CN102993310B - Fusion protein of IBD (Infectious Bursal Disease) VP2 and IL (Interleukin)-2 and application thereof - Google Patents
Fusion protein of IBD (Infectious Bursal Disease) VP2 and IL (Interleukin)-2 and application thereof Download PDFInfo
- Publication number
- CN102993310B CN102993310B CN201210506665.0A CN201210506665A CN102993310B CN 102993310 B CN102993310 B CN 102993310B CN 201210506665 A CN201210506665 A CN 201210506665A CN 102993310 B CN102993310 B CN 102993310B
- Authority
- CN
- China
- Prior art keywords
- chicken
- ibd
- infectious bursal
- bursal disease
- vaccine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 208000015181 infectious disease Diseases 0.000 title abstract description 10
- 208000027312 Bursal disease Diseases 0.000 title abstract description 8
- 230000002458 infectious effect Effects 0.000 title abstract description 8
- 102000037865 fusion proteins Human genes 0.000 title abstract description 5
- 108020001507 fusion proteins Proteins 0.000 title abstract description 5
- 102000015696 Interleukins Human genes 0.000 title abstract 2
- 108010063738 Interleukins Proteins 0.000 title abstract 2
- 241000287828 Gallus gallus Species 0.000 claims abstract description 30
- 230000004927 fusion Effects 0.000 claims abstract description 24
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 6
- 229940031626 subunit vaccine Drugs 0.000 claims description 22
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- 101500001532 Avian infectious bursal disease virus Capsid protein VP2 Proteins 0.000 claims description 3
- 101710081079 Minor spike protein H Proteins 0.000 abstract description 29
- 235000013330 chicken meat Nutrition 0.000 abstract description 29
- 108010002350 Interleukin-2 Proteins 0.000 abstract description 16
- 102000000588 Interleukin-2 Human genes 0.000 abstract description 15
- 229960005486 vaccine Drugs 0.000 abstract description 13
- 241000702626 Infectious bursal disease virus Species 0.000 abstract description 11
- 241000700605 Viruses Species 0.000 abstract description 8
- 230000001900 immune effect Effects 0.000 abstract description 3
- 238000010254 subcutaneous injection Methods 0.000 abstract description 3
- 239000007929 subcutaneous injection Substances 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 description 12
- 239000012634 fragment Substances 0.000 description 11
- 239000003921 oil Substances 0.000 description 11
- 230000003321 amplification Effects 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 210000001669 bursa of fabricius Anatomy 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 231100000614 poison Toxicity 0.000 description 4
- 239000002574 poison Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- CEGOLXSVJUTHNZ-UHFFFAOYSA-K aluminium tristearate Chemical class [Al+3].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CEGOLXSVJUTHNZ-UHFFFAOYSA-K 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000004087 circulation Effects 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 230000002434 immunopotentiative effect Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 3
- 229920000053 polysorbate 80 Polymers 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 208000030507 AIDS Diseases 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 206010062016 Immunosuppression Diseases 0.000 description 2
- 101710194807 Protective antigen Proteins 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 101150093578 VP2 gene Proteins 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 238000000265 homogenisation Methods 0.000 description 2
- 150000002460 imidazoles Chemical class 0.000 description 2
- 230000001506 immunosuppresive effect Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000010977 jade Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000004224 protection Effects 0.000 description 2
- 230000012743 protein tagging Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 241001260012 Bursa Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 206010015856 Extrasystoles Diseases 0.000 description 1
- 238000002768 Kirby-Bauer method Methods 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 101000933967 Pseudomonas phage KPP25 Major capsid protein Proteins 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000000240 adjuvant effect Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 230000027645 antigenic variation Effects 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 229940031567 attenuated vaccine Drugs 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000003711 chorioallantoic membrane Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000000951 immunodiffusion Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 229960001438 immunostimulant agent Drugs 0.000 description 1
- 239000003022 immunostimulating agent Substances 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 229940031551 inactivated vaccine Drugs 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000003307 slaughter Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 239000012646 vaccine adjuvant Substances 0.000 description 1
- 229940124931 vaccine adjuvant Drugs 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 239000002435 venom Substances 0.000 description 1
- 231100000611 venom Toxicity 0.000 description 1
- 210000001048 venom Anatomy 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention aims to provide a fusion protein of VP2 protein of an IBD (Infectious Bursal Disease) virus and an IL (Interleukin)-2, wherein the amino acid sequence of the fusion protein is SEQ ID NO: 1. According to the invention, the supervirulent VP2 protein of the IBDV (Infectious Bursal Disease Virus) is expressed with the IL-2 protein with immunological enhancement in a fusion manner, and the vaccine prepared through subcutaneous injection has a strong resistivity to resist the attach of the IBDV for chickens.
