CN102993310A - 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
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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 the 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, increased the susceptibility of body to other pathogenic agent, reduction is to the reactivity of other vaccine, and the foreign scholar claims that visually this disease is " acquired immune deficiency syndrome (AIDS) " of chicken.Although the various countries scholar has obtained great achievement in the research of IBD vaccine, all fail fundamentally to reverse the popular present situation that is on the rise of IBD.In recent ten years, because the diversity of appearance, antigenic variation and the blood serum subtype of chicken infectivity bursa of Fabricius virus IBDV highly virulent strain and variant makes present 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, and the M ﹠ M of chicken is obviously risen, and older chicken even the replacement pullet more than 18 ages in week also can be caused a disease.Traditional inactivated vaccine and attenuated vaccine have been faced with acid test.Therefore, need the mutant gene of the highly virulent strain that screening makes new advances to prepare new vaccine.
Chicken interleukin-2-2(Interleukin-2, IL-2) be important immune regulatory factor, can strengthen immunizing power for specific antigens in nonspecific mode.Numerous test-results and clinical application show that IL-2 has good adjuvant effect as vaccine adjuvant when treating the multi-infection disease.The chemical adjuvant that the immunostimulant of IL-2 is different from the past, they can regulate animal to the reaction of 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 that comprises NK cell, eosinophil and mastocyte.And with VP2 albumen and the IL-2 protein fusion expression of IBD, can strengthen the VP2 immunizing power of IBD, and the irritation cell immunity, thus the result of use of IBD VP2 subunit vaccine improved.
Summary of the invention
The purpose 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, being about to the VP2 albumen of IBDV highly virulent strain of clinical separation and the IL-2 albumen of immuno-potentiation couples together by a flexible linker, obtained a fusion rotein, this albumen can be that immune chicken is resisted the attack of the strong poison of IBDV and virulent and do not cause immunosuppression through making vaccine behind the purifying.
Fusion rotein of the present invention, its aminoacid sequence are 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 the oil phase tank, and heating and melting is to translucent, and Si Ben-80 autoclaving that adds again 6 parts is for subsequent use as oil phase; Get 4 parts of tween-80s of sterilization, add 96 parts fusion rotein, be stirred well to tween-80 and dissolve fully as water; Can prepare the 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 with 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 the pET-28a carrier; transform BL21 (DE3); the VP2-IL2 fusion rotein has obtained expression after IPTG induces; the centrifuging and taking supernatant is the VP2-IL2 protein solution after will expressing the fragmentation of thalline high-pressure homogenization; add the mineral oil adjuvant and make oil emulsion vaccine; can be used for preventing infectious bursal disease, effect is better than the 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
With the Primer5.0 software design the full gene primer of pair for amplification chicken infectivity bursa of Fabricius virus VP 2; and at upstream primer 5 ' end adding protectiveness base and BamH I restriction enzyme site; 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 that 200 μ l infect with the pathological material of disease of the doubtful IBDV of clinical separation, press the described method of RNAisoReagent specification sheets and extract 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, each 1 μ l of upstream and downstream primer (10mM), cDNA 3 μ l, 25mM MgCl2 4 μ l, Taq enzyme (5U/ μ l) 1 μ l, ddH2O 34 μ l; PCR reaction parameter: 95 ℃ of 5min, 95 ℃ of 1min, 58 ℃ of 2min, 72 ℃ of 2min, 35 circulations, 72 ℃ of 10min.Obtain a fragment about 1300bp, cut glue and reclaim this fragment, be connected with the pMD-18T carrier.PCR reclaims fragment 6 μ l, T carrier 2 μ l (after diluting 10 times), and T4 dna ligase 1 μ l, T4 buffer 2.5 μ l, water: 13.5 μ l amount to 25 μ l.16 ℃ of connections of spending the night, get 20 μ l and connect product transformed competence colibacillus e. coli jm109, selecting 10 white colonies cultivates, extract plasmid identification correct choose 3 order-checkings, the sequencing result of 3 plasmids is all consistent as a result, the nucleotide fragments that proof obtains 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 the upper Blast comparison of NCBI, has tens bases that point mutation has occured, show that this strain is the new epidemic isolates of a strain.This is because VP2 albumen is the main host protective antigen, often undergos mutation and causes antigenicity generation difference.
