CN103667198B - The structure of the restructuring duck enteritis virus vaccine of expression-secretion type duck tembusu virus E protein and application - Google Patents
The structure of the restructuring duck enteritis virus vaccine of expression-secretion type duck tembusu virus E protein and application Download PDFInfo
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Abstract
The invention provides a kind of restructuring duck enteritis virus vaccine of expression-secretion type duck tembusu virus E protein, does is its preserving number CCTCC? V201214, called after rDEV-TE-tPAS, and construction process and application.Particularly, the present invention utilizes recombinant clone technology, will SV40 promotor, duck tembusu virus E protein and tPA (Tissue be comprised? plasminogen? activator) the gene fragment SV40-E-tPA of signal peptide sequence be inserted into duck enteritis virus US7 and US8 gene between transcribed spacer in, build to obtain between US7 and US8 gene, insert the clay that SV40-E-tPA expresses framework, the restructuring duck enteritis virus vaccine CCTCC of expression-secretion type duck tembusu virus E protein is obtained by its rescue? V201214.The invention still further relates to the method building this restructuring duck enteritis virus vaccine strain, and this restructuring duck enteritis virus vaccine strain is for the preparation of the application of the vaccine of the transmissible disease of preventing duck enteritis virus and duck tembusu virus to cause.
Description
Technical field
The invention belongs to recombinant viral vaccine field, more specifically belong to restructuring duck enteritis virus vaccines arts.The invention provides a kind of restructuring duck enteritis virus vaccine strain CCTCCV201214 of expression-secretion type duck tembusu virus E protein, called after rDEV-TE-tPAS, and construction process and application.
Background technology
Duck enteritis virus (duckenteritisvirus, DEV), also known as duck enteritis virus.Duck can be caused, acute, hot, septic occurs is the deadly infectious disease of feature for goose and other Anseriformes bird.Compared with other simplexvirus, the research for DEV is less.8th time international virusology classification committee report is categorized as simplexvirus
[1], and specifically belong to classification and do not determine yet; As of late, its genom sequence has just all been checked order
[2].From 2007, this research department started the genomic examining order of DEV vaccine strain, and by building DEV vaccine strain full-length genome cosmid library, segmentation is checked order to it and analyzed, and to mid-term in 2009, is parsed by DEV vaccine strain whole genome sequence.Almost simultaneously, Li etc. also report order-checking and the analytical results of DEV vaccine strain full-length genome, and its Genome Size is about 158Kb, 78 albumen of about encoding.By forming and structural analysis DEV vaccine strain genomic gene, DEV is considered to the osculant virus in α blister sore subfamily, more close with Varicellavirus member
[2].And be all the Marek’s disease poison (MDV) of bird simplexvirus and herpes turkey virus (HTV) and belong to Marek’s disease poison and belong to, avian infectious laryngotracheitis virus (ILTV) and parrot simplexvirus (PsHV) they are infectious laryngotracheitis virus genus.
Since the TK gene successfully utilizing poxvirus to be vector expression hsv the eighties in last century, it is carrier that people start to attempt with various different DNA virus, express different foreign gene, and the recombiant vaccine of structure is used for the prevention of people and various different animals disease.A large amount of results of study shows, simplexvirus because of its genome large, the dispensable gene that can insert for foreign gene or substitute is many, is considered to a kind of virus vector of structure live recombined vaccines well.Up to now, in common livestock and poultry herpesvirus disease, existing a large amount of correlative study reports; As with Pseudorabies virus (PRV) for carrier, in the gene such as gD, gE, gG and TK, insert the foreign genes such as the E2 of swine fever (CSF) respectively, and use its immune swine, achieve good immune effect
[4-11].Be used in UL0 and UL50 of avian infectious laryngotracheitis virus (ILTV) and insert the gene constructed successful recombinant virus immunity chicken of different HA respectively, all respond well
[11-13].Equally, SakaguchiM (1993; 1994) and SonodaK (1996) etc. in US10, US3, IRL of MDV1, insert LacZ gene respectively; with its immune 1 age in days specific pathogen free chicken (SPF chicken); within 1 week, rear vMDV, vvMDV attack poison, and it is 80 ~ 100% to the protective efficacy of SPF chicken
[14-16]; In the US10 of MDV1, insert NDVF gene, in US2, insert the recombinant virus of IBDVVP2 gene to the protective efficacy of the strong poison of MDV with to contrast MDV1 suitable
[17,18]; Successful construction of recombinant virus between TsukamotoK etc. (2002) insert herpes turkey virus (HVT) as the promotor of infectious bursal disease virus (IBDV) VP2 gene UL45 and UL46 gene using Pec, is enough to the attack of resisting the strong poison of IBDV with the SPF chicken of its immunity
[19].Current, successful chicken embryo reduction living vaccine mainly studied the sixties in last century by the vaccine that China is used for duck plague prevention.As α blister sore subfamily a member, DEV also should be a kind of virus vector of structure live recombined vaccines well.