Description
Technical field
The invention belongs to genetic engineering subunit vaccine technical field, be specifically related to fusion rotein and the application thereof of IBD virus VP 2 albumen and chicken IL-2, be i.e. fusion rotein and the application of Infectious bursal disease virus VP2 and chicken IL-2.
Background technology
Infectious bursal disease (Infectious Bursal Disease, IBD) mainly encroach on the central immune organ fabricius bursa of chicken, thereby cause immunosuppression, increase the susceptibility of body to other pathogenic agent, reduce the reactivity to other vaccine, foreign scholar claims that this disease is " acquired immune deficiency syndrome (AIDS) " of chicken visually.Although various countries scholar has obtained great achievement in the research of IBD vaccine, all fail fundamentally to reverse the popular present situation being on the rise of IBD.In recent ten years, due to the diversity of appearance, antigenic variation and the blood serum subtype of chicken infectivity bursa of Fabricius virus IBDV highly virulent strain and variant, make current commercialized vaccine that enough protections can not be provided, its protection ratio only reaches 10% ~ 70%.The virulence of highly virulent strain is more than the twice of standard virus strain, makes the M & M of chicken obviously increase, and older chicken even 18 weeks replacement pullets more than age also can be caused a disease.Traditional inactivated vaccine and attenuated vaccine have been faced with acid test.The mutant gene that therefore, need to screen the highly virulent strain making new advances is prepared new vaccine.
Chicken interleukin-2-2(Interleukin-2, IL-2) be important immune regulatory factor, can strengthen the immunizing power for specific antigens in nonspecific mode.Numerous test-results and clinical application show that IL-2 has good adjuvant effect in the time treating multi-infection disease as vaccine adjuvant.The immunostimulant chemical adjuvant different from the past of IL-2, they can regulate the reaction of animal to vaccine, comprise that immunocyte moves to inoculation position, angtigen presentation, promote the generation of Th cell, ripe and the differentiation of B cell, activates the non-specific killer cell including NK cell, eosinophil and mastocyte.And by the VP2 albumen of IBD and IL-2 protein fusion expression, can strengthen the VP2 immunizing power of IBD, irritation cell immunity, thereby the result of use of raising IBD VP2 subunit vaccine.
Summary of the invention
The object of this invention is to provide fusion rotein and the application thereof of a kind of IBD virus VP 2 albumen and IL-2, relate to preparation and the application of a kind of IBDV VP2 albumen and chicken IL-2 fusion protein, the VP2 albumen of IBDV highly virulent strain and the IL-2 albumen of immuno-potentiation by clinical separation couple together by a flexible linker, obtained a fusion rotein, it can be that immune chicken is resisted the attack of the strong poison of IBDV and virulent and do not cause immunosuppression that this albumen is made vaccine after purifying.
Fusion rotein of the present invention, its aminoacid sequence is SEQ ID NO:1.
Nucleotides sequence for the above-mentioned fusion rotein of encoding is classified SEQ ID NO:2 as.
Fusion rotein of the present invention is for the preparation of subunit vaccine.
The preparation method of above-mentioned subunit vaccine is as follows: get 94 parts of injection white oils, 2 parts of aluminum stearates mix in oil phase tank, and heating and melting is to translucent, then it is for subsequent use as oil phase to add Si Ben-80 autoclaving of 6 parts; Get 4 parts of tween-80s of sterilizing, add the fusion rotein of 96 parts, be stirred well to tween-80 and dissolve completely as water; Can prepare chicken infectivity bursa of Fabricius virus VP 2 subunit vaccine in water and oil phase volume ratio 1:3 ratio mixing and emulsifying.
The subunit vaccine of above-mentioned preparation is used for preventing and treating infectious bursal disease.
The present invention is by the VP2 albumen of chicken IBDV virulent and the chicken IL-2 protein fusion expression with immuno-potentiation, and the vaccine of preparation has strong resistibility to chicken opposing infectious bursal disease strong virus attack after subcutaneous injection.
Embodiment
The major structural protein VP2 albumen of IBDV is main host protective antigen, and chicken IL-2 has obvious immuno-potentiation, the present invention couples together two kinds of genes with overlapping extension PCR method by linker, be cloned on pET-28a carrier, transform BL21 (DE3), after IPTG induction, VP2-IL2 fusion rotein has obtained expression, centrifuging and taking supernatant after the fragmentation of expression thalline high-pressure homogenization is to VP2-IL2 protein solution, add mineral oil adjuvant to make oil emulsion vaccine, can be used for preventing infectious bursal disease, effect is better than VP2 subunit vaccine.