Two, the expression of VP2 recombinant protein
To identify that a correct above-mentioned plasmid reclaims gene fragment with BamH I and Xho I double digestion, be connected with the pET-28a carrier of cutting with these two enzymes, the 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 ℃ of shaking culture 3 ~ 4 hours to OD600 be 0.6 o'clock, the adding final concentration is that the IPTG of 0.8mM induced 4 ~ 5 hours, and the SDS-PAGE electrophoresis result turns out to be secreting, expressing.
It is resuspended with 10 times of thalline weight in wet base PBS to express bacterium, the broken bacterium of high-pressure homogenization, and the centrifuging and taking supernatant is done the agar immunodiffusion, and as a result fine jade expansion is tired and is not less than 1:64.
Three, the purifying of VP2 albumen
Use ni-sepharose purification, the imidazoles wash-out of 200mM, elutriant PBS dialysed overnight.The 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 the mixing, heating is dissolved, and 116 ℃ of sterilizations 30 minutes are as the seedling oil phase; Then 4 parts of tween-80s that add sterilization add 96 parts of the VP2 recombinant protein fermented extracted liquid of above-mentioned preparation, and shake well dissolves tween-80 fully, as the seedling water; Get 3 parts of oil phases and be put in the colloidal mill, start the motor slow rotation and stir, slowly add simultaneously water l part, stirred 2～5 minutes with l0000r/min after adding again, add 1% Thiomersalate solution before stopping stirring, 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 when 21 age in days with 0.3ml/ dosage subcutaneous injection VP2 subunit vaccine only, immunity after 21 days together with every of not immune control group through the strong malicious BC6-85 strain venom of eye droppings approach inoculation 100BID infectious bursal disease, attack clinical manifestation and the death condition of observing chicken every day behind the poison, record morbidity and dead chicken number, cut open inspection and observe the pathologies such as dead chicken bursa, slaughtered the survival chicken in 72～96 hours, by only analysing, observe the pathological changes such as the fabricius bursa.8 chicken morbidities of the not immune control group of result, and the immune group chicken is without morbidity.The VP2 subunit vaccine of the above results proof the present invention preparation 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′
VP2 gene with P1 and P3 amplification IBDV, amplification system is (50 μ l): 10 * PCR Buffer, 5 μ l, dNTP (10mM each) 1 μ l, each 1 μ l of upstream and downstream primer (10mM), cDNA 3 μ l, 25mM MgCl2 4 μ l, Taq enzyme (5U/ μ l) 1 μ l, ddH2O 34 μ l.The PCR reaction conditions: 95 ℃ of denaturation 5min, 94 ℃ of sex change 1min, 52 ℃ of annealing 1min, 72 ℃ are extended 1min, circulate after 5 times, 94 ℃ of sex change 1min, 56 ℃ of annealing 1min, 72 ℃ are extended 1min, 30 circulations, 72 ℃ of extension 10min.Glue reclaims the PCR fragment that test kit reclaims 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 fragment as template, take P1 and P4 as primer, the amplification fusion gene, reaction conditions is 95 ℃ of denaturation 5min, 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations, 72 ℃ are extended 10min.Obtain the about 1700bp of fragment.The nucleotide sequencing result is SEQ ID NO:2, and the aminoacid sequence of translation is SEQ ID NO:1; Consistent with initial design all.
Six, the abduction delivering of fusion rotein
1, with the fusion gene of above-mentioned preparation and pET-28a carrier respectively through BamH I and connection after Hind III double digestion is connected, with recombinant vectors Calcium Chloride Method transformed competence colibacillus e. coli bl21 (DE3), the positive colony that filters out behind clone order-checking determines that the fragment of inserting is correct.With positive colony IPTG abduction delivering, with the albumen about the visible 65kd of expression product rear electrophoresis, expression amount is approximately 10%, agar diffusion test and western-blot method are identified and are shown, the albumen of expressing is the VP2-IL2 fusion rotein, get respectively cleer and peaceful precipitation SDS-PAGE electrophoresis behind the ultrasonication bacterium, show that albumen is solubility expression.
