CN104328135A - 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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- CN104328135A CN104328135A CN201410569393.8A CN201410569393A CN104328135A CN 104328135 A CN104328135 A CN 104328135A CN 201410569393 A CN201410569393 A CN 201410569393A CN 104328135 A CN104328135 A CN 104328135A
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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, be specifically related to fusion rotein and the application thereof of a kind of duck tembusu virus E protein and LTB.
Background technology
China is Yang Ya big country of the world, duck year production account for 70% of world's total amount, rise to 258.1 ten thousand tons in 2008 from 2000 186.6 ten thousand tons, average growth rate per annum about 3.8%, is much higher than world average level.Duck tembusu virus to take the lead in a kind of neopathy poison that finds in China for 2010, mainly cause duck to mine massively appetite and laying eggs declines to a great extent, ovarian hemorrhage, nervous symptoms etc., therefore this disease of initial stage is also called duck hemorrhagic ovaritis, egg drop syndrome etc.Resolved by virus purification, gene, current duck tembusu virus is positioned the ntaya virus group of flaviviridae, Flavivirus, carapuru virus.According to incompletely statistics, in egg duck and meat duck main producing region, about have 1.2 hundred million egg ducks and more than 1,500 ten thousand meat ducks that this disease occurs, accumulative financial loss is more than 5,000,000,000 yuan.Simultaneously as neopathy poison, there is no business-like duck tembusu virus vaccine at present and emerge.
Duck tembusu virus is single strand plus RNA virus, genome 10990bp, 95-10278bp is ORF region, to encode 3 structural protein (capsid protein C, membranin M, envelope protein E) and 7 Nonstructural 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, be the oligogene engineered vaccine exploitation candidate albumen of flavivirus.Flavivirus envelope E protein can be divided into Domain I, Domain II and Domain III tri-structural domains by its space structure, wherein Domain III structural domain can by the neutralizing antibody identification of virus, and gene is of moderate size, be more suitable for developing for genetic engineering subunit vaccine.Chu etc. construct the recombinant plasmid of expressing west nile virus E protein Domain III, and with oligodeoxynucleotide (CpG-DNA) as adjuvant, abdominal injection approach immune balb/c mice, result obtains the west nile virus neutralizing antibody of high titre, show that being aided with adjuvant west nile virus E protein Domain III can make mouse produce immunne response, has the potentiality of vaccine development.
Product Enterotoxigenic Escherichia coli (ETEC) produces heat-labile toxin (heat-labile entero toxin, LT) and has good immunogenicity and immunoadjuvant function, and it is divided into again A Asia (LTA) and B subunit (LTB).LTB is the immunogenicity position of LT, is receptor binding site, has good immunoadjuvant function, existing many sections of its immunological adjuvants for vaccine of bibliographical information.Enterotoxin LTB protein gene and foot and mouth disease VP1 protein gene are carried out amalgamation and expression by Li Runcheng etc. (2009), and find that LTB effectively can promote the immune effect of foot and mouth disease VP1 albumen, synergy is obvious.Lingering interest dragon is waited and fusion rotein prepared by LTB and pig circular ring virus ORF2 gene fusion expression is made gene engineered subunit, also obtains desirable immune effect after immunity.
Summary of the invention
The object of this invention is to provide the fusion rotein of a kind of duck tembusu virus E protein and LTB, namely the duck tembusu virus E protein Domain III part of dominant antigen epi-position is obtained by screening, and pass through Lingker(GGGSGGGS) form new fusion protein LTB-Es with enterotoxin LTB, and duck tembusu virus genetic engineering subunit vaccine is prepared using this fusion rotein as antigen.
First the present invention provides the fusion rotein LTB-Es of a kind of duck tembusu virus E protein Domain III and LTB, and the aminoacid sequence of its proteins encoded 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 is wherein above-mentioned new fusion protein LTB-Es, and its concentration is between 0.1-0.5mg/ml;
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) recombinant expression built is transformed Host Strains, construct the recombination engineering bacteria expressing new fusion protein LTB-Es; New fusion protein LTB-Es is gone out by this recombination engineering bacterium expression;
3), after purifying being carried out to recombinant expressed LTB-Es albumen, add white-oil adjuvant and make vaccine.
The present invention utilizes pET30a(+) Expressing vector constructs e. coli bl21 (DE3) Host Strains expressing duck tembusu virus new fusion protein LTB-Es.Analyze through SDS-PAGE, given expression to 26kD restructuring target protein.Be prepared into genetic engineering subunit vaccine by after recombinant protein purification, immune 14 age in days duck groups, duck group adaptive immune after immunity, can be made to protect.
