CN104328135B - Duck Tembusu virus E protein-LTB fusion protein and application thereof - Google Patents
Duck Tembusu virus E protein-LTB fusion protein and application thereof Download PDFInfo
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- CN104328135B CN104328135B CN201410569393.8A CN201410569393A CN104328135B CN 104328135 B CN104328135 B CN 104328135B CN 201410569393 A CN201410569393 A CN 201410569393A CN 104328135 B CN104328135 B CN 104328135B
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- fusion protein
- ltb
- tembusu virus
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Abstract
The invention aims to provide a duck Tembusu virus gene engineering subunit vaccine which is prepared by the following steps: screening to obtain duck Tembusu virus E protein with Domain III structure field of dominant antigen epitope, constituting a novel fusion protein LTB-Es from Lingker and enterotoxin LTB, and preparing the duck Tembusu virus gene engineering subunit vaccine by using the fusion protein as the antigen. The amino acid sequence of the coding protein of the duck Tembusu virus novel fusion protein LTB-Es is SEQ ID NO:1, and one of the nucleotide sequences is SEQ ID NO:2. The prokaryotic expression vector is utilized to construct the Escherichia coli BL21(DE3) host bacterium capable of expressing the duck Tembusu virus novel fusion protein LTB-Es. The gene engineering subunit vaccine prepared from the purified recombinant expressed protein can enable the immunized duck group to obtain immunoprotection.
Description
Technical field
The invention belongs to biological technical field and in particular to the fusion protein of a kind of duck tembusu virus e albumen and ltb and
Its application.
Background technology
China is duck culturing big country of the world, duck year production account for the 70% of world's total amount, from 186.6 ten thousand in 2000
Ton rises to 258.1 ten thousand tons in 2008, average growth rate per annum about 3.8%, is much higher than world average level.Duck tembusu virus
It is to take the lead in a kind of neopathy poison that finds in China for 2010, appetite and laying eggs declines to a great extent, ovary goes out mainly to cause duck to mine massively
This disease of blood, nervous symptoms etc., therefore initial stage is also called duck hemorrhagic ovaritis, egg drop syndrome etc..By virus purification,
Gene parses, and current duck tembusu virus is positioned the ntaya virus group of flaviviridae, Flavivirus, carapuru virus.According to not
Count completely, in egg duck and meat duck main producing region, there are about 1.2 hundred million egg ducks and more than 1,500 ten thousand meat ducks occur this disease, add up economy
Loss is more than 5,000,000,000 yuan.Simultaneously as neopathy poison, business-like duck tembusu virus vaccine is there is no to emerge at present.
Duck tembusu virus is single-stranded positive rna virus, and genome 10990bp, 95-10278bp are orf region, encode 3
Individual structural proteins (capsid protein c, memebrane protein m, envelope protein e) and 7 non-structural proteins (ns1, ns2a, ns2b, ns3,
Ns4a, ns4b, ns5), wherein envelope protein e has high immunogenicity, containing multiple important cell receptor binding sites,
And strong and lasting immune response can be caused, it is the oligogene engineered vaccine exploitation candidate albumen of flavivirus.Yellow
Peplos e albumen can be divided into domain i, domain ii and tri- domains of domain iii by its space structure, wherein
Domain iii domain can be identified by viral neutralizing antibody, and gene size is moderate, is more suitable for sub- single for genetic engineering
Position vaccine development.Chu etc. constructs the recombinant plasmid of expression west nile virus e protein structure domain iii, and uses oligodeoxynucleotide
(cpg-dna) as adjuvant, lumbar injection approach immunity balb/c mouse, in the west nile virus of result acquisition high titre and anti-
Body, shows that being aided with adjuvant west nile virus e albumen domain iii can make mouse produce immune response, have the latent of vaccine development
Power.