2, the purifying of recombinant protein
Use ni-sepharose purification, the imidazoles wash-out of 200mM, elutriant PBS dialysed overnight.The gained dialyzate is the albumen of purifying, can be used for seedling.
Seven, VP2 subunit vaccine and VP2-IL2 subunit vaccine preparation
Get 2 parts of 94 parts of injection white oils, Si Ben-80 6 part, aluminum stearates, after the mixing, heating is dissolved, and 116 ℃ of sterilizations 30 minutes are as the seedling oil phase; Get 4 parts of the tween-80s of sterilization, then add respectively 96 parts of VP2 recombinant protein, the VP2-IL2 fusion rotein fermented extracted liquid of above-mentioned preparation, shake well dissolves fully as the seedling water tween-80; Get 3 parts of oil phases and be put in the colloidal mill, start the motor slow rotation and stir, slowly add simultaneously water l part, stirred 2～5 minutes with l0000r/min after adding again, add 1% Thiomersalate solution before stopping stirring, 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
Two kinds of immune 21 age in days SPF chickens of genetic engineering subunit vaccines difference with preparation, 30 of every kind of vaccine immunities, immunity was measured respectively and is gathered anticoagulation mensuration lymphocyte transformation rate in rear 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 attacking poison together with the strong malicious BC6-85 eye droppings of 10 usefulness of not immune contrast, the result shows that lymphocyte transformation rate VP2-IL2 immune group on the 7th is apparently higher than VP2 immune group and control group; VP2 subunit vaccine 9/10 is protected after attacking poison on the 21st, 10/10 protection of fusion protein immunization group, control group 8/10 morbidity; 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 remedy the defective of existing VP2 vaccine.
Claims (6)
1. the fusion rotein of an 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. Nucleotide, described Nucleotide is used for encoding fusion rotein claimed in claim 1.
3. Nucleotide as claimed in claim 2, the sequence that it is characterized in that described Nucleotide is SEQ ID NO:2.
4. the application of fusion rotein claimed in claim 1 in the preparation subunit vaccine.
5. subunit vaccine, its preparation method is as follows: get 94 parts of injection white oils, 2 parts of aluminum stearates mix in the oil phase tank, and heating and melting is to translucent, and Si Ben-80 autoclaving that adds again 6 parts is for subsequent use as oil phase; Get 4 parts of tween-80s of sterilization, add 96 parts fusion rotein claimed in claim 1, be stirred well to tween-80 and dissolve fully as water; Namely make subunit vaccine in water and oil phase volume ratio 1:3 ratio mixing and emulsifying.
6. subunit vaccine claimed in claim 5 is used for preventing and treating infectious bursal disease.
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 |
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| Publication Number | Publication Date |
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| CN102993310A true CN102993310A (en) | 2013-03-27 |
| CN102993310B CN102993310B (en) | 2014-07-09 |
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| 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 |
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Cited By (3)
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| CN106754743A (en) * | 2016-11-17 | 2017-05-31 | 河南农业大学 | The cell adapted strain of virulent chicken infectivity bursa of Fabricius virus and its application |
| CN107488234A (en) * | 2017-09-29 | 2017-12-19 | 瑞普（保定）生物药业有限公司 | Aviadenovirus Hexon and chicken infectivity bursa of Fabricius virus VP 2 fused antigen, 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 |
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| 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 |
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| 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 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754743A (en) * | 2016-11-17 | 2017-05-31 | 河南农业大学 | The cell adapted strain of virulent chicken infectivity bursa of Fabricius virus and its application |
| 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 |
| CN107488234A (en) * | 2017-09-29 | 2017-12-19 | 瑞普（保定）生物药业有限公司 | Aviadenovirus Hexon and chicken infectivity bursa of Fabricius virus VP 2 fused antigen, subunit vaccine and preparation method 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 |
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| CN102993310B (en) | 2014-07-09 |
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