Embodiment
Applicant has the duck tembusu virus new fusion protein LTB-Es of immunological characteristic acquisition one, and obtain its gene further by the optimization of intestinal bacteria rare codon, then by pET30a(+) Prokaryotic expression vector construction goes out can e. coli bl21 (DE3) the recombination engineering bacteria of expressing protein LTB-Es, by the induction to engineering bacteria, ultrasonication, protein purification, quantitatively and the preparation such as adjuvant proportioning and production method prepared duck tembusu virus genetic engineering subunit vaccine.
The present invention is further described below in conjunction with embodiment; but what those skilled in the art should understand that is; can modify to the details of technical scheme of the present invention and form when not departing from technical scheme of the present invention or replace, these amendments and replacement all fall in scope.
the acquisition of embodiment 1 duck tembusu virus new fusion protein LTB-Es
1, biosoftware is utilized to represent strain ZJ-6(GenBank accession number to duck Tan Busu: envelope protein E JF459991.1) carries out Characterization of antigenic epitopes, selected E protein antigenicity stronger Domain III position
(303-410aa)splice with LTB sequence, medium design Lingker(GGGSGGGS) connect, obtain the fusion rotein LTB-Es of a kind of Novel duck tembusu virus E protein Domain III and LTB, the aminoacid sequence of its proteins encoded is SEQ ID NO:1, and utilize online biosoftware DNAWorks to carry out the optimization of intestinal bacteria rare codon, obtain the nucleotide sequence SEQ ID NO:2 of fusion rotein LTB-Es.
The nucleotide sequence of the fusion rotein LTB-Es newly obtained is carried out full genome synthesis, and two ends add BamH I and Hind III digestion site respectively simultaneously.
the structure of embodiment 2 engineered protein expression vector and the acquisition of engineering bacteria
1, be connected into the corresponding restriction enzyme site of pET30a carrier after the fusion rotein LTB-Es gene BamH I of above-mentioned full genome synthesis and Hind III double digestion, build pET30a/Es expression vector.
2, CaCl is used
2pET30a/Es expression vector is transformed into e. coli bl21 (DE3) by method, coats the agar plate containing 50 μ g/ml kantlex, 37 DEG C of incubated overnight.Choose 10 single bacterium colonies and extract plasmids, BamH I and the positive bacterium colony of Hind III double digestion checking check order qualification further.Positive colony after sequence verification is added in LB substratum 0.3mM IPTG during fermentation culture to 0.6 ~ 0.8 to induce 4 ~ 5 hours, collected by centrifugation thalline runs SDS-PAGE electrophoresis, sets up simultaneously and does not induce thalline in contrast.After result induction, positive colony has more a protein band than contrast bacterium at 26kD place, and consistent with recombinant protein theoretical molecular, expression amount is about more than 30%.Through the calibrating of duck tembusu virus antibody immunoblotting, display positive reaction.Prove that the positive colony obtained is the engineering bacteria of high expression engineered protein, called after LE strain.
embodiment 3 is fermented, the preparation code of purifying and 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 virulent strain detected is the smooth cloth Soviet Union SD strain of goose source.
1.2 production bacterial standards
1.2.1 form and biochemical characteristic
Incubated overnight on LB agar plate containing kantlex, culture plate presents circle, neat in edge, projection, the glossiness smooth colony of oyster white, after gramstaining, is shown as Gram-negative tyrothricin under mirror; Biochemical results be glucose fermentation+, indole test+, methyl red test+, VP-, Citric Acid utilize test-.
1.2.2 cultural characters can grow in containing the substratum of kantlex.
1.2.3 diagnostic test
1.2.3.1 PCR detects and the LB liquid culture 3 μ l of this bacterium is done template, carries out pcr amplification, should be able to amplify the fragment of about 418bp by following PCR primer.
P1: 5′- CTCTCAGAAAAAAGCGAT -3′
P2： 5 ′- CAGGATGAAGGAGTCACC-3′
The condition of amplification is as follows: 94 DEG C of 5min sex change, 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 Western-blot detects and is contained in the LB liquid nutrient medium of kantlex in 5ml by the recombinant bacterium list colony inoculation on LB solid culture flat board, is cultured to OD
600add the IPTG induction of 0.8mM when value is between 0.6 ~ 1.0, continue cultivation 4 hours, collect thalline, broken with the resuspended rear high pressure homogenization of PBS, 8000 r/min are centrifugal goes precipitation, and supernatant liquor and anti-duck tembusu virus positive serum carry out Western-blot test, should occur specific band.