Produce Enterotoxigenic Escherichia coli (etec) and produce heat-labile toxin (heat-labile entero toxin, lt)
There is good immunogenicity and immunoadjuvant function, it is divided into a Asia (lta) and b subunit (ltb) again.Ltb is the immunogene of lt
Property position, be receptor binding site, there is good immunoadjuvant function, existing many document reports its be used for the immunity of vaccine
Adjuvant.Enterotoxin ltb GFP and aftosa vp1 GFP are carried out amalgamation and expression by Li Runcheng etc. (2009), find
Ltb can effectively facilitate the immune effect of aftosa vp1 albumen, and synergy is obvious.Lingering interest dragon etc. is by ltb and pig circular ring virus orf2 base
Because the fusion protein of amalgamation and expression preparation makes gene engineered subunit, after immunity, also obtain preferable immune effect.
Content of the invention
It is an object of the invention to provide the fusion protein of a kind of duck tembusu virus e albumen and ltb, obtained by screening
The duck tembusu virus e albumen domain iii part of dominant antigen epi-position, and pass through lingker(gggsgggs) and enterotoxin
Ltb forms new fusion protein ltb-es, and it is sub- to prepare duck tembusu virus genetic engineering using this fusion protein as antigen
Subunit vaccine.
Present invention firstly provides a kind of fusion protein ltb-es of duck tembusu virus e albumen domain iii and ltb, its
The amino acid sequence of encoding proteins is seq id no:1；
A kind of nucleotides sequence of above-mentioned albumen is classified as seq id no:2；
The present invention also provides a kind of duck tembusu virus genetic engineering subunit vaccine, and antigen therein is above-mentioned new
Fusion protein ltb-es, its concentration is between 0.1-0.5mg/ml；
The duck tembusu virus genetic engineering subunit vaccine of the present invention, its preparation process is as follows:
1) new fusion protein ltb-es gene is inserted in expression vector, be built into recombinant expression；
2) by the recombinant expression building conversion Host Strains, construct the restructuring that can express new fusion protein ltb-es
Genetic engineering bacterium；Go out new fusion protein ltb-es with this recombination engineering bacterium expression；
3) recombinant expressed ltb-es albumen is carried out after purification, adding white-oil adjuvant to make vaccine.
The present invention utilizes pet30a(+) Expressing vector constructs and can express duck tembusu virus new fusion protein ltb-
Escherichia coli bl21 (de3) Host Strains of es.Through sds-page analysis, give expression to 26kd restructuring destination protein.To recombinate egg
It is prepared into genetic engineering subunit vaccine after purification in vain, immune 14 age in days duck groups, duck group's adaptive immune can be made after immunity to protect.
Specific embodiment
Applicant is obtaining a duck tembusu virus new fusion protein ltb-es with immunological characteristic, and further
Its gene being obtained by Escherichia coli rare codon optimization, then by pet30a(+) Prokaryotic expression vector construction goes out can table
Reach Escherichia coli bl21 (de3) the recombination engineering bacteria of albumen ltb-es, by the induction of engineering bacteria, ultrasonication, egg
The preparations such as white purifying, quantitation and adjuvant proportioning and production method have prepared duck tembusu virus genetic engineering subunit vaccine.
To further describe the present invention with reference to specific embodiment, but it will be understood by those skilled in the art that
In the case of without departing from technical scheme can modifying to the details of technical scheme and form or
Replace, these modifications and replacement each fall within the scope of the present invention.
The acquisition of embodiment 1 duck tembusu virus new fusion protein ltb-es
1st, using biosoftware, duck Tan Busu is represented with strain zj-6(genbank accession number: jf459991.1) coating
Albumen e carries out Characterization of antigenic epitopes, and selected e protein antigenicity stronger domain iii position (303-410aa) and ltb sequence are entered
Row splicing, medium design lingker(gggsgggs) it is attached, obtain a kind of novel duck tembusu virus e albumen domain
The fusion protein ltb-es of iii and ltb, the amino acid sequence of its encoding proteins is seq id no:1, and using biological soft online
Part dnaworks carries out Escherichia coli rare codon optimization, the nucleotide sequence seq id no of acquisition fusion protein ltb-es:
2.
