CN103667197A - Construction and application of recombinant duck viral enteritis virus vaccine for expressing secretory duck Tembusu virus M/E protein - Google Patents
Construction and application of recombinant duck viral enteritis virus vaccine for expressing secretory duck Tembusu virus M/E protein Download PDFInfo
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Abstract
The invention provides a recombinant duck viral enteritis virus vaccine for expressing secretory duck Tembusu virus M/E protein, and a construction method and application thereof. The collection number is CCTCC NO:V201215, and the name is rDEV-TME-tPAS. By using a recombinant clone technique, a gene segment rDEV-TME-tPAS comprising an SV40 promoter, duck Tembusu virus M and E proteins and a tPA (Tissue plasminogen activator) signal peptide sequence is inserted into a spacer between the US7 and US8 genes of the duck viral enteritis virus to construct a cosmid in which the rDEV-TME-tPAS expression frame is inserted between the US7 and US8 genes, thereby obtaining the recombinant duck viral enteritis virus vaccine CCTCC V201215 for expressing secretory duck Tembusu virus M/E protein. The invention also provides a method for constructing a recombinant duck viral enteritis virus vaccine strain and application of the recombinant duck viral enteritis virus vaccine strain in preparing a vaccine for preventing infectious diseases caused by duck viral enteritis virus and duck Tembusu virus.
Description
Technical field
The invention belongs to recombinant viral vaccine field, more specifically belong to restructuring duck viral enteritis virus vaccines field.The invention provides the restructuring duck viral enteritis virus vaccine strain CCTCC V201215 of a kind of expression-secretion type duck tembusu virus M albumen and E albumen, called after rDEV-TME-tPAS, and construction process and application.
Background technology
Duck enteritis virus (duck enteritis virus, DEV), claims again duck viral enteritis virus.Can cause that the deadly infectious disease acute, hot, septic is feature occurs for duck, goose and other Anseriformes bird.Compare with other simplexvirus, less for the research of DEV.The international virusology classification of Ba Ci committee report is categorized as simplexvirus
[1], and concrete genus classification is determined 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 the full genome cosmid library of DEV vaccine strain, segmentation is checked order to it and analyzed, and to mid-term in 2009, DEV vaccine strain whole genome sequence is parsed.Almost simultaneously, Li etc. have also reported the complete genomic order-checking of DEV vaccine strain and analytical results, and its Genome Size is about 158Kb, 78 albumen of approximately encoding.By DEV vaccine strain genomic gene is formed and structural analysis, DEV is considered to the osculant virus in α blister sore subfamily, more close with Varicellavirus member
[2].And the Marek’s disease poison (MDV) and the herpes turkey virus (HTV) that are all bird simplexvirus belong to Marek’s disease poison genus, avian infectious laryngotracheitis virus (ILTV) and parrot simplexvirus (PsHV) are that infectious laryngotracheitis virus belongs to.
Since successfully utilizing the TK gene that poxvirus is vector expression hsv the eighties in last century, it is carrier with various different DNA virus that people start to attempt, express different foreign genes, and the prevention for people and various different animals diseases by the recombiant vaccine building.A large amount of results of study show, simplexvirus because of its genome large, can for foreign gene insert or alternative dispensable gene many, be considered to a kind of virus vector of good structure live recombined vaccines.Up to now, in common livestock and poultry simplexvirus disease, existing a large amount of correlative study reports; As to take Pseudorabies virus (PRV) be carrier, in the genes such as gD, gE, gG and TK, insert the foreign genes such as E2 of swine fever (CSF) respectively, and use its immune swine, obtained good immune effect
[4-11].Be used in the UL0 of avian infectious laryngotracheitis virus (ILTV) and UL50 and insert respectively the gene constructed successful recombinant virus immunity chicken of different HA, all respond well
[11-13].Equally, Sakaguchi M (1993; 1994) and Sonoda K (1996) philosophy in US10, the US3 of MDV1, IRL, insert Lac Z gene; with its immune 1 age in days specific pathogen free chicken (SPF chicken); after 1 week, with vMDV, vvMDV, attack poison, its protection efficiency to SPF chicken is 80～100%
[14-16]; The recombinant virus that insert NDV F gene in the US10 of MDV1, inserts IBDV VP2 gene in US2 to the protection efficiency of the strong poison of MDV with to contrast MDV1 suitable
[17,18]; Tsukamoto K etc. (2002) are usingd Pec and are inserted successful construction of recombinant virus between the UL45 of herpes turkey virus (HVT) and UL46 gene as the promotor of infectious bursal disease virus (IBDV) VP2 gene, with its immune SPF chicken, are enough to resist the strong malicious attack of IBDV
[19].Current, China is mainly to study successful chicken embryo the sixties in last century to weaken living vaccine for the vaccine of duck plague prevention.As α blister sore subfamily a member, DEV should be also a kind of virus vector of good structure live recombined vaccines.
