CN1754959A - Preparation process and application of genetic engineering subunit vaccine of infectious bursal disease - Google Patents
Preparation process and application of genetic engineering subunit vaccine of infectious bursal disease Download PDFInfo
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Abstract
The invention relates to a bacterium for the production of genetically-engineered subunit oil-adjuvant vaccines against infectious bursal disease, i.e. Escherichia coli BL21/pET-28a-VP2, CCTCC No: M204038. The process for constructing the bacterium consists of inserting VP2 protein gene of the infectious bursal disease virus onto expression plasmid pET-28a to construct prokaryotic expression plasmid, then using the prokaryotic expression plasmid to transform the escherichia coli BL21, using lactose as inducer to induce Escherichia coli BL21/pET-28a-VP2 expression VP2 protein, purifying and charging oil-adjuvant to obtain the vaccines.
Description
Technical field
The present invention relates to a kind of preparation method and application of genetic engineering subunit vaccine of infectious bursal disease, belong to the genetically engineered field.
Background technology
(Infectious bursal disease IBD) is virus disease acute, the height contagiousness to infectious bursal disease.It is by infectious bursa of Fabricius virus (Infectious bursal disease virus, the immunosuppression sexually transmitted disease of the chicken that IBDV) causes.The infectivity of infectious bursal disease is extremely strong, can bamboo telegraph in a chicken group.
This sick pathogenic agent IBDV has very strong anti-deactivation.Therefore, the use of vaccine is necessary except adopting strict hygiology measure under the situation of height infection pressure, also is a compulsory measure.The main purpose of doing like this is the protection chick avoids IBDV in the several weeks after hatching infection.Reaching this purpose has two important measures, at first will induce the maternal antibody of high titre between the whole laying period of kind of chicken of laying eggs by oil-emulsion inactivated vaccinating agent.In addition, hatch back inoculation attenuated live vaccine in good time chick.The time of living vaccine inoculation is very crucial.Because, in the maternal antibody energy that in the chicken body, exists and living vaccine.Yet, 1986 highly virulent strain (vvIBDV) appearance make the situation of epidemic prevention that very big variation take place.Because the virulence of vvIBDV is stronger and the variation of antigenic characteristic, makes it can break through the protection and the postvaccinal protection of Paracox of maternal antibody.Therefore, traditional vaccine has lost effect to vvIBDV.The exploitation novel vaccine becomes inevitable.Novel vaccine is the complete mesogenic living vaccine of attenuation not, and its main purpose provides enough strong immunostimulation with the provide protection of generation to highly virulent strain.Yet these mesogenic living vaccines itself may cause immunosuppression and disturb the immunization of other vaccine, as newcastle disease vaccine and marek's disease vaccine.And the case of mesogenic living vaccine immunity postoperative infection highly virulent strain still constantly occurs.The effective ways of another antagonism vvIBDV are inactivated vaccines, but this inactivated vaccine must have quite high virus antigen titre effect are arranged.At the inactivated vaccine the most effective in high infected area is to prepare antigen with the fabricius bursa that infects.This antigenic preparation must be used the vvIBDV infected chicken, slaughters infected chicken then and extract required antigen from the fabricius bursa.This process need butchers a large amount of chickens thereby production cost is very high.At now forcing people to explore new improvement vaccine and exploitation novel vaccine, infect with control IBDV and reduce financial loss with the defective of IBDV vaccine.
Owing to the perfect day by day and practicability of gene clone and recombinant technology, many scientists have researched and developed many experimental reorganization IBD vaccines in recent years.Mostly these vaccines are with virus to be the vaccine of live vector.Main vector virus has: fowl poxvirus, herpevirus of turkey, fowl adenovirus, Marek ' sdisease virus and Semlidk Forest virus.In addition, vivoexpression VP2 and generation IBDV viruslike particle also have been proved immunogenicity.The live vector vaccine and the subunit vaccine that with the bacterium are expression vector are also studied for many years.Recently, there is report to produce the IBDV subunit vaccine, in laboratory study and field experiment, obtains effect preferably with yeast expression system.Research with escherichia expression system research and development subunit vaccine and viable bacteria carrier vaccine has also been carried out for many years, but known result of study is, escherichia coli expression IBDV VP2 albumen non-immunogenicity or form insoluble inclusion body and can't be used as vaccine.
Summary of the invention
The method and the application that the object of the present invention is to provide a kind of E.coli.BL21/pET-28a-VP2 bacterial classification and produce the IBD genetic engineering subunit vaccine and produce IBD genetically engineered viable bacteria carrier vaccine with this bacterial classification.