The nucleotide sequence of the new fusion protein ltb-es obtaining is carried out full genome synthesis, two ends add respectively simultaneously
Bamh i and hind iii restriction enzyme site.
The structure of embodiment 2 engineered protein expression vector and the acquisition of engineering bacteria
1st, the fusion protein ltb-es gene of above-mentioned full genome synthesis is connected into after bamh i and hind iii double digestion
The corresponding restriction enzyme site of pet30a carrier, builds pet30a/es expression vector.
2nd, use cacl2Pet30a/es expression vector is transformed into Escherichia coli bl21 (de3) by method, coats containing 50 μ g/ml
The agar plate of kanamycins, 37 DEG C of incubated overnight.Choose 10 single bacterium colonies and extract plasmid, bamh i and hind iii double digestion
The positive further sequencing identification of bacterium colony of checking.By the positive colony after sequence verification in lb culture medium fermented and cultured to 0.6
Add 0.3mm iptg to induce when～0.8 4～5 hours, thalline is collected by centrifugation and runs sds-page electrophoresis, set up non-induction bacterium simultaneously
Body is as comparison.After result induction, positive colony has more a protein band than comparison bacterium at 26kd, theoretical with recombinant protein
Molecular weight is consistent, and expression is about more than 30%.Through the calibrating of duck tembusu virus antibody immunoblotting, show positive reaction.Prove
The positive colony obtaining is the engineering bacteria of high efficient expression engineered protein, is named as le strain.
Embodiment 3 ferments, purify preparation code with duck tembusu virus genetic engineering subunit vaccine
1 bacterium kind
1.1 manufacture bacterial classifications are that duck tembusu virus genetic engineering subunit vaccine produces bacterium le strain；The strong poison of detection
Strain is the sd strain of the smooth cloth in goose source Soviet Union.
1.2 production bacterial standards
1.2.1 form and biochemical characteristic
Incubated overnight on lb agar plate containing kanamycins, culture plate presents circle, neat in edge, projection,
The glossiness smooth colony of milky, after Gram's staining, is shown as Gram-negative brevibacterium under mirror；Biochemical results are equal
For glucose fermentation+, indole test+, methyl red test+, vp-, citric acid using test-.
1.2.2 cultural character can grow in the culture medium containing kanamycins.
1.2.3 diagnostic test
The lb liquid culture 3 μ l of this bacterium is done template by 1.2.3.1 pcr detection, carries out pcr expansion with following pcr primer
Increase, should be able to amplify 418bp about fragment.
p1:5′- ctctcagaaaaaagcgat -3′
P2:5 '-caggatgaaggagtcacc-3 '
The condition of amplification is as follows: 94 DEG C of 5min denaturation, 94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min, 30 circulations, and 72
DEG C extend 10min.
1.2.3.2 the recombinant bacterium single bacterium colony on lb solid culture flat board is inoculated in 5ml and contains by western-blot detection
Have in the lb fluid nutrient medium of kanamycins, cultivate to od600Add the iptg induction of 0.8mm when value is between 0.6～1.0, hold
Continuous culture 4 hours, collects thalline, crushed with the resuspended rear high pressure homogenization of pbs, 8000 r/min centrifugations go to precipitate, supernatant with anti-
Duck tembusu virus positive serum carries out western-blot test, specific band should.
1.2.4 purely use appropriate media inspection, should be purely.
1.2.6 basic bacteria generation f5 ~ f8 generation.
1.2.7 freeze-drying lactobacillus are preserved, -20 DEG C, storage life is 2 years.