The genomic constitution of DEV and structure and MDV etc. have larger difference, and the genome structure as MDV is TRL-UL-IRL-IRS-US-TRS, and the genomic structure of DEV is UL-IRS-US-TRS.And DEV with grow with other several livestock and poultry simplexviruss of subfamily the Biological characteristics copied in animal body and be also not quite similar.Therefore, in PRV, MDV, ILTV, the site of Absorbable organic halogens insertion foreign gene is not necessarily applicable to DEV.Owing to relatively lagging behind to the research of DEV, the research report both at home and abroad about Nonessencial region in DEV gene is still blank.The method being usually used in building simplexvirus recombinant virus has three kinds.One is homologous recombination; The second inserts in BAC by viral genome, then on BAC, builds sudden change, go out recombinant virus with the corresponding cellular rescue of its transfection; The third is inserted in clay respectively the hsv gene fragment containing overlapped district, and build sudden change in its respective section, then go out recombinant virus with the corresponding cellular rescue of its cotransfection.But for the virus of this fundamental research shortage of DEV, unclear classification Chu, dispensable gene the unknown, with first or second method construction of recombinant virus workload large, and efficiency is low.And the difficult point of the third method is the foundation of many clays infections clone, if the success of this platform construction, can construction of recombinant virus fast and effectively.So far, this infections clone constructing technology comparative maturity of simplexvirus, and be seen in report
[20-27].
This research department, in early-stage Study, identifies the Nonessencial region can stablizing insertion for foreign gene in the genome of duck enteritis virus.On this basis; the protective gene E gene expression construct of duck tembusu virus PTD2010 strain inserts between genomic US7 and the US8 gene of DEV by this research; SPF duck can be made to produce the antibody of good antagonism duck tembusu virus with its immune specific pathogen free duck (SPF duck), and not affect the immune effect of DEV.
Summary of the invention
The invention provides a kind of restructuring duck enteritis virus vaccine strain of expression-secretion type duck tembusu virus E protein, its deposit number is CCTCCV201214, called after rDEV-TE-tPAS, and construction process and application.
Particularly, the present invention utilizes recombinant clone technology, the gene fragment SV40-E (SEQIDNO:1) comprising duck tembusu virus E gene and SV40 promoter sequence is inserted into duck enteritis virus (duckenteritisvirus, DEV) in the transcribed spacer (nucleotide sequence of the transcribed spacer between US7 and US8 gene is shown in SEQIDNO:5) between US7 and US8 gene, build the clay pFOS5us78SV40E obtaining and insert SV40E expression cassette between US7 and US8 gene, the restructuring duck enteritis virus vaccine strain CCTCCV201214 of expression-secretion type duck tembusu virus E protein is obtained by its rescue, called after rDEV-TE-tPAS.The invention still further relates to the method building this restructuring duck enteritis virus vaccine strain, and this restructuring duck enteritis virus vaccine strain is for the preparation of the application of the vaccine of the transmissible disease of preventing duck enteritis virus and duck tembusu virus to cause.
In one embodiment of the invention, the invention provides a kind of restructuring duck enteritis virus vaccine strain of expression-secretion type duck tembusu virus E protein, its deposit number is CCTCCV201214, called after rDEV-TE-tPAS, it is preserved in China typical culture collection center (CCTCC on April 16th, 2012, Wuhan, China, Wuhan University, postcode: 430072).The transcribed spacer (SEQIDNO:5) of restructuring duck enteritis virus vaccine strain CCTCCV201214 between genomic US7 and the US8 gene of duck enteritis virus DEV of described expression-secretion type duck tembusu virus E protein is middle inserts the gene fragment SV40-E (SEQIDNO:1) comprising duck tembusu virus E gene and SV40 promoter sequence.
In one embodiment of the invention, the invention provides the method for the restructuring duck enteritis virus vaccine strain CCTCCV201214 of construction expression secretor type duck tembusu virus E protein, described method comprises the steps:
(1) duck enteritis virus (DEV) genomic Fosmid library is built, and therefrom select the 5 clay combined systems for saving duck enteritis virus, by them difference called after pFOS1, pFOS2, pFOS3, pFOS4, pFOS5, wherein pFOS5 clay comprises US7 and the US8 gene in duck enteritis virus genome and the transcribed spacer between them;
(2) the clay pFOS5 comprising US7 and the US8 gene in DEV genome and the transcribed spacer between them obtained in step (1) is utilized, between US7 and the US8 gene of this clay, insert the gene fragment (SEQIDNO:1) comprising duck tembusu virus E protein and SV40 promoter sequence, build recombination mutation clay; With
(3) utilize pFOS1, pFOS2, pFOS3 and pFOS4 cotransfection in the 5 clay combined systems obtained in the recombination mutation clay and step (1) obtained in step (2) secondary to chick embryo fibroblast CEF, save out recombinant virus CCTCCV201214, by its called after rDEV-TE-tPAS.
In preferred embodiments, FseI-SbfI-PmeI joint is all contained at the duck enteritis virus DNA fragmentation two ends that 5 Cosmid clones obtained in above-mentioned steps (1) comprise, can be overlapped, and can splice covering duck enteritis virus full-length genome (their overlap and replace mode can see Fig. 9).
In preferred embodiments, duck tembusu virus E protein in above-mentioned steps (2) derives from duck tembusu virus PTD2010 strain, duck tembusu virus PTD2010 strain to be separated in 2010 by the present inventor and to obtain from Putian, fujian Province duck group, was preserved by Vet Biotechnology National Key Laboratory and animal influenza key lab of the Ministry of Agriculture; SV40 promoter sequence in above-mentioned steps (2) derives from the plasmid comprising SV40 promotor, such as, and pSI plasmid (purchased from Promega company) etc.