1.2.4 pure appropriate media inspection, should be pure.
1.2.6 basic bacteria generation F5 ~ F8 generation.
1.2.7 preserve freeze-drying lactobacillus ,-20 DEG C, preservation period is 2 years.
vaccine manufactures and the inspection of semifinished product
2.1 production Spawn preparation
2.1.1 freeze-drying lactobacillus is inoculated in the LB liquid nutrient medium containing kantlex by first order seed breeding and qualification respectively, 37 DEG C of shaking culture 8 ~ 10 hours, then streak inoculation in containing kantlex LB solid medium on 37 DEG C cultivate 16 ~ 18 hours, as first order seed.2 ~ 8 DEG C of preservations, be no more than 14 days; Substratum goes down to posterity, should be no more than for 2 generations.
2.1.2 secondary seed breeding is chosen the colonies typical 10 meeting 1.2.1 item standard and is mixed in a small amount of LB nutrient solution in first order seed, is inoculated in the LB nutrient solution containing kantlex, cultivates 8 ~ 10 hours, purely checks for 37 DEG C, should be pure.Put 2 ~ 8 DEG C of preservations, 3 days should be no more than.
2.2 seedling substratum are improvement LB substratum, containing Tryptones 10g in every 1000 ml substratum, and yeast leaching powder 5g, sodium-chlor 10 g, glucose 5g, MgSO
47H
2o 5g.
2.3 prepared by antigen for vaccine liquid
2.3.1 bacterium liquid is cultivated and is used culture tank aerated culture, loads appropriate substratum (about 70%) and defoamer by culture tank volume, and by the 5% inoculation secondary seed bacterium liquid cultivating base unit weight after sterilizing, 37 DEG C of aerated culture, treat the OD of bacterium liquid
600when value reaches 7.0, add the alpha-lactose induction of 8g/L, then continue cultivation 6 ~ 8 hours.Use 20%NaOH to regulate pH 7.0, control dissolved oxygen more than 20% by rotating speed association.When dissolved oxygen rises rapidly, flow feeding.
2.3.2 after broken bacterium is cultivated and terminates, collected by centrifugation thalline.The thalline PBS collected cleans 2 times, by the suspension that the thalline PBS of collection makes 10%, uses high pressure homogenizer disrupt bacteria at 4 DEG C.Bacterium liquid after fragmentation, 8000r/min, centrifugal 15 minutes, collects supernatant liquor.
2.3.3 dialysis tubing put into by the recombinant protein elutriant that nickel column chromatography purification is collected, and PBS liquid, as extracellular fluid dialysis, namely obtains recombinant protein liquid after dialysis desalting.
2.3.4 the deactivation formaldehyde solution that will add 10% in the supernatant liquor of purifying in proportion, the ultimate density of formaldehyde solution is 0.2%, 37 DEG C of deactivations 12 hours, with the intestinal bacteria that deactivation is remaining.The sample that takes a morsel carries out the inspection of semifinished product.2 ~ 8 DEG C of preservations, are no more than 7 days; Less than-15 DEG C preservations, are no more than 60 days.
2.4 the inspection of semifinished product
2.4.1 albumen Es content detection BCA method detects Supernatant protein concentration, is diluted to final concentration 0.5mg/ml, sterile filtration, stand-by.
2.4.2 steriling test is tested by existing " Chinese veterinary pharmacopoeia ", answers asepsis growth.
2.4.3 endotoxin content detects and carries out intracellular toxin detection by tachypleus amebocyte lysate method, and endotoxin content can not be used for seedling higher than 1000EU/ml person.
2.5 vaccine preparations
2.5.1 oil phase preparation gets high-quality injection white oil 94 parts, aluminum stearate 2 parts.Mix in oil phase tank, heating and melting is to translucent, then Jia Siben-80 6 parts, maintains 30 minutes, be cooled to room temperature for subsequent use when temperature reaches 125 ~ 130 DEG C.
2.5.2 the tween-80 4 parts of sterilizing is got in aqueous phase preparation, adds the work in-process 96 parts be up to the standards, is stirred well to tween-80 and dissolves completely.