The genomic constitution of DEV and structure and MDV etc. have larger difference, and if the genome structure of MDV is TRL-UL-IRL-IRS-US-TRS, and the genomic structure of DEV is UL-IRS-US-TRS.And DEV and being also not quite similar with other several livestock and poultry simplexviruss of subfamily Biological characteristics copying of growing in animal body.Therefore, in PRV, MDV, ILTV, can stablize the site of inserting foreign gene and not necessarily be applicable to DEV.Because the research to DEV relatively lags behind, the research report about Nonessencial region in DEV gene is still blank both at home and abroad.The method that is usually used in building simplexvirus recombinant virus has three kinds.A kind of is homologous recombination; The second is that viral genome is inserted in BAC, then on BAC, builds sudden change, with the corresponding cellular rescue of its transfection, goes out recombinant virus; The third is that the hsv gene fragment that contains overlapped district is inserted in clay respectively, and builds sudden change in its respective section, then goes out recombinant virus with the corresponding cellular rescue of its cotransfection.Yet 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 this platform construction success, fast and effectively construction of recombinant virus.So far, this infections clone constructing technology of simplexvirus is comparative maturity, and has been seen in report
[20-27].
This research department is in early-stage Study, and in the genome of duck viral enteritis virus, identifying can be for the stable Nonessencial region inserting of foreign gene.On this basis; this research is inserted the protective gene M of duck tembusu virus PTD2010 strain and E gene expression construct between the genomic US7 of DEV and US8 gene; with its immune specific pathogen free duck (SPF duck), can make SPF duck produce the antibody of good antagonism duck tembusu virus, and not affect the immune effect of DEV.
Summary of the invention
The restructuring duck viral enteritis virus vaccine strain that the invention provides a kind of expression-secretion type duck tembusu virus M/E albumen, its deposit number is CCTCC V201215, called after rDEV-TME-tPAS, and construction process and application.
Particularly, the present invention utilizes recombinant clone technology, the gene fragment SV40-TME that comprises duck tembusu virus M and E gene and SV40 promoter sequence (SEQ ID NO:1) is inserted into duck viral enteritis virus (duck enteritis virus, DEV) in the transcribed spacer between US7 and US8 gene (nucleotide sequence of the transcribed spacer between US7 and US8 gene is shown in SEQ ID NO:5), build and obtain the clay pFOS5us78SV40 TME that inserts SV40TME expression cassette between US7 and US8 gene, by its rescue, obtained the restructuring duck viral enteritis virus vaccine strain CCTCC V201215 of expression-secretion type duck tembusu virus M and E albumen, called after rDEV-TME-tPAS.The invention still further relates to the method that builds this restructuring duck viral enteritis virus vaccine strain, and this restructuring duck viral enteritis virus vaccine strain is for the preparation of the application of preventing duck viral enteritis and duck tembusu virus.