The present invention's duck enteritis virus used is DEV vaccine strain virus (CVCCAV1222) (GeneBankEU082088) (Chinese veterinary microorganism culture presevation administrative center (CVCC), catalog number AV1222; Purchased from China Veterinery Drug Inspection Office).
In this research, the present inventor finds, the on position of E protein does not affect the immune effect (data do not show) of constructed recombinant vaccine strain to duck enteritis virus.But E protein inserts other positions, the protected effect that whether can affect its anti-duck enteritis virus needs experiment to prove.
In one embodiment of the invention, the invention provides the application of the restructuring duck enteritis virus vaccine strain CCTCCV201214 of described expression-secretion type duck tembusu virus E protein, the vaccine of its transmissible disease caused for the preparation of prevention duck enteritis virus and duck tembusu virus.
In a preferred embodiment of the invention, the transmissible disease that described duck enteritis virus and duck tembusu virus cause comprises the transmissible disease that duck enteritis virus and duck tembusu virus cause in poultry, such as, the duck viral enteritis caused by duck enteritis virus DEV, the duck tembusu virus caused by duck tembusu virus is sick.
In one embodiment of the invention, the invention provides a kind of vaccine, it comprises the restructuring duck enteritis virus vaccine strain CCTCCV201214 of expression-secretion type duck tembusu virus E protein of the present invention, and medicinal adjuvant, vehicle etc.Those skilled in the art, according to the application purpose of described vaccine, the factor such as bird of carrying out immunity, easily can select the medicinal adjuvant, vehicle etc. that are applicable to.
In a preferred embodiment of the invention, described vaccine can be effective to the transmissible disease preventing to be caused in poultry by duck enteritis virus and duck tembusu virus, such as, be effective to the duck viral enteritis preventing to be caused by duck enteritis virus DEV, the duck tembusu virus caused by duck tembusu virus is sick.Described vaccine can be used for poultry, such as, and duck, goose, chicken etc.
Therefore, the invention provides following:
1. the restructuring duck enteritis virus vaccine strain of expression-secretion type duck tembusu virus E protein, its deposit number is CCTCCV201214, called after rDEV-TE-tPAS.
2. the restructuring duck enteritis virus vaccine strain of the expression-secretion type duck tembusu virus E protein according to the 1st, inserts the gene fragment (SEQIDNO:1) comprising duck tembusu virus E protein and SV40 promoter sequence in the transcribed spacer wherein between genomic US7 and the US8 gene of duck enteritis virus DEV.
3. the restructuring duck enteritis virus vaccine strain of the expression-secretion type duck tembusu virus E protein according to the 1st or the 2nd, wherein said duck tembusu virus E protein is increased by duck tembusu virus PTD2010 strain and obtains.
4. the restructuring duck enteritis virus vaccine strain of the expression-secretion type duck tembusu virus E protein according to the 2nd, wherein said SV40 promoter sequence derives from the plasmid comprising SV40 promotor.
5. the restructuring duck enteritis virus vaccine strain of the expression-secretion type duck tembusu virus E protein according to the 4th, the plasmid of the wherein said SV40 of comprising promotor comprises pSI plasmid.
6. build the method for the restructuring duck enteritis virus vaccine strain of the expression-secretion type duck tembusu virus E protein described in the 1st, described method comprises the steps:
(1) the genomic Fosmid library of duck enteritis virus DEV is built, and therefrom select the 5 clay combined systems for saving duck enteritis virus, by them difference called after pFOS1, pFOS2, pFOS3, pFOS4, pFOS5, wherein pFOS5 clay comprises US7 and the US8 gene in duck enteritis virus genome and the transcribed spacer between them;
(2) the clay pFOS5 comprising US7 and the US8 gene in duck enteritis virus genome and the transcribed spacer between them obtained in step (1) is utilized, insert the gene fragment (SEQIDNO:1) comprising duck tembusu virus E protein and SV40 promoter sequence in transcribed spacer between US7 and the US8 gene of this clay, build recombination mutation clay; With
(3) utilize pFOS1, pFOS2, pFOS3 and pFOS4 cotransfection in the 5 clay combined systems obtained in the recombination mutation clay and step (1) obtained in step (2) secondary to chick embryo fibroblast CEF, save out recombinant virus CCTCCV201214, by its called after rDEV-TE-tPAS.
7. the method according to the 6th, the DEVDNA fragment two ends that each Cosmid clones in the 5 clay combined systems wherein obtained in step (1) comprises are containing FseI-SbfI-PmeI joint, overlapped, and splicing covers duck enteritis virus full-length genome.
8. the application of the restructuring duck enteritis virus vaccine strain of the expression-secretion type duck tembusu virus E protein described in the 1st, the vaccine of its transmissible disease caused for the preparation of prevention duck enteritis virus and duck tembusu virus.
9. the application according to the 8th, the transmissible disease that wherein said duck enteritis virus and duck tembusu virus cause comprises duck viral enteritis and duck tembusu virus is sick.