2.5.3 emulsification is got oil phase 3 parts and is placed in high-speed shearing machine, starts motor stirring at low speed, slowly adds aqueous phase 1 part simultaneously, then with 3600r/min emulsification 40 minutes, before termination is stirred, add 1% Thiomersalate solution, final concentration reached 0.01%.After emulsification, sampling 10ml adds centrifuge tube, with 3000r/min centrifugal 15 minutes, separates out aqueous phase and should be no more than 0.5ml at the bottom of pipe.
2.5.4 packing quantitative separating, sealing bottleneck.
inspection after construction
3.1 physical behavior
Outward appearance is oyster white emulsion.
Formulation is water-in-oil-type.Get a clean suction pipe, draw a small amount of vaccine and drip in cold water, except the 1st, all should indiffusion.
Stability is drawn vaccine 10ml and is added in centrifuge tube, through 3000r/min centrifugal 15 minutes, and the aqueous phase of separating out at the bottom of pipe should be no more than 0.5ml.
Viscosity is undertaken by existing " Chinese veterinary pharmacopoeia ", conforms with the regulations.
Loading quantity inspection is undertaken by existing " Chinese veterinary pharmacopoeia ", conforms with the regulations.
3.2 steriling tests are undertaken by existing " Chinese veterinary pharmacopoeia ", asepsis growth.
3.3 safety verifications get the healthy susceptible duck 10 of 7-14 age in days, and muscle or neck subcutaneous injection vaccine 1ml/ only, observe 14 days, do not occur the local that caused by vaccine and systemic adverse reactions.
3.4 efficacy test
Get the healthy susceptible duck 20 of 7-14 age in days and be only equally divided into two groups, often organize 10.First group is immune group, and the duck tembusu virus genetic engineering subunit vaccine prepared with the present invention carries out that neck is subcutaneous or intramuscular injection is immune, and only, second group is saline control group to 0.5ml/, the stroke-physiological saline solution of injection equal volume.Immunity is respectively organized intramuscular injection duck Tan Busu virulent strain SD strain for 21 days afterwards and is carried out attacking poison, attacks poison and cuts open inspection in latter 5 days, get spleen and carry out isolated viral.Control group should at least 8 isolated viral positives, test group then at least 7 be separated negative.
3.5 formaldehyde, the antiseptic mercurials determination of residual amount are undertaken by existing " Chinese veterinary pharmacopoeia ", all conform with the regulations.
clinical trial after the immunity of embodiment 4 duck tembusu virus genetic engineering subunit vaccine
Healthy for 14 ages in days susceptible duck 60 is only equally divided into three groups, often organizes 20.First group is immune group, the duck tembusu virus genetic engineering subunit vaccine prepared with the present invention carries out that neck is subcutaneous or intramuscular injection is immune, 0.5ml/ only, second group is positive controls, namely be prepared into genetic engineering subunit vaccine with recombinant expressed independent duck Tan Busu E protein Domain III with reference to embodiment 4 method, neck is subcutaneous or intramuscular injection is immune, and 0.5ml/ only, 3rd group is saline control group, the stroke-physiological saline solution of injection equal volume.Immunity is respectively organized intramuscular injection duck Tan Busu virulent strain SD strain for 21 days afterwards and is carried out attacking poison, attacks to add up afterwards for malicious 14 days and attacks malicious protection ratio (morbidity number of elements accounts for the percentage of total number of elements).Wherein to fall ill standard: clinically occur mental symptom, cut open the enlargement of inspection spleen, and duck tembusu virus can be isolated at spleen.
Result showed duck tembusu virus genetic engineering subunit vaccine prepared by the present invention to the passive protection phase of duck group more than 21 days; duck group after immunity 14 days attack malicious protection ratio 100%; and positive controls is attacked malicious protection ratio and is only 60%, the malicious protection ratio of attacking of saline control group is 0%.Prove that vaccine of the present invention has good clinical protection effect to duck group, can carry out clinical expansion and application.
