CN102993311B - Infusion protein of IDB (Infectious Bursal Disease) virus VP2 protein and LTB (Leukotrienes B) and application thereof - Google Patents
Infusion protein of IDB (Infectious Bursal Disease) virus VP2 protein and LTB (Leukotrienes B) and application thereof Download PDFInfo
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Abstract
The invention relates to an infusion protein of an IDB (Infectious Bursal Disease) virus VP2 protein and an LTB (Leukotrienes B) of enterotoxin of escherichia coli, wherein the amino acid sequence of the infusion 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 LTB protein of enterotoxin of escherichia coli with a mucosal immunologic adjuvant in a fusion manner, and an expression product is emulsified to prepare a vaccine to make SPF chickens which are 21 days old immune. An immune reference and a non-immune reference for a subunit vaccine of VP2 are set. After 7 days of immunity, the lymphocyte transformation rate is determined, and after 21 days of immunity, blood is collected to determine the valence of antibody of immunodiffusion of serum agar and toxic substances are counteracted together with the non-immune reference. The result shows that vaccine immunity groups can resist virulent attach. The lymphocyte transformation rate and the serum valence of antibody are remarkably higher than the immune reference group of VP2, so that the infusion protein has an excellent immunogenicity.
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 LTB, be i.e. fusion rotein and the application of Infectious bursal disease virus VP2 and enterotoxins of Escherichia coli LTB.
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,
E.coli LT (heat-liable enterotoxin, LT) is by a kind of extracellular toxin of enterotoxigenic E.Coli (Enterotoxi-genetic E.coli, ETEC) secretion, has very strong immunological adjuvant characteristic.Escherichia coli heat labile enterotoxin is six glycoprotein polyprotein precursors, and the A subunit that is 28 ku by 1 molecular mass and 5 B subunit monomer compositions that molecular mass is 11 ku form AB5 type structure.A subunit has toxic action, and B subunit has stronger immunoadjuvant function, and can avoid the toxic action of A subunit, and the studied person of B subunit nontoxic and that have an adjuvanticity pays close attention to.
Research finds that IBD is conventionally by infecting through the mucosal route such as digestive tube and respiratory tract, good local mucous membrane immunity is the important defence line that this disease of opposing infects, so by the natural infection approach of simulation pathogenic agent, strengthen the initial respiratory tract of invading of pathogenic agent and the specific antibody of the local generation of gastrointestinal mucosal, and then cause that general systemic immunity replys, significant to preventing and treating this disease.Therefore, setting up a kind of genetic engineering subunit vaccine that can prevent infectious bursal disease just has important practical significance and market development value.
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 LTB, relate to preparation and the application of a kind of IBDV VP2 albumen and E.coli LT B fusion rotein, VP2 albumen by the IBDV highly virulent strain of clinical separation couples together by linker with the E.coli LT B albumen with mucosal adjuvant effect, 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.
A kind of preparation method of 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 be by the VP2 albumen of chicken IBDV virulent with there is mucosal adjuvant E.coli LT B protein fusion expression, 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 E.coli LT LTB is good mucosal adjuvant, 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-LTB fusion rotein has obtained expression, centrifuging and taking supernatant after the fragmentation of expression thalline high-pressure homogenization is to VP2-LTB 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, cause the aminoacid sequence of translation also to have the invention of several sites to change.Show that this strain is a new epidemic isolates.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 E.coli LT B gene
Adopt overlapping extension PCR method amplification fusion gene.Use respectively following primer.Wherein P5 introduces the restriction enzyme site of Not I.
P?1：5′-GGATCCGCGTCGACATGACAA?ACCTGCAAGATC?-?3′
P?3：5′-?AACGCCGTAAATAAAACCATGCCAGAGCCGCCAGAGCC?-?3′
P?4：5′-GGCTCTGGCGGCTCTGGCATGGTTTTATTTACGGCGTT?-?3′
P?5：5′-GCGGCCGCCTAGTTTTCCATACTGATTG?-?3′
With the VP2 gene (SEQ ID NO:4) 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.
With P4 and P5 amplification E.coli LT B gene, from enterotoxigenic escherichia coli, extract plasmid with plasmid extraction kit, carry out pcr amplification taking plasmid as template, PCR 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), plasmid DNA 3 μ l, 25mM MgCl2 4 μ l, (5U/ μ is 1 μ l l), ddH2O 34 μ l for TaqT 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 350bp, and its aminoacid sequence is SEQ ID NO:5.
Again with P1 and P5 taking the VP2 PCR fragment that reclaims and LTB PCR fragment as template, 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 Not I 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 7 ~ 8%, agar diffusion test and the qualification of western-blot method show, the albumen of expressing is VP2-LTB 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-LTB 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 respectively 96 parts of VP2 recombinant protein, the VP2-LTB 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-LTB subunit vaccine.
Eight, the immune effect of VP2 subunit vaccine and VP2-LTB 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-LTB immune group on the 7th is apparently higher than VP2 immune group and control group; After within 21st, attacking poison, two immune group all can obtain 10/10 protection, and control group 9/10 is fallen ill; The geometric mean VP2-LTB immune group of the agar diffusion antibody titer of serum apparently higher than VP2 immune group.The above results shows that the prepared fusion rotein of the present invention has good effect as vaccine.
Claims (4)
1. a fusion rotein of Infectious bursal disease virus VP2 and enterotoxins of Escherichia coli LTB, 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 IDNO:2.
4. fusion rotein claimed in claim 1 is in the application of preparing in subunit vaccine.
Priority Applications (1)
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| CN103483449A (en) * | 2013-08-20 | 2014-01-01 | 东北农业大学 | Two ScFv (Single Chain Variable Fragment ) antibodies, encoding genes and application thereof for preparing preparation for treating or preventing infectious bursal disease of chicken |
| 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 |
| CN107485712A (en) * | 2017-08-09 | 2017-12-19 | 扬州优邦生物药品有限公司 | A kind of PRV subunit vaccine and its preparation method and application |
| CN109134668A (en) * | 2018-09-19 | 2019-01-04 | 天康生物股份有限公司 | Fusion protein of chicken infectivity bursa of Fabricius virus and preparation method thereof, application, expression system and vaccine |
| CN114107227B (en) * | 2021-11-04 | 2022-09-06 | 扬州优邦生物药品有限公司 | Recombinant turkey herpesvirus live vector vaccine for simultaneously expressing classical strain infectious bursal disease virus VP2 protein and variant strain infectious bursal disease virus VP2 protein |
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