10. a vaccine, it comprises the restructuring duck enteritis virus vaccine strain CCTCCV201214 of the expression-secretion type duck tembusu virus E protein described in the 1st, and medicinal adjuvant, vehicle.
Accompanying drawing explanation
Below in conjunction with in the detailed description of accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:
Fig. 1: pCC1Fos clay collection of illustrative plates;
Fig. 2: the viral dDEV of rescue and parent DEV vaccine strain viral genome cut with BamHI, EcoRI, BbvCI enzyme respectively after pulse electrophoresis collection of illustrative plates, wherein DEV: parent DEV vaccine strain (namely for the parental virus vaccine strain of construction of recombinant virus), dDEV: three strain virus that the 5 clay systems (see embodiment 1-3) being built by the present inventor and screen are saved out, M1: low scope PFG molecule marker (LowRangePFGMarker); M2:DL15000 molecule marker; M3: λ-HindIII digests molecule marker (λ-HindIIIdigestMarker);
Fig. 3: pUCccdBkan plasmid map;
Fig. 4: pFOS5us78KanccdB clay collection of illustrative plates;
Fig. 5: pENTRMCS plasmid map;
Fig. 6: pSIE plasmid map;
Fig. 7: pENTRsv40-E plasmid map;
Fig. 8: pFOS5us78SV40E clay collection of illustrative plates;
Fig. 9: duck enteritis virus infections clone rescue recombinant virus schematic diagram;
Figure 10: the expression Immunofluorescence test of recombinant virus E protein in CEF (A and B, B are negative control) and western blotting (westernblot) (C) detected result figure;
Figure 11: PCR detects the situation of heterogenous expression framework in rDEV-TE-tPAS;
Figure 12: SEQIDNO:1:SV40-E expresses framework, and wherein italic thickened portion is duck tembusu virus E gene.
Embodiment
Come by the following examples to illustrate the present invention further.But should be appreciated that, described embodiment is illustrational object, is not intended to limit scope and spirit of the present invention.
The structure in embodiment 1.DEV vaccine strain genome Fosmid library
The genomic Fosmid library of DEV is built by EPICENTRE company " CopyControlFosmidLibraryProductionKit " test kit specification sheets.
Method is as follows: by DEV vaccine strain virus (CVCCAV1222) (GeneBankEU082088) (Chinese veterinary microorganism culture presevation administrative center, catalog number AV1222; Purchased from China Veterinery Drug Inspection Office) DNA physical method namely use No. 25 syringe needles (purchased from Shanghai Zhi Yu Medical Devices Co., Ltd.) aspirate repeatedly carry out cut-outs process, with T4DNA polysaccharase (T4DNAPolymerase, purchased from NewEnglandBiolabs) and alkaline phosphatase (AlkalinePhosphatase, purchased from NewEnglandBiolabs) end smoothing and dephosphorylation process are carried out to DNA fragmentation, pulse electrophoresis is (with Bio-Rad company CHEFMapper
xAPulsedField system carries out pulse electrophoresis, and the condition of pulse electrophoresis is: electrophoretic buffer is 0.5xTBE, and agarose gel concentration is 1%, and program is 2K-80K), reclaim the DNA fragmentation between 38kbp-48kbp.DEVDNA segment two ends T4 ligase enzyme after reclaiming is connected FseI-SbfI-PmeI joint, is connected on pCC1Fos (purchased from EPICENTRE, Fig. 1 is shown in by collection of illustrative plates) carrier after refining, 4 DEG C of connections of spending the night.Mixed liquid is packed, transfection Escherichia coli EPI300-T1 (purchased from EPICENTRE).Confirm library titre, its process of the test is as follows: packaged mixed liquid is carried out 10 times of gradient dilutions, gets 10 respectively
-2, 10
-4, 10
-5, 10
-6four dilution diluent 10 μ l infect 100 μ lEPI300-T1 cells, and the LB be applied to by this bacterium containing 12.5 μ g/ml paraxin is dull and stereotyped, 37 DEG C of incubated overnight, statistics colony counts, and calculate its titre, and result is 3.8x10
5cfu/lib.Namely the fosmid library of DEV is successfully built.
Embodiment 2. is for saving the selection of DEV viral cosmid
After library construction success, picking 286 clone extracts clay, uses alkaline lysis
[5]extract clay, send the precious biotech firm in Dalian to check order to the DEVDNA fragment ends inserted in pCC1Fos, sequencing primer sequence is as follows:
Primer1：5’-TAATACGACTCACTATAGGG-3’
Primer2：5’-GCCAAGCTATTTAGGTGAGA-3’
Through the analysis of end sequencing, obtain the clone 250 that Insert Fragment two ends are all connected with complete FseI-SbfI-PmeI joint altogether.Many groups are chosen for saving the 5 clay combinations of DEV from these 250 clones.FseI-SbfI-PmeI joint is all contained at the DEVDNA fragment two ends of wherein often cloning in group, can be overlapped, and can splice the full DEV genome of covering.
Embodiment 3. virus rescue
The DNA of the clay selected by test kit extraction is extracted by amount in Qiagen company.With FseI, SbfI or PmeI restriction endonuclease (all purchased from NewEnglandBiolabs), linearization process is carried out to selected clay, reaction conditions is as follows: SbfI restriction endonuclease 20U (also can use FseI or PmeI restriction endonuclease), clay 10 μ g, 37 DEG C act on 1 hour, phenol/chloroform, alcohol settling prepares transfection DEVDNA.