Technical scheme of the present invention is:
1, the extraction of total RNA
Total RNA uses living worker UNIQ-10Trizol RNA extraction test kit to carry out.Concise and to the point process is as follows: the fabricius bursa is taken from the IBD chicken that dies of illness, and grinds and homogenate.With cell freeze thawing 3 times.Get 50 milligrams of tissue homogenates and place a polyethylene centrifuge tube, add 0.5 milliliter of Trizol reagent under the room temperature.Seal and thermal agitation 15 seconds.Add chloroform/primary isoamyl alcohol 100 microlitres, thermal agitation 30 seconds.Put under 4 ℃ of conditions 12, centrifugal 15 minutes of 000rpm.The colourless aqueous solution of upper strata water is changed in the centrifuge tube of an other no RNA enzyme, and per 450 microlitre liquid add the gentle jolting of dehydrated alcohol 150 microlitres.Whole solution is changed in UNIQ-10 post and the collection tube.Room temperature was placed 2 minutes.Under the room temperature 12, centrifugal 1 minute of 000rpm.Post is taken out from collection tube, discard waste liquid in the collection tube.Post is reentered in the same collection tube.Add 450 microlitre RW liquid in the UNIQ-10 post, room temperature was placed 2 minutes.Room temperature 12, centrifugal 1 minute of 000rpm.From collection tube, take out post, discard the waste liquid in the collection tube, post is put into same collection tube.Repeat previous step once.With post 12, under the 000rpm centrifugal 1 minute.Post is changed in the centrifuge tube of no RNA enzyme.The water that adds the no RNA enzyme that 50 microlitre DEPC handled on the film of post central authorities, 70 ℃ of insulations 2 minutes.Pipe was put under the 12000rpm centrifugal 1 minute.Total be exactly the solution fabricius bursa in the centrifuge tube and the RNA of IBDV.
2, VP2 gene clone
According to VP2 gene 5 ' and 3 ' the terminal synthetic oligonucleotide primer of conserved regions sequence, 5 ' end of each primer comprises a convenience with the restriction site of gene clone to the carrier.This is used to carry out the RT-PCR amplification to primer and produces cDNA.Get total RNA liquid 8 microlitres, add 70～80 ℃ of sex change of 1 microlitre, 40 μ mol/L reverse primers 5 minutes, ice bath is 3 minutes immediately.Add 4 μ 1M-MLV, 5 * RTbuffer subsequently successively, 5 μ l 4mmol/L dNTPs, 40u Rnasin, 200u M-MLV, mix, centrifugal, add in the specimen tube of step 1 every pipe 11 microlitres, make cumulative volume reach 20 μ 1, after the centrifugal several seconds, 37 ℃ of water-baths 1 hour, 95 ℃ of 10 minutes deactivation RT enzymes.
The pcr amplification of cDNA: 10 μ l cDNA liquid, 5 μ l, 10 * Ex Taq damping fluid, the 4 TaKaRaEX Taq of unit (a kind of high-fidelity DNA polymerase can not held at the thing of expanding production simultaneously to provide an extra A and be used for the T carrier and connect), two kind of 40 each 0.5 μ l of μ mol primer.4 μ l dNTP mixed solutions (every pipe 2.5mM), cumulative volume is 50 μ l.PCR temperature cycle program is: 94 ℃, and 5 minutes; (94 ℃ 60 seconds, 64 ℃ 120 seconds, 72 ℃ 120 seconds) circulation 35 times; 72 ℃, 10 minutes.Separate amplified fragments with 1% agarose gel electrophoresis, the corresponding nucleic acids band is cut, and extracts test kit with a small amount of glue and carry out purifying.Isolating VP2 fragment is connected on the pMD18-T carrier.The DNA plasmid Transformed E .coli DH5 α that connects, the white bacterial strain that is grown on the LBG plate that contains penbritin, X-Gal and IPTG is separated.Extract plasmid DNA, analyze existence and the closure of VP2 with restriction enzyme and PCR method.
3, the structure of expression vector
The VP2 gene fragment that will contain correct direction and sequence is downcut with restriction enzyme, and the method that the VP2 gene fragment reclaims after with agarose gel electrophoresis same as described above reclaims.Subclone is to linearizing pET-28a carrier then, and linearizing process is used identical restriction enzyme.Novel plasmid called after pET-28a-VP2.It is used for Transformed E .coli.BL21.Transformed bacteria is identified with PCR and restriction restriction endonuclease.Separate the cDNA clone and the order-checking that contain VP2.
