supernatant and cell pyrolysis liquid, carrying out SDS-PAGE electrophoresis and Western blot analyzes, the molecular weight size that found that the reorganization chicken IL-18 of vivoexpression is that (see accompanying drawing 9, the left side is the SDS-PAGE electrophoresis to 19.5Kda, and the right side is that Western blot analyzes.1 expression albumen marker; Swimming lane 2 and 5 is lymphocyte deposits of pCI-ChIL-18-F transfection; Swimming lane 4 and 7 is lymphocyte deposits of pCI-ChIL-18-M transfection; Swimming lane 3 and 5 is control cells of pCI transfection).
(2) expression in chicken interleukin-2 18 (IL-18) the gene prokaryotic plasmid pGEX-4T-2-IL-18 escherichia coli
(1) a small amount of of ChIL-18 fusion rotein is expressed
1) pGEX-4T-2-ChIL-18 positive colony and matched group pGEX-4T-2 are inoculated in the LB fluid medium that 5ml contains ampicillin (50 μ g/ml), put 37 ℃ of shaking tables, shake with the 300rpm rotating speed and spend the night.
2) culture that will spend the night adds the LB fluid medium that contains ampicillin (50 μ g/ml) with 1: 100 ratio, puts 37 ℃ of shaking tables, cultivates with the 250rpm rotating speed.
3) treat bacterial growth to OD600=0.4-1.0, add IPTG, making final concentration is 0.1mM-1mM.
4) cultivate 1h-5h for 28-37 ℃, collect antibacterial.
5) 5000 * g, centrifugal 15min.
6) abandon supernatant, with the resuspended bacterial precipitation of an amount of 1 * PBS.
7) the centrifugal 15min of 5000 * g abandons supernatant, removes culture fluid fully.
8) with the resuspended precipitation of 200 μ l PBS.Use the ultrasonic treatment antibacterial, become translucent until solution.
Collect supernatant and carry out SDS-PAGE, observe the protein expression situation with the antibacterial sediment.
(2) SDS-PAGE and Western-blot analysis fusioning protein expression
The result is referring to Figure 10, SDS-PAGE electrophoresis and Western-blot analyze, the molecular weight size that found that the warm chicken IL-18 of reorganization of vivoexpression is about 45Kda (in the accompanying drawing 10, the left side is the SDS-PAGE electrophoresis, the right side is analyzed for Western blot, the 1st, albumen marker, 2 and 4 is that SDS-PAGE analyzes ChIL-18 expressing fusion protein situation, the 3rd, the pGEX-4T-2 negative control, 5 and 7western-blot analyze the ChIL-18 expressing fusion protein, the 6th, the western-blot of pGEX-4T-2 negative control analyzes).
(3) reorganization chicken interleukin-2 18 active detections
The active detection of reorganization chicken IL-18 undertaken by mtt assay, and spleen lymphocyte is diluted to 8 * 10
6Individual/ml, after adding the ConA cultivation 24h of 10mg/L, collect lymphocyte, wash twice with PBS, the RPMI 1640 that reuse contains 0.05mol/L α-Metnylamannoside (can available from sigma company) cultivates 30min, PBS washing three times is resuspended among the RPMI 1640, behind the viable count, add IL-18 sample to be checked, each sample repeats 5 repeating holes, continues to cultivate 48 hours, adds 5g/L MTT solution (can available from sigma company) 10 μ l, continue to cultivate 4 hours, every hole adds 10%SDS 100 μ l, reacts 2 hours, measures the arithmetical average of the OD value at λ=570nm place and represents.
The result detects the active result of chicken IL-18 referring to accompanying drawing 11:MTT method and shows: detected IL-18 activity is apparently higher than the inductive T lymphocyte of ConA supernatant in the Vero of pCI-IL-18 transfection group cell pyrolysis liquid; Also can detect the IL-18 activity in the Vero cell conditioned medium liquid of pCI-IL-18 transfection group, illustrate that the chicken IL-18 that expresses is a secreted protein; The cell pyrolysis liquid of negative control group and cell conditioned medium liquid all do not have specific IL-18 activity (the inductive T lymphocyte of 1 expression ConA supernatant in the accompanying drawing 11; Supernatant behind the B pCI-IL-18 transfection Vero cell; C pCI-IL-18 transfection Vero cell lysate; The D negative control; E pCI transfection Vero cell conditioned medium liquid; F pCI transfection Vero cell lysate).
Present embodiment has been described the immunologic function that chicken interleukin-2 18 (IL-18) albumen provided by the invention and chicken interleukin-2 18 eukaryon expression plasmid pCI-IL-18 strengthen the chicken body, and then has strengthened the immune effect of various bird vaccines and the chicken body resistivity to various cause of diseases.These bird vaccines comprise attenuated vaccine, recombinant vaccine and dna vaccinations such as chicken infectivity bursa of Fabricius virus, infectious bronchitis virus, Avian pneumo-encephalitis virus, marek's disease virus, infectious laryngotracheitis virus, bird flu virus, chicken anemia factor, chicken coccidiosis.