With reference to the calcium phosphate procedure of ReddySM (2002) by 5 sections of DEVDNA cotransfections time for chick embryo fibroblast (CEF)
[28], through repeatedly repeating, wherein have 3 group of 5 clay combination transfection 4-6 days afterwards visible CEF there is DEV virus typical cytopathic, choose the good one group of 5 clay combination of repeatability and carry out subsequent experimental.Gathering in the crops the virus that this organizes 5 clay cotransfection rescues, called after dDEV, inoculating by this dDEV and parent DEV virus (namely for building the parental virus of this infections clone) secondary to CEF respectively.
The preparation method of CEF is as follows: get 9-10 age in days SPF chicken embryo, after cotton ball soaked in alcohol sterilization, with tincture of iodine wiping air chamber position, aseptic taking-up chicken embryo after de-iodine, be placed in the plate filling Hank ' s liquid (purchased from HyClone) and wash, and remove head, four limbs and internal organ, shred with scissors.Pancreatin (4mL/ embryo) with 0.25% digests 4-5 minute in 37 DEG C of water-baths, discards pancreatin, washs 2 times with Hank ' s liquid.Add the appropriate M199 nutritive medium (purchased from HyClone) containing serum and dual anti-(penicillin 100u/mL, Streptomycin sulphate 100mg/mL), piping and druming makes cell dispersal, makes 10 with after four layers of filtered through gauze
6-10
7the cell suspension of cells/ml, is finally sub-packed in and cultivates 37 DEG C of rotating and culturing in rolling bottle.After 36-48 hour, by seed culture of viruses: cell culture fluid volume ratio be 1: 1000 by virus inoculation in CEF.When cytopathy reaches 100%, collect nutrient solution; Centrifugal 10 minutes of 4 DEG C of 6000g, remove cell debris; Get supernatant 50000g centrifugal 2 hours enriching virus; Then through 20% and 60% sucrose density gradient centrifugation, centrifugal 2 hours of 50000g, reclaims 20% and 60% middle layer; After obtain the good virus of purifying through 50000g ultracentrifugation desugar process in centrifugal 2 hours.Extract viral complete genome DNA
[29], use BamHI, EcoRI and BbvCI (all purchased from NewEnglandBiolabs) to carry out enzyme to former vaccine strain DEV and dDEV respectively and cut.Reaction conditions is as follows: get each 20U of BamHI, EcoRI and BbvCI respectively, mixes respectively with DEV genomic dna 8 μ g, and in 50 μ l systems, 37 DEG C act on 1 hour.With Bio-Rad company CHEFMapper
xAPulsedField system carries out pulse electrophoresis, and the condition of pulse electrophoresis is: running buffer liquid level 0.5xTBE, and agarose gel concentration is 1%, and program is 2K-70K.
Save the viral restriction enzyme mapping obtained identical with parental virus, as shown in Figure 2.5 clay composition merit rescue DEV viruses selected by explanation.The present inventor is by 5 selected clay group memberships called after pFOS1, pFOS2, pFOS3, pFOS4, pFOS5 respectively, FseI-SbfI-PmeI joint is all contained at the DEVDNA fragment two ends that this 5 Cosmid clones comprises, can be overlapped, and can splice and cover full DEV genome (their overlap and replace mode can see Fig. 9), and wherein pFOS5 comprises genomic US7 and the US8 gene of DEV and the transcribed spacer between them (nucleotide sequence of the transcribed spacer between US7 and US8 gene is see SEQIDNO:5).About this 5 clay system, the present inventor applies for a patent, and application number is 201010207207.8, and exercise question is " duck enteritis virus vaccine strain infections clone system and construction process and application " thereof, and the date of application is on June 13rd, 2010.
The structure that SV40-E expresses the recombination mutation clay of framework (SEQIDNO:1) is inserted in the transcribed spacer of embodiment 4. between genomic US7, US8 gene of DEV
Based on above-described embodiment 1-3 result, in transcribed spacer (SEQIDNO:5) in 5 selected clay group membership pFOS5 between genomic US7 and the US8 gene of DEV, particularly, transcribed spacer between US7 and US8 gene is 223bp altogether, in this research, this transcribed spacer has lacked wherein four Nucleotide of the 108 to 111, (nucleotides sequence that SV40-E expresses framework is classified as SEQIDNO:1 to replace insertion SV40-E expression framework, wherein comprise SV40 promotor, see Figure 12, wherein italic thickened portion is E gene (SEQIDNO:4)), build 1 recombination mutation clay, pFOS5us78SV40E (collection of illustrative plates of this sudden change clay as shown in Figure 8, its forming types can see Fig. 9).In this research, the present inventor finds, the on position of E gene does not affect its immune effect to duck enteritis virus.But E gene inserts other positions, whether can affect it needs experiment to prove to the protected effect of duck enteritis virus.
The building process of pFOS5us78SV40E clay is summarized as follows:
The structure of 4.1pUCccdBkan:
Respectively multiplexed PCR amplification is carried out to " RfA " that provide in Invitrogen company GatewayVectorConversionSystemwithOneShotccdBSurvival2T1C ompetentCells test kit (wherein gene is aatR1-paraxin-ccdB-aatR2) gene (SEQIDNO:2) with pair primer of three shown in table 1 (being synthesized by TaKaRa company).