Below in conjunction with specific embodiment, fusion rotein of the present invention is described in detail.
One, the screening of VP2 gene
By Primer5.0 software design the full gene primer of pair for amplification chicken infectivity bursa of Fabricius virus VP 2; and add protectiveness base and BamH I restriction enzyme site at upstream primer 5 ' end; add protectiveness base and Xho I restriction enzyme site at 5 of downstream primer ' end, primer sequence is:
Primer?1：5′-GCG?TCG?ACA?TGA?CAA?ACC?TGC?AAG?ATC?-?3′
Primer?2：5′-CCC?TCG?AGT?TAT?CCT?TAT?GGC?CCG?GAT?T?-3′
Get the egg inoculation chorioallantoic membrane suspension supernatant liquor of the pathological material of disease infection of the doubtful IBDV of the clinical separation of 200 μ l, the method described in RNAisoReagent specification sheets of pressing is extracted viral RNA, carry out the synthetic cDNA of reverse transcription by reverse transcription test kit working instructions, carry out pcr amplification, amplification system is (50 μ l): 10 × PCR Buffer, 5 μ l, dNTP (10mM each) 1 μ l, the each 1 μ l of upstream and downstream primer (10mM), cDNA 3 μ l, 25mM MgCl2 4 μ l, (5U/ μ is 1 μ l l), ddH2O 34 μ l for Taq enzyme; PCR reaction parameter: 95 DEG C of 5min, 95 DEG C of 1min, 58 DEG C of 2min, 72 DEG C of 2min, 35 circulations, 72 DEG C of 10min.Obtain the fragment of a 1300bp left and right, cut glue and reclaim this fragment, be connected with pMD-18T carrier.PCR reclaims fragment 6 μ l, T carrier 2 μ l (after diluting 10 times), T4 DNA ligase 1 μ l, T4 buffer 2.5 μ l, water: 13.5 μ l, amount to 25 μ l.16 DEG C of connections of spending the night, get 20 μ l and connect product transformed competence colibacillus e. coli jm109, selecting 10 white colonies cultivates, what extraction plasmid identification was correct chooses 3 order-checkings, the sequencing result of 3 plasmids is all consistent as a result, prove that the nucleotide fragments obtaining does not produce mistake in the process of amplification, its nucleotides sequence is classified SEQ ID NO:4 as, and the aminoacid sequence of translation is SEQ ID NO:3.
This nucleotides sequence is listed in to the upper Blast comparison of NCBI, has tens bases that point mutation has occurred, show that this strain is the new epidemic isolates of a strain.This is because VP2 albumen is main host protective antigen, often undergos mutation and causes antigenicity generation difference.
Two, the expression of VP2 recombinant protein
An above-mentioned plasmid correct qualification is reclaimed to gene fragment with BamH I and Xho I double digestion, with be connected with the pET-28a carrier that these two enzymes are cut, transformed competence colibacillus e. coli jm109, extract the correct rear BL21(DE3 that transforms receptor of plasmid identification), picking list bacterium colony, switching 5mlLB tubule substratum, 37 DEG C of shaking culture 3 ~ 4 hours to OD600 be 0.6 o'clock, adding final concentration is the IPTG induction 4 ~ 5 hours of 0.8mM, and SDS-PAGE electrophoresis result turns out to be secreting, expressing.
To express bacterium resuspended with 10 times of thalline weight in wet base PBS, the broken bacterium of high-pressure homogenization, centrifuging and taking supernatant, does agar immunodiffusion, and the expansion of result fine jade is tired and is not less than 1:64.
Three, the purifying of VP2 albumen
With ni-sepharose purification, the imidazoles wash-out of 200mM, elutriant PBS dialysed overnight.Gained dialyzate is the albumen of purifying, can be used for seedling.
Four, the preparation of VP2 subunit vaccine
Get 2 parts of 94 parts of injection white oils, Si Ben-80 6 part, aluminum stearates, after mixing, heating is dissolved, and 116 DEG C of sterilizings 30 minutes are as seedling oil phase; Add 4 parts of the tween-80s of sterilizing, then add 96 parts of the VP2 recombinant protein fermented extracted liquid of above-mentioned preparation, shake well, dissolves tween-80 completely, as seedling water; Get 3 parts of oil phases and be put in colloidal mill, start motor slow rotation and stir, slowly add water l part simultaneously, after adding, stir 2～5 minutes with l0000r/min again, before termination is stirred, add 1% Thiomersalate solution, making its ultimate density is 0.01%.