The expressed sequence sequencing result:
atg aca aac ctg caa gat caa acc caa cag att gtt ccg ttc ata cgg 48
Met Thr Asn Leu Gln Asp Gln Thr Gln Gln Ile Val Pro Phe Ile Arg
1 5 10 15
agc ctt ctg atg cca aca acc gga ccg gcg tcc att ccg gac gac acc 96
Ser Leu Leu Met Pro Thr Thr Gly Pro Ala Ser Ile Pro Asp Asp Thr
20 25 30
cta gag aag cac act ctc agg tca gag acc tcg acc tac aat ttg act 144
Leu Glu Lys His Thr Leu Arg Ser Glu Thr Ser Thr Tyr Asn Leu Thr
35 40 45
gtg ggg gac aca ggg tca ggg cta att gtc ttt ttc cct ggt ttc cct 192
Val Gly Asp Thr Gly Ser Gly Leu Ile Val Phe Phe Pro Gly Phe Pro
50 55 60
ggc tca att gtg ggc gct cac tac aca ctg cag agc aat ggg aac tac 240
Gly Ser Ile Val Gly Ala His Tyr Thr Leu Gln Ser Asn Gly Asn Tyr
65 70 75 80
aag ttc gat cag atg ctc ctg act gcc cag aac cta ccg gcc agc tac 288
Lys Phe Asp Gln Met Leu Leu Thr Ala Gln Asn Leu Pro Ala Ser Tyr
85 90 95
aac tac tgc agg cta gtg agt cgg agt ctc aca gtg agg tca agc aca 336
Asn Tyr Cys Arg Leu Val Ser Arg Ser Leu Thr Val Arg Ser Ser Thr
100 105 110
ctc cct ggt ggc gtt tat gca cta aat ggc acc ata aac gcc gtg acc 384
Leu Pro Gly Gly Val Tyr Ala Leu Asn Gly Thr Ile Asn Ala Val Thr
115 120 125
ttc caa gga agc ctg agt gaa ctg aca gat gtt agc tac aat ggg ttg 432
Phe Gln Gly Ser Leu Ser Glu Leu Thr Asp Val Ser Tyr Asn Gly Leu
130 135 140
atg tct gca aca gcc aac atc aac gac aaa att ggg aac gtc cta gta 480
Met Ser Ala Thr Ala Asn Ile Asn Asp Lys Ile Gly Asn Val Leu Val
145 150 155 160
ggg gag ggg gta acc gtc ctc agc tta ccc aca tca tat gat ctt ggg 528
Gly Glu Gly Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly
165 170 175
tat gtg aga ctc ggt gac ccc att ccc gct ata ggg ctc gac cca aaa 576
Tyr Val Arg Leu Gly Asp Pro Ile Pro Ala Ile Gly Leu Asp Pro Lys
180 185 190
atg gta gca aca tgt gac agc agt gac agg ccc aga gtc tac act ata 624
Met Val Ala Thr Cys Asp Ser Ser Asp Arg Pro Arg Val Tyr Thr Ile
195 200 205
act gca gcc gat gat tac caa ttc tca tca cag tac caa gca ggt ggg 672
Thr Ala Ala Asp Asp Tyr Gln Phe Ser Ser Gln Tyr Gln Ala Gly Gly
210 215 220
gta aca atc aca ctg ttc tca gct aat atc gat gcc atc aca agc ctc 720
Val Thr Ile Thr Leu Phe Ser Ala Asn Ile Asp Ala Ile Thr Ser Leu
225 230 235 240
agc atc ggg gga gaa ctt gtg ttt caa aca agc gtc caa ggc ctt ata 768
Ser Ile Gly Gly Glu Leu Val Phe Gln Thr Ser Val Gln Gly Leu Ile
245 250 255
ctg ggt gct acc atc tac ctt ata ggc ttt gat ggg act gcg gta atc 816
Leu Gly Ala Thr Ile Tyr Leu Ile Gly Phe Asp Gly Thr Ala Val Ile
260 265 270
acc aga gct gtg gcc gcg gac aat ggg cta acg gcc ggc act gac aac 864
Thr Arg Ala Val Ala Ala Asp Asn Gly Leu Thr Ala Gly Thr Asp Asn
275 280 285
ctt atg cca ttc aat att gtg att cca acc agc gag ata acc cag cca 912
Leu Met Pro Phe Asn Ile Val Ile Pro Thr Ser Glu Ile Thr Gln Pro
290 295 300
atc aca tcc atc aaa ctg gag ata gtg acc tcc aaa agt ggt ggt cag 960
Ile Thr Ser Ile Lys Leu Glu Ile Val Thr Ser Lys Ser Gly Gly Gln
305 310 315 320
gcg ggg gat cag atg tca tgg tca gca agt ggg agc cta gca gtg acg 1008
Ala Gly Asp Gln Met Ser Trp Ser Ala Ser Gly Ser Leu Ala Val Thr
325 330 335
atc cac ggt ggc aac tat cca ggg gcc ctc cgt ccc gtc aca cta gta 1056
Ile His Gly Gly Asn Tyr Pro Gly Ala Leu Arg Pro Val Thr Leu Val
340 345 350
gcc tac gaa aga gtg gca aca ggg tct gtc gtt acg gtc gcc ggg gtg 1104
Ala Tyr Glu Arg Val Ala Thr Gly Ser Val Val Thr Val Ala Gly Val
355 360 365
agc aac ttc gag ctg atc cca aat cct gaa eta gca aag aac ctg gtc 1152
Ser Asn Phe Glu Leu Ile Pro Asn Pro Glu Leu Ala Lys Asn Leu Val
370 375 380
aca gaa tac ggc cga ttt gac cca ggg gcc atg aac tac aca aaa ttg 1200
Thr Glu Tyr Gly Arg Phe Asp Pro Gly Ala Met Asn Tyr Thr Lys Leu
385 390 395 400
ata ctg agt gag agg gac cgt ctt ggc atc aag acc gta tgg cca aca 1248
Ile Leu Ser Glu Arg Asp Arg Leu Gly Ile Lys Thr Val Trp Pro Thr
405 410 415
agg gag tac act gac ttt cgc gag tac ttc atg gag gtg gcc gac ctc 1296
Arg Glu Tyr Thr Asp Phe Arg Glu Tyr Phe Met Glu Val Ala Asp Leu
420 425 430
aac tct ccc ctg aag att gca gga gca ttt ggc ttc aaa gac ata atc 1344
Asn Ser Pro Leu Lys Ile Ala Gly Ala Phe Gly Phe Lys Asp Ile Ile
435 440 445
cgg gcc ata agg aaa gct tgc ggc cgc act cga gca cca cca cca cca 1392
Arg Ala Ile Arg Lys Ala Cys Gly Arg Thr Arg Ala Pro Pro Pro Pro
450 455 460
cca ctg aga tcc ggc tgc taa 1413
Pro Leu Arg Ser Gly Cys End
465 470
4, the expression process in the intestinal bacteria
The single bacterium colony of picking e. coli bl21/pET-28a-VP2 is inoculated in the LB+Kan nutrient solution of 10ml, and 37 ℃ of shaking culture are to OD