Vaccine manufactures and the inspection of semifinished product
2.1 prepared by production seed
2.1.1 freeze-drying lactobacillus are inoculated in the lb fluid nutrient medium containing kanamycins by first order seed breeding and identification respectively
In, 37 DEG C of shaken cultivation 8 ~ 10 hours, then streak inoculation on the lb solid medium containing kanamycins 37 DEG C of cultures 16 ~
18 hours, as first order seed.Preserve at 2～8 DEG C, less than 14 days；Culture medium passes on, should be less than for 2 generations.
2.1.2 10 mixing of colonies typical meeting 1.2.1 item standard are chosen in secondary seed breeding in first order seed
In a small amount of lb nutrient solution, it is inoculated in the lb nutrient solution containing kanamycins, cultivates 8～10 hours for 37 DEG C, carry out purely checking,
Should be purely.Put 2～8 DEG C of preservations, should be less than 3 days.
2.2 seedling culture mediums are improvement lb culture medium, 10g containing tryptone in every 1000 ml culture mediums, and yeast soaks
Powder 5g, sodium chloride 10 g, glucose 5g, mgso4∙ 7h2o 5g.
2.3 antigen for vaccine liquid preparations
2.3.1 bacterium solution culture with culture tank ventilate culture, by culture tank volume load appropriate culture medium (70% about) and
Defoamer, presses 5% inoculation secondary seed bacterium solution of culture base unit weight, 37 DEG C of ventilation cultures, treats the od of bacterium solution after sterilizing600Value reaches
When 7.0, add the alpha-lactose induction of 8g/l, be cultivated for 6～8 hours.Adjust ph using 20%naoh 7.0, by turning
Speed association controls dissolved oxygen more than 20%.When dissolved oxygen rises rapidly, flow feeding.
2.3.2, after broken bacterium culture terminates, thalline is collected by centrifugation.The thalline collected is cleaned with pbs 2 times, the bacterium that will collect
Body pbs makes 10% suspension, uses high-pressure homogenization crusher machine bacterium at 4 DEG C.Bacterium solution after broken, 8000r/min, centrifugation
15 minutes, collect supernatant.
2.3.3 bag filter put into by the recombinant protein eluent of nickel column chromatography purified pool, pbs liquid as extracellular fluid dialysis,
Recombinant protein liquid is obtained final product after dialysis desalting.
2.3.4 inactivate and 10% formalin, the ultimate density of formalin in the supernatant of purifying, will be proportionally added into
For 0.2%, 37 DEG C inactivate 12 hours, to inactivate the Escherichia coli of remaining.A small amount of sample is taken to carry out the inspection of semifinished product.2～8 DEG C of guarantors
Deposit, less than 7 days；Less than -15 DEG C preserved, less than 60 days.
2.4 the inspection of semifinished product
2.4.1 albumen es content detection detects Supernatant protein concentration with bca method, is diluted to final concentration 0.5mg/ml,
Aseptic filtration, stand-by.
2.4.2 steriling test is tested by existing " Chinese veterinary pharmacopoeia ", answers asepsis growth.
2.4.3 endotoxin content detection carries out endotoxin detection by TAL method, and endotoxin content is not higher than
1000eu/ml person can be used for seedling.
2.5 vaccine preparations
2.5.1 oil phase preparation takes 94 parts of high-quality injection white oil, 2 parts of aluminum stearate.Oil phase tank mixes, plus
Heat is melted to translucent, then 6 parts of Jia Siben -80, reaches to temperature and maintains 30 minutes when 125 ~ 130 DEG C, is cooled to room temperature standby
With.
2.5.2 aqueous phase preparation takes 4 parts of the Tween-80 of sterilizing, plus 96 parts of the semi-finished product that inspection is qualified, is stirred well to
Tween-80 is completely dissolved.
2.5.3 emulsification takes 3 parts of oil phase to be placed in high-speed shearing machine, starts motor stirring at low speed, is slowly added into water simultaneously
1 part of phase, then emulsified 40 minutes with 3600r/min, add 1% thimerosal solution before terminating stirring, final concentration reaches 0.01%.Breast
After change, sampling 10ml adds centrifuge tube, is centrifuged 15 minutes with 3000r/min, and ttom of pipe separates out aqueous phase and should be less than 0.5ml.