The immune effect of VP2 subunit vaccine:
20 SPF chickens are divided into two groups at random, every group 10, wherein one group in the time of 21 age in days with 0.3ml/ dosage subcutaneous injection VP2 subunit vaccine only, immunity was inoculated the strong malicious BC6-85 strain venom of 100BID infectious bursal disease together with every of not immune control group through eye droppings approach after 21 days, attack clinical manifestation and the death condition of after poison, observing chicken every day, record morbidity and dead chicken number, cut open inspection and observe the pathologies such as dead chicken bursa, within 72～96 hours, slaughter survival chicken, by only analysing, observe the pathological changes such as the fabricius bursa.8 chicken morbidities of the not immune control group of result, and immune group chicken is without morbidity.The above results proves that VP2 subunit vaccine prepared by the present invention has good immunogenicity.
Five, the fusion of VP2 albumen and chicken IL-2 albumen
Adopt overlapping extension PCR method amplification fusion gene.Use respectively following primer.Wherein P5 introduces the restriction enzyme site of Hind III.
P?1：5′-GGATCCGCGTCGACATGACAA?ACCTGCAAGATC?-?3′
P?3：5′-?AACGCCGTAAATAAAACCATGCCAGAGCCGCCAGAGCC?-?3′
P?4：5′-AAGCTT?ttatttttgcagatatctca-?3′
With the VP2 gene of P1 and P3 amplification IBDV, amplification system is (50 μ l): 10 × PCR Buffer, 5 μ l, dNTP (10mM each) 1 μ l, the each 1 μ l of upstream and downstream primer (10mM), cDNA 3 μ l, 25mM MgCl2 4 μ l, (5U/ μ is 1 μ l l), ddH2O 34 μ l for Taq enzyme.PCR reaction conditions: 95 DEG C of denaturation 5min, 94 DEG C of sex change 1min, 52 DEG C of annealing 1min, 72 DEG C are extended 1min, circulate after 5 times, 94 DEG C of sex change 1min, 56 DEG C of annealing 1min, 72 DEG C are extended 1min, 30 circulations, 72 DEG C are extended 10min.Glue recovery test kit reclaims the PCR fragment of about 1400bp.
The fragment of synthetic chicken IL-2 gene mature peptide, its aminoacid sequence is SEQ ID NO:5; The VP2 of this fragment and IBDV is reclaimed to fragment as template, taking P1 and P4 as primer, amplification fusion gene, reaction conditions is 95 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C are extended 30s, 30 circulations, 72 DEG C are extended 10min.Obtain the about 1700bp of fragment.Nucleotide sequencing result is SEQ ID NO:2, and the aminoacid sequence of translation is SEQ ID NO:1; All consistent with initial design.
Six, the abduction delivering of fusion rotein
1, the fusion gene of above-mentioned preparation is connected respectively with pET-28a carrier after BamH I and the recovery of Hind III double digestion, by recombinant vectors Calcium Chloride Method transformed competence colibacillus e. coli bl21 (DE3), the positive colony filtering out after clone order-checking determines that the fragment of inserting is correct.By positive colony IPTG abduction delivering, by the albumen of visible expression product rear electrophoresis 65kd left and right, expression amount is approximately 10%, agar diffusion test and the qualification of western-blot method show, the albumen of expressing is VP2-IL2 fusion rotein, after ultrasonication bacterium, get respectively cleer and peaceful precipitation SDS-PAGE electrophoresis, show that albumen is solubility expression.
2, the purifying of recombinant protein
With ni-sepharose purification, the imidazoles wash-out of 200mM, elutriant PBS dialysed overnight.Gained dialyzate is the albumen of purifying, can be used for seedling.
Seven, VP2 subunit vaccine and the preparation of VP2-IL2 subunit vaccine
Get 2 parts of 94 parts of injection white oils, Si Ben-80 6 part, aluminum stearates, after mixing, heating is dissolved, and 116 DEG C of sterilizings 30 minutes are as seedling oil phase; Get 4 parts of the tween-80s of sterilizing, then add respectively 96 parts of VP2 recombinant protein, the VP2-IL2 fusion rotein fermented extracted liquid of above-mentioned preparation, shake well, dissolves completely as seedling water tween-80; Get 3 parts of oil phases and be put in colloidal mill, start motor slow rotation and stir, slowly add water l part simultaneously, after adding, stir 2～5 minutes with l0000r/min again, before termination is stirred, add 1% Thiomersalate solution, making its ultimate density is 0.01%.The vaccine of preparation is respectively VP2 subunit vaccine and VP2-IL2 subunit vaccine.