600=0.6～1.0, to make final concentration be 1mmol/L or add alpha-lactose to final concentration 20mmol/L to add IPTG, cultivated 5 hours for 37 ℃ again.Take out back 5000rpm * 10min.Bacterial sediment is resuspended in the PBS liquid of 1/10 volume (1ml).Add N,O-Diacetylmuramidase to 100 μ g/ml, add the TritonX-100 of 1/10 volume simultaneously, PMSF 3 μ l, 37 ℃ are incubated 1 hour.Multigelation 3 times is handled with the ultrasonic cell-break device and to be sheared DNA to solution thickness no longer.12, centrifugal 15 minutes of 000g separates soluble part and insoluble part.Soluble part is carried out the antigenic activity inspection.
5, the preparation of vaccine
The preparation of IBD subunit oil emulsion vaccine: the ammonium sulfate precipitation of the supernatant of above-mentioned lysate being used the 20-45% saturation ratio.Collecting precipitation heavily is dissolved in an amount of PBS liquid, changes in the dialysis tubing PBS is dialysed.With solution dilution to the AGP antigen titre of VP2 is 1/16.Add 2 parts of oil-emulsion by 1 part of VP2 protein solution, and carry out decentralized system and become oil emulsion vaccine (preparation of oil-emulsion: mineral oil: Si Ben-80=9: 1, fully add 4% tween-80 behind the mixing, 2% aluminum stearate, thorough mixing, standby behind the high pressure steam sterilization).
E. coli bl21/pET-28a-VP2IBD viable bacteria carrier vaccine preparation: its process is identical with aforesaid " structure of expression vector " process, does not handle but do not make bacteriolyze.It is standby to collect an amount of stablizer freeze-drying processing back of thalline adding.
The preparation of IBD mesogenic living vaccine B87: the IBD living vaccine is that the Chinese Hangzhou amount of recommending veterinary biologics company limited produces, and by specification requires with sterile saline dilution back standby.
6, immunization experiment
(1) test chicken is bought 200 1 age in days Ai Weiyin meat sold on the market chicks from Jing Zhou Kang Ermei kind chicken house.During the 18th (12) age in days,, detect maternal antibody with agar gel immunodiffusion test (AGP) to every chicken blood sampling.Reject the positive chicken of maternal antibody, the negative chicken of maternal antibody is divided into 5 groups at random, be respectively A, B, C, D, E, group.
(2) inoculation method is intramuscular injection.
(3) grouping and immunization
Table 1 chicken immune experiment grouping and immunization
| Group | The vaccination type | Inoculation for the first time | Inoculation for the second time | ||
| Number of elements | Dosage (ml) | Number of elements | Dosage (ml) | ||
| A | Negative blank group | 6 | —— | 6 | —— |
| B | Positive blank group | 17 | —— | 17 | —— |
| C | E.coli.BL21/pET-VP2 | 14 | 0.1 | 14 | 0.1 |
| D | VP2 subunit oil-emulsion vaccine | 14 | 0.3 | 14 | —— |
| E | IBD mesogenic living vaccine | 6 | 0.1 | 6 | 0.1 |
(4) challenge test
IBDV virulent strain BC 6/85 is available from China Veterinary Drugs Supervisory Inst., with 2 * 10
4Chicken embryo median infective dose (EID
50)/when ml attacks poison, every chicken muscle injection 0.1ml.Attacked for the second time poison after the immunization in 10 days.
(5) immune effect is determined foundation
Attack the poison back and cutd open test chicken extremely on the 10th day.Check the fabricius bursa, and observe its appearance luster.Get a part of fabricius bursa and make tissue slice, with the tissues observed pathological change.
Fabricius bursa histology ANOMALOUS VARIATIONS is analyzed foundation, sees Table 2.
Table 2 IBDV infects back fabricius bursa damaged tissue and learns points-scoring system
| The damage scoring | Histological characteristic |
| 012345 | The equal not damaged of any lymph follicle, medullary substance and the cortex boundary clear of the fabricius bursa.Accidental lymph follicle is slightly downright bad, and fabricius bursa one-piece construction is kept normally.＜50% lymph follicle has the lymph follicle of serious lymphocyte loss＞50% to have serious lymphocyte loss only to have the profile of lymph follicle to exist, connective tissue proliferation, blister cavitiesization.Whole lymph follicle structure forfeiture, fibrosis. |
(7) the interior anti-IBDV antibody production of chicken body after the immunization: see Table 3.