2.5.4 dispense quantitative separating, seal bottleneck.
Product inspection
3.1 physical behavior
Outward appearance is milky emulsion.
Formulation is water-in-oil type.Take a cleaning suction pipe, draw a small amount of vaccine and drip in cold water, in addition to the 1st, all should not
Diffusion.
Stability is drawn vaccine 10ml and is added in centrifuge tube, is centrifuged 15 minutes through 3000r/min, the aqueous phase that ttom of pipe separates out
0.5ml should be less than.
Viscosity is carried out by existing " Chinese veterinary pharmacopoeia ", meets regulation.
Loading quantity inspection is carried out by existing " Chinese veterinary pharmacopoeia ", meets regulation.
3.2 steriling tests are carried out by existing " Chinese veterinary pharmacopoeia ", asepsis growth.
3.3 safety verifications only take the susceptible duck of 7-14 age in days health 10, muscle or neck hypodermic injection vaccine 1ml/, see
Examine 14 days, occur without the locally and systemically bad reaction being caused by vaccine.
3.4 efficacy test
The susceptible duck 20 of 7-14 age in days health is taken only to be equally divided into two groups, every group 10.First group is immune group, uses the present invention
The duck tembusu virus genetic engineering subunit vaccine of preparation carries out that neck is subcutaneous or intramuscular injection is immune, 0.5ml/ only, the
Two groups is saline control group, the SPSS of injection equal volume.The smooth cloth of each group intramuscular injection duck after immune 21 days
Soviet Union's velogen strain sd strain carries out attacking poison, attacks malicious latter 5 days cut open inspections, takes spleen to carry out isolated viral.Control group should at least 8 isolated virals
The positive, then at least 7 separation are negative for test group.
3.5 formaldehyde, the antiseptic mercurials determination of residual amount are carried out by existing " Chinese veterinary pharmacopoeia ", all meet regulation.
Clinical trial after the immunity of embodiment 4 duck tembusu virus genetic engineering subunit vaccine
Susceptible for 14 age in days health duck 60 is only equally divided into three groups, every group 20.First group is immune group, uses system of the present invention
Standby duck tembusu virus genetic engineering subunit vaccine carries out that neck is subcutaneous or intramuscular injection is immune, 0.5ml/ only, second
Organize as positive controls, be prepared into reference to embodiment 4 method with recombinant expressed independent duck smooth cloth Soviet Union e albumen domain iii
Genetic engineering subunit vaccine, neck is subcutaneous or intramuscular injection is immune, and only, the 3rd group is saline control group to 0.5ml/,
The SPSS of injection equal volume.Each group intramuscular injection duck Tan Busu velogen strain sd strain after immune 21 days carries out attacking poison,
After attacking malicious 14 days, statistics attacks malicious protective rate (morbidity number of elements accounts for the percentage of total number of elements).Wherein morbidity standard: clinical spirit
Symptom, cut open inspection spleen enlargement, and duck tembusu virus can be isolated in spleen.
Result shows passive protection phase to duck group for the duck tembusu virus genetic engineering subunit vaccine of present invention preparation
More than 21 days, duck group after immunity 14 days attack malicious protective rate 100%, and positive controls are attacked malicious protective rate and are only
60%, the malicious protective rate of attacking of saline control group is 0%.Prove that the vaccine of the present invention has good clinical protection to duck group
Effect, can carry out clinical expansion and application.