Eight, the immune effect of VP2 subunit vaccine and VP2-IL2 bigeminy subunit vaccine
By two kinds of genetic engineering subunit vaccines of preparation immune 21 age in days SPF chickens respectively, 30 of every kind of vaccine immunities, immunity is measured respectively and is gathered anticoagulation mensuration lymphocyte transformation rate for latter 7 days, the separation of serum of taking a blood sample after immune 21 days is measured the expansion of fabricius bursa fine jade and is tired, and attack poison together with the strong malicious BC6-85 eye droppings of 10 use of not immune contrast, result shows that lymphocyte transformation rate VP2-IL2 immune group on the 7th is apparently higher than VP2 immune group and control group; After within 21st, attacking poison, VP2 subunit vaccine 9/10 is protected, and fusion protein immunization group 10/10 is protected, and control group 8/10 is fallen ill; The geometric mean of the agar diffusion antibody titer of VP2 subunit vaccine immune group serum is 1:19.7, and the geometric mean of the obvious antibody titer of VP2-IL2 immune group is 1:34.3, apparently higher than VP2 subunit vaccine immune group.The above results shows that the prepared fusion rotein of the present invention has good effect as vaccine, and, because VP2 of the present invention is new allelotrope, can effectively make up the defect of existing VP2 vaccine.
Claims (4)
1. a fusion rotein for Infectious bursal disease virus VP2 and chicken interleukin-2-2, is characterized in that, the aminoacid sequence of described fusion rotein is SEQ ID NO:1.
2. a Nucleotide, the described Nucleotide fusion rotein claimed in claim 1 that is used for encoding.
3. Nucleotide as claimed in claim 2, is characterized in that the sequence of described Nucleotide is SEQ ID NO:2.
4. fusion rotein claimed in claim 1 is in the application of preparing in subunit vaccine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210506665.0A CN102993310B (en) | 2012-12-03 | 2012-12-03 | Fusion protein of IBD (Infectious Bursal Disease) VP2 and IL (Interleukin)-2 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210506665.0A CN102993310B (en) | 2012-12-03 | 2012-12-03 | Fusion protein of IBD (Infectious Bursal Disease) VP2 and IL (Interleukin)-2 and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102993310A CN102993310A (en) | 2013-03-27 |
| CN102993310B true CN102993310B (en) | 2014-07-09 |
Family
ID=47922469
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210506665.0A Active CN102993310B (en) | 2012-12-03 | 2012-12-03 | Fusion protein of IBD (Infectious Bursal Disease) VP2 and IL (Interleukin)-2 and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102993310B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754743B (en) * | 2016-11-17 | 2020-06-02 | 河南农业大学 | Super-strong-toxicity chicken infectious bursal disease virus cell adaptive strain and application thereof |
| CN107488234B (en) * | 2017-09-29 | 2021-04-30 | 瑞普（保定）生物药业有限公司 | Fusion antigen of avian adenovirus Hexon and chicken infectious bursal disease virus VP2, subunit vaccine and preparation method thereof |
| CN108329394A (en) * | 2018-01-30 | 2018-07-27 | 长春西诺生物科技有限公司 | A kind of short beak runting syndrome vaccine of duck and preparation method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102688487A (en) * | 2012-06-18 | 2012-09-26 | 青岛易邦生物工程有限公司 | Production method of newcastle disease (ND), infectious bronchitis (IB), infectious bursal disease virus (IBD) and viral arthritis (VA) four-joint inactivated vaccine |
-
2012
- 2012-12-03 CN CN201210506665.0A patent/CN102993310B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN102993310A (en) | 2013-03-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107432930B (en) | Group I4 avian adenovirus DNA vaccine and preparation method and application thereof | |
| CN107899008B (en) | Sick three subunit vaccines of a kind of pig epidemic diarrhea, transmissible gastroenteritis of swine, pig fourth type coronavirus | |
| CN102993311B (en) | Infusion protein of IDB (Infectious Bursal Disease) virus VP2 protein and LTB (Leukotrienes B) and application thereof | |
| CN102675471A (en) | Pig foot-and-mouth disease virus O-type broad spectrum multi-epitope recombination antigen and application thereof | |