Table 1: for the PCR primer of clone's " Rfkan " (wherein gene is aatR1-kantlex-ccdB-aatR2) gene
Its detailed process is summarized as follows: use tR1 and tR2 respectively, and these two pairs of primers of ccdB1 and ccdB2 increase and obtain aatR1 fragment and ccdB-aatR2 fragment from ReadingFrameCassetteA, its reaction conditions is: 95 DEG C 5min-35 circulation (94 DEG C 45s-54 DEG C 45s-72 DEG C of 45s)-72 DEG C of 10min.Then this increases to kalamycin resistance gene from pMOD6 plasmid (purchased from EPICENTRE company) to primer to use P6K1 and P6K2, and its reaction conditions is: 95 DEG C of 5min-35* (94 DEG C 45s-54 DEG C 45s-72 DEG C of 45s)-72 DEG C of 10min.These three sheet segment DNAs of purifying respectively, and using these three fragments jointly as template, using tR1 and ccdB2 as primer, amplification obtains RfKan gene (SEQIDNO:3), namely its gene is aatR1-kantlex-ccdB-aatR2, and its reaction conditions is: 95 DEG C 5min-35 circulation (94 DEG C 45s-54 DEG C 45s-72 DEG C of 1.5min)-72 DEG C of 10min.
And " RfKan " fragment obtained utilized XbaI and HindIII to be cloned in pUC18 carrier (purchased from TaKaRa company), obtain pUCccdBkan, as shown in Figure 3.
The structure of 4.2pFOS5us78KanccdB clay:
Increase from the pUCccdBkan of above-mentioned structure with the ccdB gene of restructuring arm with primer US78ccd1 and US78ccd2 (being synthesized by TaKaRa company) shown in table 2, its PCR reaction conditions is: 95 DEG C 5min-35 circulation (94 DEG C 45s-54 DEG C 45s-72 DEG C of 2min)-72 DEG C of 10min.With the Counter-SelectionBACModificationKit test kit of GeneBridges company, increased fragment is cloned in pFOS5 clay, obtain pFOS5us78KanccdB clay, namely between US7 and the US8 gene of pFOS5 clay, ccdB and kalamycin resistance gene is inserted, as shown in Figure 4.
Table 2: for increasing from pUCccdBkan with the primer of the ccdB gene of restructuring arm
The structure of 4.3pENTRMCS plasmid:
For convenience of follow-up test, the pENTR-gus plasmid (purchased from Invitrogen company) provided in Invitrogen company GatewayVectorConversionSystemwithOneShotccdBSurvival2T1C ompetentCells test kit has been done following transformation by this research: by the gus gene elmination in pENTR-gus, and add BamHI, BglII, EcoRI, EcoRV, SacI, SalI, KpnI and XbaI eight restriction enzyme sites.With primer MCS1 and MCS2 of two shown in table 3, with point mutation Cloning Kit (purchased from Invitrogen company), pENTR-gus is transformed into pENTRMCS, as shown in Figure 5.
Table 3: for pENTR-gus being transformed into the primer of pENTRMCS
The structure of 4.4pSIE plasmid:
With primer pEf and pEr shown in table 4, amplification E gene (SEQIDNO:4), this E gene source is in duck tembusu virus PTD2010 strain, it is separated in 2010 by the present inventor from Putian, fujian Province duck group, is preserved by Vet Biotechnology National Key Laboratory and animal influenza key lab of the Ministry of Agriculture.Cut with BamHI and EcoRV (purchased from NewEnglandBiolabs) enzyme and E gene is connected in pSI plasmid (purchased from Promega company), successfully build pSIE, as shown in Figure 6.Wherein pSI plasmid comprises SV40 promotor (see Fig. 6).
Table 4: for building the primer of pSIE
The structure of 4.5pENTRsv40-E plasmid:
With primer entry1 and entry2 shown in table 5, increase sv40-E gene from the above-mentioned pSIE successfully constructed, and be cloned in the pENTRMCS built, and obtains pENTRsv40-E, as shown in Figure 7.
Table 5: for building the primer of pENTRsv40-E
| Primer | Sequence |
| entry1 | 5’-TGA GGA TCC GGG CGG AGC CTA TGG AAAA-3’ |
| entry2 | 5’-CGA GGA TCC CAG CCC GGA TCC TTA TCGA-3’ |
The structure of 4.6pFOS5us78SV40E clay:
PFOS5us78KanccdB and pENTRsv40-E is utilized the effect of Invitrogen company GatewayVectorConversionSystemwithOneShotccdBSurvival2T1C ompetentCells test kit, make the kanccdB gene in the sv40-E expression framework replacement pFOS5us78KanccdB in pENTRsv40-E, thus obtain the clay pFOS5us78SV40E inserting sv40-E expression cassette in the transcribed spacer between US7 and US8, as shown in Figure 8.Particularly, described sv40-E expresses the tetranucleotide fragment of the 108 to 111 that framework replaces the transcribed spacer (SEQIDNO:5) between described DEV genome US7 and US8.