Protest test after the immunity of table 1 duck tembusu virus genetic engineering subunit vaccine
Base sequence:
gcgccgcagaccatcaccgagctctgctctgaataccgtaacacccagatctacaccatcaacgacaaa
atcctgtcttacaccgaatctatggcgggtaaacgtgaaatggttatcatcaccttcaaatctggtgaaaccttcca
ggttgaagtcccgggttctcaacacatcgactctcagaaaaaagcgatcgaacgtatgaaagacaccctgcgtatca
cctacctgaccgaaaccaaaatcgacaaactgtgcgtttggaacaacaaaaccccgaattctatcgcggcgatctct
atgaaaaacgtgccgggtgttggtggtggctctggtggcggttctccgatgtgctctaacaccttctctctggttaa
aaacccgaccgacaccggtcacggtactgtggttgtcgaactctcttacgcgggtaccgacggtccgtgccgtgttc
ctatcagcatgtctgccgacctgaacgacatgaccccggttggtcgtctgatcaccgttaacccgtacgtttctacc
tcttctaccggtgcgaaaatcatggttgaggttgagccgccgttcggtgactccttcatcctggttggttccggtaa
aggtcagatccgttaccagtggcaccgttctggctctaccatcggtaaggcgttcacctct
Amino acid sequence:
apqtitelcseyrntqiytindkilsytesmagkremviitfksgetfqvevpgsqhidsqkkaiermk
dtlrityltetkidklcvwnnktpnsiaaismknvpgvgggsgggspmcsntfslvknptdtghgtvvvelsyagtd
gpcrvpismsadlndmtpvgrlitvnpyvstsstgakimveveppfgdsfilvgsgkgqiryqwhrsgstigkafts
Claims (6)
1. a kind of duck tembusu virus new fusion protein ltb-es is it is characterised in that the ammonia of described fusion protein ltb-es
Base acid sequence is seq id no:1.
2. a kind of coding claim 1 described in duck tembusu virus new fusion protein ltb-es gene it is characterised in that
The nucleotides sequence of described gene is classified as seq id no:2.
3. application in preparing vaccine for the duck tembusu virus new fusion protein ltb-es described in claim 1.
4. a kind of duck tembusu virus subunit vaccine is it is characterised in that the antigen of described vaccine is described in claim 1
Duck tembusu virus new fusion protein ltb-es.
5. vaccine as claimed in claim 4 is it is characterised in that duck tembusu virus new fusion protein in described vaccine
The concentration of ltb-es is 0.1-0.5mg/ml.
6. the preparation method of the vaccine described in claim 4 is it is characterised in that described preparation method includes following step
Rapid:
1) ltb-es gene is connected in prokaryotic expression carrier, is built into recombinant expression plasmid；
2) by the recombinant expression plasmid building conversion Host Strains, structure can express duck tembusu virus new fusion protein ltb-es
Recombination engineering bacteria, go out new fusion protein ltb-es with this recombination engineering bacterium expression；
3) recombinant expressed new fusion protein ltb-es is carried out after purification, adding white-oil adjuvant to make vaccine.
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| CN201410569393.8A CN104328135B (en) | 2014-10-23 | 2014-10-23 | Duck Tembusu virus E protein-LTB fusion protein and application thereof |
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| CN104328135B true CN104328135B (en) | 2017-01-18 |
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| CN106046174B (en) * | 2016-06-17 | 2019-06-04 | 杨麟 | Tocilizumab/CH12 is coupled mimic epitope peptide |
| CN111607605B (en) * | 2020-05-27 | 2023-10-24 | 重庆医科大学 | Construction method of multivalent epitope and subunit vaccine |
| CN113980146B (en) * | 2021-11-11 | 2022-09-27 | 扬州优邦生物药品有限公司 | Trimerization duck flavivirus E protein domainIII, and preparation method and application thereof |
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| WO2006011151A2 (en) * | 2004-07-28 | 2006-02-02 | Gavish - Galilee Bio Applications Ltd. | Vaccine comprising recombinant ct or lt toxin |
| CN103626878B (en) * | 2013-12-09 | 2015-03-18 | 青岛农业大学 | Newcastle disease virus F protein and enterotoxin LTB fusion protein and application thereof |
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