Anti-IBDV antibody A GP detected result goes out rate in the table 3 experimental chicken body
| Group | The vaccination type | Chicken number (only) | Antibody positive number (only) | Antibody positive rate (%) |
| A | Negative blank group | 6 | 0 | 0 |
| B | Positive blank group | 17 | 0 | 0 |
| C | E.coli.BL21/pET28a-VP2 | 14 | 14 | 100.0 |
| D | VP2 subunit finish seedling | 14 | 14 | 100.0 |
| E | IBD mesogenic living vaccine | 6 | 2 | 33.3 |
By table 3 as seen, the antibody positive of C group (E.coli.BL21/pET28a-VP2 live vaccine group) and D group (VP2 subunit finish seedling group) all reaches 100%.And E group commercially available vaccine antibody positive recall rate is 33.3%.
(8) attack the fabricius bursa histological examination table 4 as a result of poison back testing chicken on the 10th day
Table 4 histological examination fabricius bursa damage appraisal result
| Group | Vaccine | Quantity | Experimental chicken fabricius bursa damage scoring number | Protection ratio (%) | |||||
| 0 | 1 | 2 | 3 | 4 | 5 | ||||
| A | Negative blank | 6 | 6 | 0 | 0 | 0 | 0 | 0 | |
| B | Positive blank | 17 | 0 | 0 | 0 | 0 | 8 | 9 | 0/17 (0.0) |
| C | E.coli.BL21/pET28a-V P2 live vaccine | 14 | 12 | 0 | 1 | 0 | 1 | 0 | 12/14 (85.7) |
| D | VP2 subunit finish seedling | 14 | 8 | 5 | 1 | 0 | 0 | 0 | 13/14 (92.8) |
| E | IBD mesogenic living vaccine | 6 | 0 | 2 | 2 | 0 | 2 | 0 | 2/6 (33.3) |
The present invention has the following advantages and positively effect:
(1) the expression IBDV VP2 albumen that this bacterial classification can be reliable and stable.This expression product is a soluble proteins in the cytosol, has the proteic antigenic activity of natural VP2, and production cost is lower.
(2) this bacterial classification can be used to producer gene engineering subunit vaccine and genetically engineered viable bacteria carrier vaccine, is used for the prevention of IBD.
(3) be used for the standard antigen preparation of ELISA with can be used as gene engineering antigen behind the VP2 protein purification of expressing, or the preparation of other standard antigen such as AGP.
China typical culture collection center is used for the culture collection of patented procedure and accepts letter of information (receipt):
Classification name Escherichia coli BL21/pET-28a-VP2
Preservation date on May 24th, 2004
Depositary institution China Wuhan Wuhan University postcode 430072
Deposit number CCTCC NO:M204038
Embodiment
The VP2 gene of coding IBDV highly virulent strain is connected to formation expression plasmid pET-28a-VP2 on the pET-28a plasmid, and Transformed E scherichia coli BL21 obtains genetically engineered bacterial classification Escherichia coliBL21/pET-28-VP2.This bacterial classification can be used for the production of genetic engineering subunit vaccine, for the prevention of IBD provides a novel method.This bacterial classification also can be used for the production of VP2 gene engineering antigen, is used for the anti-IBD detection of antibodies of chicken serum.