protest test after the immunity of table 1 duck tembusu virus genetic engineering subunit vaccine
Base sequence:
GCGCCGCAGACCATCACCGAGCTCTGCTCTGAATACCGTAACACCCAGATCTACACCATCAACGACAAAATCCTGTCTTACACCGAATCTATGGCGGGTAAACGTGAAATGGTTATCATCACCTTCAAATCTGGTGAAACCTTCCAGGTTGAAGTCCCGGGTTCTCAACACATCGACTCTCAGAAAAAAGCGATCGAACGTATGAAAGACACCCTGCGTATCACCTACCTGACCGAAACCAAAATCGACAAACTGTGCGTTTGGAACAACAAAACCCCGAATTCTATCGCGGCGATCTCTATGAAAAACGTGCCGGGTGTTGGTGGTGGCTCTGGTGGCGGTTCTCCGATGTGCTCTAACACCTTCTCTCTGGTTAAAAACCCGACCGACACCGGTCACGGTACTGTGGTTGTCGAACTCTCTTACGCGGGTACCGACGGTCCGTGCCGTGTTCCTATCAGCATGTCTGCCGACCTGAACGACATGACCCCGGTTGGTCGTCTGATCACCGTTAACCCGTACGTTTCTACCTCTTCTACCGGTGCGAAAATCATGGTTGAGGTTGAGCCGCCGTTCGGTGACTCCTTCATCCTGGTTGGTTCCGGTAAAGGTCAGATCCGTTACCAGTGGCACCGTTCTGGCTCTACCATCGGTAAGGCGTTCACCTCT
Aminoacid sequence:
APQTITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGETFQVEVPGSQHIDSQKKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISMKNVPGVGGGSGGGSPMCSNTFSLVKNPTDTGHG
TVVVELSYAGTDGPCRVPISMSADLNDMTPVGRLITVNPYVSTSSTGAKIMVEVEPPFGDSFILVGSGKGQIRYQWHRSGSTIGKAFTS
Claims (7)
1. a duck tembusu virus new fusion protein LTB-Es gene, is characterized in that, the aminoacid sequence of the proteins encoded of described gene is SEQ ID NO:1.
2. gene as claimed in claim 1, it is characterized in that, the nucleotides sequence of described gene is classified as SEQ ID NO:2.
3. a duck tembusu virus new fusion protein LTB-Es, is characterized in that, the aminoacid sequence of described albumen is SEQ ID NO:1.
4. duck tembusu virus new fusion protein LTB-Es gene according to claim 1 is preparing the application in vaccine.
5. a duck tembusu virus subunit vaccine, is characterized in that, the antigen of described vaccine is duck tembusu virus new fusion protein LTB-Es according to claim 3.
6. vaccine as claimed in claim 5, it is characterized in that, in described vaccine, the concentration of duck tembusu virus new fusion protein LTB-Es is 0.1-0.5mg/ml.
7. the preparation method of vaccine according to claim 5, is characterized in that, described preparation method includes following step:
1) LTB-Es gene is connected in prokaryotic expression carrier, is built into recombinant expression plasmid;
2) recombinant expression plasmid built is transformed Host Strains, build the recombination engineering bacteria expressing duck tembusu virus new fusion protein LTB-Es, go out new fusion protein LTB-Es by this recombination engineering bacterium expression;
3), after purifying being carried out to recombinant expressed new fusion protein LTB-Es, add white-oil adjuvant and make vaccine.
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| CN106046174A (en) * | 2016-06-17 | 2016-10-26 | 杨麟 | Tocilizumab/CH12 coupled simulated epitope |
| CN111607605A (en) * | 2020-05-27 | 2020-09-01 | 重庆医科大学 | Construction method of multivalent epitope and subunit vaccine |
| CN113980146A (en) * | 2021-11-11 | 2022-01-28 | 扬州优邦生物药品有限公司 | Trimerization duck flavivirus E protein domainIII, and preparation method and application thereof |
| CN115896151A (en) * | 2022-11-18 | 2023-04-04 | 广东海洋大学 | Construction method and application of food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106046174A (en) * | 2016-06-17 | 2016-10-26 | 杨麟 | Tocilizumab/CH12 coupled simulated epitope |
| CN106046174B (en) * | 2016-06-17 | 2019-06-04 | 杨麟 | Tocilizumab/CH12 is coupled mimic epitope peptide |
| CN111607605A (en) * | 2020-05-27 | 2020-09-01 | 重庆医科大学 | Construction method of multivalent epitope and subunit vaccine |
| CN111607605B (en) * | 2020-05-27 | 2023-10-24 | 重庆医科大学 | Construction method of multivalent epitope and subunit vaccine |
| CN113980146A (en) * | 2021-11-11 | 2022-01-28 | 扬州优邦生物药品有限公司 | Trimerization duck flavivirus E protein domainIII, and preparation method and application thereof |
| 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 |
| CN115896151A (en) * | 2022-11-18 | 2023-04-04 | 广东海洋大学 | Construction method and application of food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector |
| CN115896151B (en) * | 2022-11-18 | 2024-08-02 | 广东海洋大学 | Construction method and application of food-grade recombinant duck tembusu virus truncated E gene lactococcus lactis vector |
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