(1) chicken interleukin-2 18 (IL-18) albumen has strengthened the immunologic function of chicken body
The non-immunity of 14 ages in days comes 40 of Hangzhoupro chickens, be divided into two groups, every group 20, promptly inject the protein groups and the normal control group of chicken interleukin-2 18 (IL-18), each is organized to cut open in chicken 4 weeks after injection and kills, aseptic collection spleen, thymus, fabricius bursa, peripheral blood are made single lymphocyte suspension, and mtt assay is measured the breeder reaction of spleen, thymus, peripheral blood T proliferation of lymphocytes and fabricius bursa B cell, and compare with the normal control group.The lymphocyte of getting the above-mentioned preparation of 50 μ l adds 50 μ l ConA solution (40 μ l/ml), 5%CO respectively
2Incubator was cultivated 48 hours for 40 ℃; Above-mentioned culture all adds 10 μ l MTT (5mg/ml), continues to cultivate to add 10%SDS-0.01%HCl solution 100 μ l after 3 hours, and mixing continued to cultivate after 2 hours, with the control wells zeroing, measured the OD value at λ=570nm place, and each sample is established 5 repeating holes.Lymphproliferation response in certain time is with the OD of 5 chickens
570Average is represented.Carry out statistical procedures by method of analysis of variance on cell proliferation response data, significant level is P＜0.05.
The result is referring to Figure 12, and A represents to inject the splenic T proliferation of lymphocytes after 4 weeks of chicken IL-18 albumen in accompanying drawing 12; B represents the splenic T proliferation of lymphocytes of normal control chicken (not injecting chicken IL-18 albumen) after 4 weeks; C represents to inject the thymus T proliferation of lymphocytes after 4 weeks of chicken IL-18 albumen; D represents the thymus T proliferation of lymphocytes of normal control chicken (not injecting chicken IL-18 albumen) after 4 weeks; E represents to inject the peripheral blood T proliferation of lymphocytes after 4 weeks of chicken IL-18 albumen; F represents the peripheral blood T proliferation of lymphocytes of normal control chicken (not injecting chicken IL-18 albumen) after 4 weeks; G represents to inject the breeder reaction of the fabricius bursa bone-marrow-derived lymphocyte after 4 weeks of chicken IL-18 albumen; H represents the breeder reaction of the fabricius bursa bone-marrow-derived lymphocyte of normal control chicken (not injecting chicken IL-18 albumen) after 4 weeks.Referring to accompanying drawing 12: the breeder reaction of spleen, thymus, peripheral blood T proliferation of lymphocytes and fabricius bursa B cell behind the albumen of chicken body injection chicken interleukin-2 18 (IL-18) all is significantly higher than normal control group (P＜0.05), illustrate that chicken interleukin-2 18 (IL-18) albumen has the effect of enhancing chicken body immunity function merit.Therefore, chicken interleukin-2 18 (IL-18) albumen can improve progeny's effect of birds attenuated vaccine, inactivated vaccine and recombinant vaccine, and these bird vaccines comprise attenuated vaccine, recombinant vaccine and dna vaccinations such as chicken infectivity bursa of Fabricius virus, infectious bronchitis virus, Avian pneumo-encephalitis virus, marek's disease virus, infectious laryngotracheitis virus, bird flu virus, chicken anemia factor, chicken coccidiosis.
(2) chicken interleukin-2 18 (IL-18) plasmid has strengthened the immunologic function of chicken body
The non-immunity of 14 ages in days comes 40 of Hangzhoupro chickens, be divided into two groups, every group 20, promptly inject the plasmid group and the normal control group of chicken interleukin-2 18 (IL-18), each is organized to cut open in chicken 4 weeks after injection and kills, aseptic collection spleen, thymus, fabricius bursa, peripheral blood are made single lymphocyte suspension, and mtt assay is measured the breeder reaction of spleen, thymus, peripheral blood T proliferation of lymphocytes and fabricius bursa B cell, and compare with the normal control group.The lymphocyte of getting the above-mentioned preparation of 50 μ l adds 50 μ l ConA solution (40 μ l/ml) respectively, and the 5%CO2 incubator was cultivated 48 hours for 40 ℃; Above-mentioned culture all adds 10 μ l MTT (5mg/ml), continues to cultivate to add 10%SDS-0.01%HCl solution 100 μ l after 3 hours, and mixing continued to cultivate after 2 hours, with the control wells zeroing, measured the OD value at λ=570nm place, and each sample is established 5 repeating holes.Lymphproliferation response in certain time is represented with the OD570 average of 5 chickens.Carry out statistical procedures by method of analysis of variance on cell proliferation response data, significant level is P＜0.05.