Gene order
Organization Applicant
City: Jing Zhou, Hubei
Country: China
PostalCode：434025
PhoneNumber：07168496979
EmailAddress：rongjunl@public.js.hb.cn
＜110〉OrganizationName: Changjiang University
＜120〉Title: a kind of preparation method of genetic engineering subunit vaccine of infectious bursal disease and application
＜130〉AppFileReference: a kind of preparation method of genetic engineering subunit vaccine of infectious bursal disease and application
<140>CurrentAppNumber：cn
<141>CurrentFilingDate：2004-08-28
<170>PatentIn version 3.1
<210>1
<211>Length：1413
SequenceName：IBDV subunit vaccine VP2 gene sequence
<212>Type：DNA
<213>OrganismName：Infectious bursal disease virus
Feature
<221>FeatureKey：CDS
<222>LocationFrom：1
<222>LocationTo：1413
<400>PreSequenceString：1
atgacaaacc tgcaagatca aacccaacag attgttccgt tcatacggag ccttctgatg 60
ccaacaaccg gaccggcgtc cattccggac gacaccctag agaagcacac tctcaggtca 120
gagacctcga cctacaattt gactgtgggg gacacagggt cagggctaat tgtctttttc 180
cctggtttcc ctggctcaat tgtgggcgct cactacacac tgcagagcaa tgggaactac 240
aagttcgatc agatgctcct gactgcccag aacctaccgg ccagctacaa ctactgcagg 300
ctagtgagtc ggagtctcac agtgaggtca agcacactcc ctggtggcgt ttatgcacta 360
aatggcacca taaacgccgt gaccttccaa ggaagcctga gtgaactgac agatgttagc 420
tacaatgggt tgatgtctgc aacagccaac atcaacgaca aaattgggaa cgtcctagta 480
ggggaggggg taaccgtcct cagcttaccc acatcatatg atcttgggta tgtgagactc 540
ggtgacccca ttcccgctat agggctcgac ccaaaaatgg tagcaacatg tgacagcagt 600
gacaggccca gagtctacac tataactgca gccgatgatt accaattctc atcacagtac 660
caagcaggtg gggtaacaat cacactgttc tcagctaata tcgatgccat cacaagcctc 720
agcatcgggg gagaacttgt gtttcaaaca agcgtccaag gccttatact gggtgctacc 780
atctacctta taggctttga tgggactgcg gtaatcacca gagctgtggc cgcggacaat 840
gggctaacgg ccggcactga caaccttatg ccattcaata ttgtgattcc aaccagcgag 900
ataacccagc caatcacatc catcaaactg gagatagtga cctccaaaag tggtggtcag 960
gcgggggatc agatgtcatg gtcagcaagt gggagcctag cagtgacgat ccacggtggc 1020
aactatccag gggccctccg tcccgtcaca ctagtagcct acgaaagagt ggcaacaggg 1080
tctgtcgtta cggtcgccgg ggtgagcaac ttcgagctga tcccaaatcc tgaactagca 1140
aagaacctgg tcacagaata cggccgattt gacccagggg ccatgaacta cacaaaattg 1200
atactgagtg agagggaccg tcttggcatc aagaccgtat ggccaacaag ggagtacact 1260
gactttcgcg agtacttcat ggaggtggcc gacctcaact ctcccctgaa gattgcagga 1320
gcatttggct tcaaagacat aatccgggcc ataaggaaag cttgcggccg cactcgagca 1380
ccaccaccac caccactgag atccggctgc taa 1413
Sequence
<210>2
<211>Length：470
<212>Type：PRT
<213>OrganismName：Infectious bursal desease virus
<221>FeatureKey：VP2
<222>LocationFrom：1
<222>LocationTo：470
<400>PreSequenceString：2
MTNLQDQTQQ IVPFIRSLLM PTTGPASIPD DTLEKHTLRS ETSTYNLTVG DTGSGLIVFF 60
PGFPGSIVGA HYTLQSNGNY KFDQMLLTAQ NLPASYNYCR LVSRSLTVRS STLPGGVYAL 120
NGTINAVTFQ GSLSELTDVS YNGLMSATAN INDKIGNVLV GEGVTVLSLP TSYDLGYVRL 180
GDPIPAIGLD PKMVATCDSS DRPRVYTITA ADDYQFSSQY QAGGVTITLF SANIDAITSL 240
SIGGELVFQT SVQGLILGAT IYLIGFDGTA VITRAVAADN GLTAGTDNLM PFNIVIPTSE 300
ITQPITSIKL EIVTSKSGGQ AGDQMSWSAS GSLAVTIHGG NYPGALRPVT LVAYERVATG 360
SVVTVAGVSN FELIPNPELA KNLVTEYGRF DPGAMNYTKL ILSERDRLGI KTVWPTREYT 420
DFREYFMEVA DLNSPLKIAG AFGFKDIIRA IRKACGRTRA PPPPPLRSGC 470
Claims (5)
1, a kind of bacterial classification that is used for the production of infectious bursal disease gene engineered subunit oil emulsion vaccine is characterized in that: Escherichia coli BL21/pET-28a-VP2, CCTCC NO:M204038.