The rescue of embodiment 5. recombinant virus
PFOS1, pFOS2, pFOS3, pFOS4 (built by embodiment 1-3 and screen) and pFOS5us78SV40E (being built by embodiment 4) five cosmid DNAs are extracted with extraction reagent kit in Qiagen company.With FseI or SbfI restriction endonuclease (purchased from NewEnglandBiolabs company) by clay linearization process used, its reaction conditions is as follows: SbfI restriction endonuclease 20U (also can use FseI or PmeI restriction endonuclease), clay 10 μ g, 37 DEG C act on 1 hour, phenol/chloroform, alcohol settling, preparation transfection DEVDNA.With reference to ReddySM (2002) method respectively by five clay cotransfections time for chick embryo fibroblast CEF
[28].PFOS1, pFOS2, pFOS3, pFOS4 and the genomic relation of pFOS5us78SV40E and DEV are as shown in Figure 9.
Wherein the preparation method of CEF is as follows: get 9-10 age in days SPF chicken embryo, after cotton ball soaked in alcohol sterilization, with tincture of iodine wiping air chamber position, aseptic taking-up chicken embryo after de-iodine, be placed in the plate filling Hank ' s liquid (purchased from HyClone) and wash, and remove head, four limbs and internal organ, shred with scissors.Pancreatin (4mL/ embryo) with 0.25% digests 4-5min in 37 DEG C of water-baths, discards pancreatin, washs 2 times with Hank ' s liquid.Add the appropriate M199 nutritive medium (purchased from HyClone) containing serum and twin antibiotic (penicillin 100u/mL, Streptomycin sulphate 100mg/mL, the two available from Sigma), piping and druming makes cell dispersal, makes 10 with after four layers of filtered through gauze
6-10
7the cell suspension of cells/ml, is finally sub-packed in 37 DEG C of cultivations in culturing bottle.Then, with reference to ReddySM (2002) method respectively by secondary for above-mentioned five clay cotransfections for CEF
[3], after transfection 6-9 days, can be observed the appearance that the contracting of cell circle waits typical cytopathic.Save out recombinant virus, called after rDEV-TE-tPAS, this restructuring duck enteritis virus vaccine strain is preserved in China typical culture collection center (CCTCC, Wuhan, China on April 16th, 2012, Wuhan University), deposit number is CCTCCV201214.
Embodiment 6. recombinant virus E protein marking protein trace (westernblot) is identified
The recombinant virus rDEV-TE-tPAS of rescue and parental virus DEV is inoculated respectively secondary to CEF.Detect the expression of E protein by western blotting (westernblot) detection method when pathology appears in 80% cell.
Western blotting step is summarized as follows: collect respectively 80% cell occur pathology infected recombinant virus rDEV-TE-tPAS and TMUV time for CEF.Carry out SDS-page electrophoresis, with transferring film immersion bubble nylon membrane (purchased from Sartorius company) and filter paper 10 minutes, carry out wet turn, transferring film condition is voltage 20mA/cm2, and 4 DEG C are spent the night.Film is washed 2 times, each 5 minutes with PBS.Be placed on by nylon membrane in plate, add 5% skimming milk and close immersion film, 37 DEG C are swayed 1 hour.Add with 1%BSA confining liquid by the anti-E protein antibody of the rabbit of the dilution proportion of 1: 100 (prepared by the Vet Biotechnology National Key Laboratory at the present inventor place and animal influenza key lab of the Ministry of Agriculture and preserved) (as primary antibodie), the ratio adding 0.1ml in every square centimeter adds a resistant to liquids, and room temperature sways 1 hour.Film is washed 3 times, each 10 minutes with PBST.Add with 1%BSA confining liquid by 1: 5000 the anti-rabbit IgG antibody (available from Sigma) (resisting as two) of infrared fluorescent labels of dilution proportion, the ratio adding 0.1ml in every square centimeter adds two resistant to liquids, and room temperature sways 1 hour.Film is washed 3 times, each 10 minutes with PBST.Finally take a picture in infrared scanner.
Embodiment 7. experimentation on animals
RDEV-TE-tPAS and parent DEV will be recombinated respectively by 10
6tCID
50the SPF duck (being provided by Harbin veterinary institute Animal House) 8 in 16 week age is provided.100DLD is used respectively after 3 weeks
50the strong poison (CVCCAV1222, purchased from China Veterinery Drug Inspection Office) of DEV; (be separated to from Putian, fujian Province duck group in 2010 by the present inventor with duck tembusu virus PTD2010 strain; preserved by Vet Biotechnology National Key Laboratory and animal influenza key lab of the Ministry of Agriculture) attack, observe the protected effect of recombinant virus to the strong poison of DEV and duck tembusu virus.
Result
1.E protein expression detects
After infecting CEF with the 8th generation recombinant virus rDEV-TE-tPAS, when pathology appears in 80% cell, detect the expression of E protein by western blotting detection method.Its result as shown in Figure 10.Recombinant virus can be good in CEF expression E protein.
2. genetic stability detects
Recombinant virus was passed for 20 generations continuously, extracts the DNA of parent DEV virus and recombinant virus, identify by PCR method.As shown in figure 11.And sequencing analysis (sequencing result does not show) is carried out to it, have no the generation of disappearance or sudden change.