The result is referring to accompanying drawing 13, and A represents to inject the splenic T proliferation of lymphocytes after 4 weeks of chicken IL-18 plasmid in the accompanying drawing 13; B represents the splenic T proliferation of lymphocytes of normal control chicken (not injecting chicken IL-18 plasmid) after 4 weeks; C represents to inject the thymus T proliferation of lymphocytes after 4 weeks of chicken IL-18 plasmid; D represents the thymus T proliferation of lymphocytes of normal control chicken (not injecting chicken IL-18 plasmid) after 4 weeks; E represents to inject the peripheral blood T proliferation of lymphocytes after 4 weeks of chicken IL-18 plasmid; F represents the peripheral blood T proliferation of lymphocytes of normal control chicken (not injecting chicken IL-18 plasmid) after 4 weeks; G represents to inject the breeder reaction of the fabricius bursa bone-marrow-derived lymphocyte after 4 weeks of chicken IL-18 plasmid; H represents the breeder reaction of the fabricius bursa bone-marrow-derived lymphocyte of normal control chicken (not injecting chicken IL-18 plasmid) after 4 weeks.Referring to accompanying drawing 13: the breeder reaction of spleen, thymus, peripheral blood T proliferation of lymphocytes and fabricius bursa B cell behind the plasmid of chicken body injection chicken interleukin-2 18 (IL-18) all is significantly higher than normal control group (P＜0.05), illustrate that chicken interleukin-2 18 (IL-18) plasmid has the effect of enhancing chicken body immunity function merit.Therefore, chicken interleukin-2 18 (IL-18) albumen can improve progeny's effect of birds attenuated vaccine, inactivated vaccine and recombinant vaccine, and these bird vaccines comprise attenuated vaccine, recombinant vaccine and dna vaccinations such as chicken infectivity bursa of Fabricius virus, infectious bronchitis virus, Avian pneumo-encephalitis virus, marek's disease virus, infectious laryngotracheitis virus, bird flu virus, chicken anemia factor, chicken coccidiosis.
SEQ?No?1：
＜110〉Zhejiang University
＜120〉preparation of chicken interleukin-2 18 immunostimulants
<130>1
<160>1
<170>PatentIn?version?3.1
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Claims (10)
1. chicken interleukin-2 18 bird vaccine immunostimulants, it is characterized in that: it is for the aqueous solution of chicken leukocyte eukaryon expression plasmid, its concentration is 1～20 μ g/ μ l, this eukaryon expression plasmid contains the recombinant vector of interleukin-18 gene of the coded sequence of SEQ No 1, and the preservation of this recombinant vector number is CGMCCNo.1599, and the length of this gene is 591 nucleotide, type is a nucleotide, topological structure is a line style, and chain is a strand, and total length is the coding region.
2. chicken interleukin-2 18 bird vaccine immunostimulants according to claim 1 is characterized in that: this eukaryon expression plasmid for pCI as carrier for expression of eukaryon, contain the aqueous solution of the described chicken leukocyte of claim 1 eukaryon expression plasmid.
3. chicken interleukin-2 18 bird vaccine immunostimulants according to claim 1 and 2, it is characterized in that: it contains described eukaryon expression plasmid expressed chicken interleukin-2 18 albumen in eukaryotic cell or animal body, and chicken interleukin-2 18 shared weight ratios are 1-20%.
4. chicken interleukin-2 18 bird vaccine immunostimulants according to claim 1 are used in the immunostimulant of preparation chicken infectivity bursa of Fabricius virus dna vaccination.
5. chicken interleukin-2 18 bird vaccine immunostimulants according to claim 1 are used in the immunostimulant of preparation infectious bronchitis virus dna vaccination.
6. chicken interleukin-2 18 bird vaccine immunostimulants according to claim 1 are used in the immunostimulant of preparation Avian pneumo-encephalitis virus dna vaccination.
7. chicken interleukin-2 18 bird vaccine immunostimulants according to claim 1 are used in the immunostimulant of preparation iltv dna vaccine.
8. chicken interleukin-2 18 bird vaccine immunostimulants according to claim 1 are used in the immunostimulant of preparation bird flu virus dna vaccination.
9. chicken interleukin-2 18 bird vaccine immunostimulants according to claim 1 are used in the immunostimulant of preparation chicken anemia factor dna vaccination.
10. chicken interleukin-2 18 bird vaccine immunostimulants according to claim 1 are used in the immunostimulant of preparation chicken coccidiosis dna vaccination.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104804099A (en) * | 2015-04-01 | 2015-07-29 | 广州谱泰生物技术有限公司 | Recombinant H9N2 subtype avian influenza enhanced multi-epitope vaccine |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104804099A (en) * | 2015-04-01 | 2015-07-29 | 广州谱泰生物技术有限公司 | Recombinant H9N2 subtype avian influenza enhanced multi-epitope vaccine |
| CN104804099B (en) * | 2015-04-01 | 2019-01-25 | 广州谱泰生物技术有限公司 | A kind of reinforced polyepitope vaccines of recombination H9N2 subtype avian influenza |
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Open date: 20061115 |