2, bacterial classification according to claim 1 is characterized in that being connected to the infectious bursal disease virus VP 2 gene sequence and the deduced amino acid of the modified on the pET-28a in preparation process, this sequence is:
atg aca aac ctg caa gat caa acc caa cag att gtt ccg ttc ata cgg 48
Met Thr Asn Leu Gln Asp Gln Thr Gln Gln Ile Val Pro Phe Ile Arg
1 5 10 15
agc ctt ctg atg cca aca acc gga ccg gcg tcc att ccg gac gac acc 96
Ser Leu Leu Met Pro Thr Thr Gly Pro Ala Ser Ile Pro Asp Asp Thr
20 25 30
cta gag aag cac act ctc agg tca gag acc tcg acc tac aat ttg act 144
Leu Glu Lys His Thr Leu Arg Ser Glu Thr Ser Thr Tyr Asn Leu Thr
35 40 45
gtg ggg gac aca ggg tca ggg cta att gtc ttt ttc cct ggt ttc cct 192
Val Gly Asp Thr Gly Ser Gly Leu Ile Val Phe Phe Pro Gly Phe Pro
50 55 60
ggc tca att gtg ggc gct cac tac aca ctg cag agc aat ggg aac tac 240
Gly Ser Ile Val Gly Ala His Tyr Thr Leu Gln Ser Asn Gly Asn Tyr
65 70 75 80
aag ttc gat cag atg ctc ctg act gcc cag aac cta ccg gcc agc tac 288
Lys Phe Asp Gln Met Leu Leu Thr Ala Gln Asn Leu Pro Ala Ser Tyr
85 90 95
aac tac tgc agg cta gtg agt cgg agt ctc aca gtg agg tca agc aca 336
Asn Tyr Cys Arg Leu Val Ser Arg Ser Leu Thr Val Arg Ser Ser Thr
100 105 110
ctc cct ggt ggc gtt tat gca cta aat ggc acc ata aac gcc gtg acc 384
Leu Pro Gly Gly Val Tyr Ala Leu Asn Gly Thr Ile Asn Ala Val Thr
115 120 125
ttc caa gga agc ctg agt gaa ctg aca gat gtt agc tac aat ggg ttg 432
Phe Gln Gly Ser Leu Ser Glu Leu Thr Asp Val Ser Tyr Asn Gly Leu
130 135 140
atg tct gca aca gcc aac atc aac gac aaa att ggg aac gtc cta gta 480
Met Ser Ala Thr Ala Asn Ile Asn Asp Lys Ile GIy Asn Val Leu Val
145 150 155 160
ggg gag ggg gta acc gtc ctc agc tta ccc aca tca tat gat ctt ggg 528
Gly Glu Gly Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly
165 170 175
tat gtg aga ctc ggt gac ccc att ccc gct ata ggg ctc gac cca aaa 576
Tyr Val Arg Leu Gly Asp Pro Ile Pro Ala Ile Gly Leu Asp Pro Lys
180 185 190
atg gta gca aca tgt gac agc agt gac agg ccc aga gtc tac act ata 624
Met Val Ala Thr Cys Asp Ser Ser Asp Arg Pro Arg Val Tyr Thr Ile
195 200 205
act gca gcc gat gat tac caa ttc tca tca cag tac caa gca ggt ggg 672
Thr Ala Ala Asp Asp Tyr Gln Phe Ser Ser Gln Tyr Gln Ala Gly Gly
210 215 220
gta aca atc aca ctg ttc tca gct aat atc gat gcc atc aca agc ctc 720
Val Thr Ile Thr Leu Phe Ser Ala Asn Ile Asp Ala Ile Thr Ser Leu
225 230 235 240
agc atc ggg gga gaa ctt gtg ttt caa aca agc gtc caa ggc ctt ata 768
Ser Ile Gly Gly Glu Leu Val Phe Gln Thr Ser Val Gln Gly Leu Ile
245 250 255
ctg ggt gct acc atc tac ctt ata ggc ttt gat ggg act gcg gta atc 816
Leu Gly Ala Thr Ile Tyr Leu Ile Gly Phe Asp Gly Thr Ala Val Ile
260 265 270
acc aga gct gtg gcc gcg gac aat ggg cta acg gcc ggc act gac aac 864
Thr Arg Ala Val Ala Ala Asp Asn Gly Leu Thr Ala Gly Thr Asp Asn
275 280 285
ctt atg cca ttc aat att gtg att cca acc agc gag ata acc cag cca 912
Leu Met Pro Phe Asn Ile Val Ile Pro Thr Ser Glu Ile Thr Gln Pro
290 295 300
atc aca tcc atc aaa ctg gag ata gtg acc tcc aaa agt ggt ggt cag 960
Ile Thr Ser Ile Lys Leu Glu Ile Val Thr Ser Lys Ser Gly Gly Gln
305 310 315 320
gcg ggg gat cag atg tca tgg tca gca agt ggg agc cta gca gtg acg 1008
Ala Gly Asp Gln Met Ser Trp Ser Ala Ser Gly Ser Leu Ala Val Thr
325 330 335
atc cac ggt ggc aac tat cca ggg gcc ctc cgt ccc gtc aca cta gta 1056
Ile His Gly Gly Asn Tyr Pro Gly Ala Leu Arg Pro Val Thr Leu Val
340 345 350
gcc tac gaa aga gtg gca aca ggg tct gtc gtt acg gtc gcc ggg gtg 1104
Ala Tyr Glu Arg Val Ala Thr Gly Ser Val Val Thr Val Ala Gly Val
355 360 365
agc aac ttc gag ctg atc cca aat cct gaa cta gca aag aac ctg gtc 1152
Ser Asn Phe Glu Leu Ile Pro Asn Pro Glu Leu Ala Lys Asn Leu Val
370 375 380
aca gaa tac ggc cga ttt gac cca ggg gcc atg aac tac aca aaa ttg 1200
Thr Glu Tyr Gly Arg Phe Asp Pro Gly Ala Met Asn Tyr Thr Lys Leu
385 390 395 400
ata ctg agt gag agg gac cgt ctt ggc atc aag acc gta tgg cca aca 1248
Ile Leu Ser Glu Arg Asp Arg Leu Gly Ile Lys Thr Val Trp Pro Thr
405 410 415
agg gag tac act gac ttt cgc gag tac ttc atg gag gtg gcc gac ctc 1296
Arg Glu Tyr Thr Asp Phe Arg Glu Tyr Phe Met Glu Val Ala Asp Leu
420 425 430
aac tct ccc ctg aag att gca gga gca ttt ggc ttc aaa gac ata atc 1344
Asn Ser Pro Leu Lys Ile Ala Gly Ala Phe Gly Phe Lys Asp Ile Ile
435 440 445
cgg gcc ata agg aaa gct tgc ggc cgc act cga gca cca cca cca cca 1392
Arg Ala Ile Arg Lys Ala Cys Gly Arg Thr Arg Ala Pro Pro Pro Pro
450 455 460
cca ctg aga tcc ggc tgc taa 1413
Pro Leu Arg Ser Gly Cys End
465 470
3, bacterial classification according to claim 1 and 2, it is characterized in that the proteic method of abduction delivering infectious bursal disease virus VP 2, this expressing protein is the albumen that solubility has antigenic activity, can be used for preparing gene engineering subunit vaccine of chicken's infectious bursal disease.