| CN107337718A (en) | A kind of gene for encoding carrying Cap gene of porcine circovirus type 2 and its application | |
| CN114149493A (en) | Group I4 avian adenovirus fiber-2 protein antigen and method for preparing genetic engineering subunit vaccine and application thereof | |
| CN102993310B (en) | Fusion protein of IBD (Infectious Bursal Disease) VP2 and IL (Interleukin)-2 and application thereof | |
| CN102994520B (en) | Infectious bursal disease VP2 gene and application thereof | |
| CN104610455B (en) | A kind of duck tembusu virus genetic engineering subunit vaccine | |
| CN107488234A (en) | Aviadenovirus Hexon and chicken infectivity bursa of Fabricius virus VP 2 fused antigen, subunit vaccine and preparation method thereof | |
| CN107936123B (en) | Swine transmissible gastroenteritis virus fusion protein and preparation method and application thereof | |
| CN104530230B (en) | A kind of duck hepatitis A virus VP1 GFPs and its application | |
| CN104328135A (en) | Duck Tembusu virus E protein-LTB fusion protein and application thereof | |
| CN103007271B (en) | Chicken subunit four-combination vaccine and preparation and application thereof | |
| CN104448005B (en) | The fusion rotein antigen of duck hepatitis A virus (HAV) 3 type VP1 albumen and LTB and application thereof | |
| CN108530522A (en) | A kind of OmpK multi-epitopes polypeptide, construction method and its application of recombination | |
| CN104250304A (en) | Fusion protein, coding vaccine composition and application thereof | |
| CN102973930B (en) | Chicken subunit bivalent vaccine, as well as preparation and application thereof | |
| CN103626878B (en) | Newcastle disease virus F protein and enterotoxin LTB fusion protein and application thereof | |
| CN110013549B (en) | Subunit vaccine for hepatitis E | |
| CN113855795A (en) | Avian hepatitis E virus ORF2 subunit vaccine | |
| WO2015039270A1 (en) | Use of protein a1g_07050 in immunoprotection of anti-rickettsia rickettsii | |
| CN102614506A (en) | Recombinant HPV16L2 protein vaccine and preparation method thereof | |
| CN108586617B (en) | Method for improving immune response of PCV2VLPs vaccine by displaying TFlg at C terminal | |
| CN102206258A (en) | NmpC subunit vaccine of salmonella paratyphi A and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent of invention or patent application | ||
| CB02 | Change of applicant information |
Address after: 266111 Shandong city of Qingdao province Chengyang Qingda Industrial Park, the first northbound dual Applicant after: QINGDAO VLAND BIOTECH Inc. Applicant after: QINGDAO BOITE BIOPHARMACEUTICAL Co.,Ltd. Address before: 266061 Shandong city of Qingdao province high tech park of Laoshan district by the No. 29 Shandong Road, building 6F high speed Applicant before: QINGDAO KDN PHARMACEUTICAL Co.,Ltd. Applicant before: QINGDAO BOITE BIOPHARMACEUTICAL Co.,Ltd. |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: APPLICANT; FROM: QINGDAO KDN PHARMACEUTICAL CO., LTD. TO: QINGDAO WEILAN BIOLOGY CO., LTD. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C56 | Change in the name or address of the patentee | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 266111 Shandong city of Qingdao province Chengyang Qingda Industrial Park, the first northbound dual Patentee after: QINGDAO VLAND BIOTECH Inc. Patentee after: QINGDAO VLAND BIOTECH GROUP CO.,LTD. Address before: 266111 Shandong city of Qingdao province Chengyang Qingda Industrial Park, the first northbound dual Patentee before: QINGDAO VLAND BIOTECH Inc. Patentee before: QINGDAO BOITE BIOPHARMACEUTICAL Co.,Ltd. |
|
| CP01 | Change in the name or title of a patent holder |
Address after: 266111 north head of Shuangyuan Road, Qingda Industrial Park, Chengyang District, Qingdao City, Shandong Province Patentee after: QINGDAO VLAND BIOTECH Inc. Patentee after: Qingdao Blue Animal Health Group Co.,Ltd. Address before: 266111 north head of Shuangyuan Road, Qingda Industrial Park, Chengyang District, Qingdao City, Shandong Province Patentee before: QINGDAO VLAND BIOTECH Inc. Patentee before: QINGDAO VLAND BIOTECH GROUP CO.,LTD. |
|
| CP01 | Change in the name or title of a patent holder |