3. results of animal
RDEV-TE-tPAS and parent DEV will be recombinated respectively by 10
6tCID
50the SPF duck (being provided by Harbin veterinary institute Animal House) 8 in 16 week age is provided.100DLD is used respectively after 3 weeks
50the strong poison (CVCCAV1222, purchased from China Veterinery Drug Inspection Office) of DEV; Attack with duck tembusu virus PTD2010 strain.No matter result is for duck viral enteritis or for duck tembusu virus, obtain 100% protection with the duck of recombinant virus rDEV-TE-tPAS immunity.Its result is as shown in table 6.
Table 6: results of animal table
Note: wherein TMUV:PT2010; DEV is poison by force: CVCCAV1222; The representation (*/8) of protection result is the number obtaining the SPF duck protected in 8 tested SPF ducks; Wherein PBS is used as negative control.
Discuss
(namely this research by utilizing the platform of duck enteritis virus infections clone, the DEV virus 5 clay group that the present inventor builds, see embodiment 1-3, also can be the application for a patent for invention of 201010207207.8 see the application number of the present inventor), success obtains the duck enteritis virus of restructuring duck tembusu virus E gene, at home and abroad still belongs to the first time.
By to recombinant virus rDEV-TE-tPAS in vivo with the detection of growth characteristics of cultured, this research proves that the transcribed spacer between genomic US7 and US8 of DEV can stablize that to insert with SV40 be the duck tembusu virus E gene expression construct of promotor first.This recombinant virus can good representation E protein in vitro.The immune effect suitable with former vaccine strain DEV not only can be provided after primary immune response being carried out to duck with it, and E protein can obtain good expression in SPF duck body, can induce good immune effect, the duck after immunity can resist the attack of the strong poison of duck tembusu virus completely.
As can be seen here, the restructuring duck enteritis virus vaccine strain CCTCCV201214 (its called after rDEV-TE-tPAS) of the expression-secretion type duck tembusu virus E protein that the present invention obtains may be used for preparing the vaccine of the transmissible disease of preventing duck enteritis virus and duck tembusu virus to cause, for the transmissible disease of preventing duck enteritis virus and duck tembusu virus to cause in poultry.
Therefore, the present invention also provides the vaccine of restructuring duck enteritis virus vaccine strain CCTCCV201214 (that is, rDEV-TE-tPAS of the present invention) and medicinal adjuvant, the vehicle etc. comprising expression-secretion type duck tembusu virus E protein.Those skilled in the art, according to the application purpose of described vaccine, the factor such as bird of carrying out immunity, easily can select the medicinal adjuvant, vehicle etc. that are applicable to.。
Suddenly there is a kind of declining to a great extent to lay eggs as feature, the ovarian hemorrhage acute infectious disease that is cardinal symptom the minority duck group of China East China, South China in spring in 2010, rapid spread is to national most area afterwards.Through domestic multiple laboratory diagnosis, this disease is caused by a kind of flavivirus; Initial stage is called duck hemorrhagic ovaritis, egg drop syndrome, Duckling flavivirus disease etc., sick along with renaming duck tembusu virus as to the parsing of cause of disease full genome and the development later stage unification of this course of disease.This disease can betide the egg duck of multiple kind, also can betide meat duck and wild duck.Sick duck starts to occur that food consumption and egg productivity sharply decline, and generates heat subsequently, One's spirits are drooping, draws green loose stool.Initial stage pathology mainly occurs in ovary, show as ovarian dysgenesis, ovarian follicle sex change, distortion, follicular theca is congested, hemorrhage, atrophy, later isolates can cause duck and occur nervous symptoms, both legs paralysis, astasia, some strain also can cause heart, liver hemorrhage in addition, and mortality ratio 5%-30% is not etc.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 duck morbidities, accumulative financial loss is more than 5,000,000,000 yuan.Because the morbidity of this disease is unexpected, also there is no effective Prevention and Curation measure at present, the present invention utilizes ripe duck enteritis virus attenuated vaccine to be vector construction to express the recombinant virus of duck tembusu virus E protein, this recombinant virus is used for the generation of preventing duck tembusu virus disease, has good application prospect and using value.
Should be appreciated that, although with reference to the embodiment that it is exemplary, the present invention shown particularly and describe, but will be understood by those skilled in the art that, under the condition not deviating from the spirit and scope of the present invention defined by accompanying claim, the change of various forms and details can be carried out wherein, the arbitrary combination of various embodiment can be carried out.
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Claims (4)
1. the restructuring duck enteritis virus vaccine strain of expression-secretion type duck tembusu virus E protein, its deposit number is CCTCCNo.V201214.
2. the application of the restructuring duck enteritis virus vaccine strain of expression-secretion type duck tembusu virus E protein according to claim 1, the vaccine of its transmissible disease caused for the preparation of prevention duck enteritis virus and duck tembusu virus.
3. application according to claim 2, the transmissible disease that wherein said duck enteritis virus and duck tembusu virus cause is selected from duck viral enteritis and duck tembusu virus is sick.
4. a vaccine, it comprises the restructuring duck enteritis virus vaccine strain CCTCCNo.V201214 of expression-secretion type duck tembusu virus E protein according to claim 1, and medicinal adjuvant, vehicle.
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