4, bacterial classification according to claim 1 and 2 is characterized in that through abduction delivering, can directly be used as viable bacteria carrier vaccine after the lyophilize, the prevention infectious bursal disease.
5, according to right 3 described bacterial classifications, it is characterized in that the VP2 albumen that abduction delivering comes out, can be used as gene engineering antigen, be used for the antibody test of infectious bursal disease through behind the purifying.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102973935A (en) * | 2012-12-03 | 2013-03-20 | 青岛康地恩药业股份有限公司 | Chicken subunit triple vaccine, as well as preparation and application thereof |
| CN103341164A (en) * | 2013-07-19 | 2013-10-09 | 天津瑞普生物技术股份有限公司 | Infectious bursal disease antigen-antibody complex as well as preparation and preparation method thereof |
| CN103849631A (en) * | 2012-11-28 | 2014-06-11 | 普莱柯生物工程股份有限公司 | Infectious bursal disease virus subunit antigen-containing vaccine composition, preparation method and application thereof |
| CN104513298A (en) * | 2014-12-09 | 2015-04-15 | 山东信得科技股份有限公司 | Method for expression of chicken infectious bursal soluble VP 2-4-3 polypeptide |
| CN106148455A (en) * | 2015-03-25 | 2016-11-23 | 普莱柯生物工程股份有限公司 | A kind of derivant and application thereof |
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| CN101792742A (en) * | 2010-04-07 | 2010-08-04 | 西北农林科技大学 | Recombinant baculovirus expressing IBDV (Infectious Bursal Disease Virus) immunogen gene and preparation method and application thereof |
| CN102688486A (en) * | 2012-06-18 | 2012-09-26 | 青岛易邦生物工程有限公司 | Production method of triple inactivated vaccine for newcastle disease, infectious bursal disease and viral arthritis |
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| US4530831A (en) * | 1982-11-02 | 1985-07-23 | Akzo N.V. | Infectious Bursal Disease vaccine |
| US5064646A (en) * | 1988-08-02 | 1991-11-12 | The University Of Maryland | Novel infectious bursal disease virus |
| CN1099295C (en) * | 1998-09-04 | 2003-01-22 | 中国科学院上海生物化学研究所 | Infectious cloacal bursa virus vaccine for chicken and its preparing process and application |
| US6468984B1 (en) * | 1999-06-08 | 2002-10-22 | Innovo Biotechnologies Ltd. | DNA vaccine for protecting an avian against infectious bursal disease virus |
| CN1102409C (en) * | 1999-12-23 | 2003-03-05 | 中国兽药监察所 | Triple live chicken vaccine and its production process |
| CN1522760A (en) * | 2003-09-04 | 2004-08-25 | 长江大学 | Bivalent gene engineering vaccine of fowl infectious bursal disease and pasteurellosis |
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| CN103849631A (en) * | 2012-11-28 | 2014-06-11 | 普莱柯生物工程股份有限公司 | Infectious bursal disease virus subunit antigen-containing vaccine composition, preparation method and application thereof |
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| CN102973935A (en) * | 2012-12-03 | 2013-03-20 | 青岛康地恩药业股份有限公司 | Chicken subunit triple vaccine, as well as preparation and application thereof |
| CN103341164A (en) * | 2013-07-19 | 2013-10-09 | 天津瑞普生物技术股份有限公司 | Infectious bursal disease antigen-antibody complex as well as preparation and preparation method thereof |
| CN103341164B (en) * | 2013-07-19 | 2015-06-24 | 天津瑞普生物技术股份有限公司 | Infectious bursal disease antigen-antibody complex as well as preparation and preparation method thereof |
| CN104513298A (en) * | 2014-12-09 | 2015-04-15 | 山东信得科技股份有限公司 | Method for expression of chicken infectious bursal soluble VP 2-4-3 polypeptide |
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| CN106148455A (en) * | 2015-03-25 | 2016-11-23 | 普莱柯生物工程股份有限公司 | A kind of derivant and application thereof |
| CN108624611A (en) * | 2018-05-15 | 2018-10-09 | 武汉科前生物股份有限公司 | The preparation and its application of infectious bursa of Fabricius virus virus-like particle |
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| CN100360662C (en) | 2008-01-09 |
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