In one embodiment of the invention, the invention provides a kind of restructuring duck viral enteritis virus vaccine strain of expression-secretion type duck tembusu virus M/E albumen, its deposit number is CCTCC V201215, called after rDEV-TME-tPAS, described vaccine strain expression-secretion type duck tembusu virus M albumen (also referred to as prM albumen) and E albumen, it is preserved in Chinese Typical Representative culture collection center (CCTCC, Wuhan, China on April 16th, 2012, Wuhan University, postcode: 430072).In the transcribed spacer (SEQ ID NO:5) of the restructuring duck viral enteritis virus vaccine strain CCTCC V201215 of described expression-secretion type duck tembusu virus M/E albumen between the genomic US7 of duck enteritis virus DEV and US8 gene, insert the gene fragment SV40-TME (SEQ ID NO:1) that comprises duck tembusu virus M and E gene and SV40 promoter sequence.
In one embodiment of the invention, the invention provides the method for the restructuring duck viral enteritis virus vaccine strain CCTCC V201215 of construction expression secretor type duck tembusu virus M/E albumen, described method comprises the steps:
(1) build duck viral enteritis virus (DEV) genomic Fosmid library, and therefrom select for saving 5 clay combined systems of duck viral enteritis virus, they are distinguished to called after pFOS1, pFOS2, pFOS3, pFOS4, pFOS5, and wherein pFOS5 clay comprises US7 and US8 gene and the transcribed spacer between them in duck viral enteritis viral genome;
(2) utilize obtain in step (1) comprise US7 in DEV genome and the clay pFOS5 of US8 gene and the transcribed spacer between them, between the US7 of this clay and US8 gene, insert the gene fragment (SEQID NO:1) that comprises duck tembusu virus M and E albumen and SV40 promoter sequence, build recombination mutation clay; With
(3) utilize the recombination mutation clay and pFOS1, pFOS2, pFOS3 and the pFOS4 cotransfection in the middle 5 clay combined systems that obtain of step (1) that in step (2), obtain inferior to chick embryo fibroblast CEF, save out recombinant virus CCTCC V201215, by its called after rDEV-TME-tPAS.
In preferred embodiments, Fse I-Sbf I-Pme I joint is all contained at the duck viral enteritis viral dna fragment two ends that the 5 clay clones that obtain in above-mentioned steps (1) comprise, can be overlapped, and can splice and cover the viral genome entirely of duck viral enteritis (their overlapping and replace mode can referring to Fig. 9).
In preferred embodiments, duck tembusu virus M in above-mentioned steps (2) and E dietary protein origin are in PTD2010 strain virus, it is separated in October, 2010 by the inventor from Putian, fujian Province duck group, by Vet Biotechnology National Key Laboratory and animal influenza key lab of the Ministry of Agriculture, is preserved; SV40 promoter sequence in above-mentioned steps (2) derives from the plasmid that comprises SV40 promotor, for example, and pSI plasmid (purchased from Promega company) etc.
The present invention's duck viral enteritis virus used is DEV vaccine strain virus (CVCC AV1222) (GeneBank EU082088) (Chinese veterinary microorganism culture presevation administrative center (CVCC), catalog number AV1222; Purchased from China Veterinery Drug Inspection Office).
In this research, inventor's discovery, the on position of M and E albumen does not affect constructed recombinant vaccine strain to the immune effect of duck viral enteritis virus (data do not show).But M and E albumen insert other positions, the protection effect that whether can affect its anti-duck viral enteritis virus need to test to prove.
In one embodiment of the invention, the invention provides the application of the restructuring duck viral enteritis virus vaccine strain CCTCC V201215 of described expression-secretion type duck tembusu virus M/E albumen, its vaccine viral for the preparation of preventing duck viral enteritis and the transmissible disease that duck tembusu virus causes.
In a preferred embodiment of the invention, the transmissible disease that described duck viral enteritis virus and duck tembusu virus cause comprises the transmissible disease that duck viral enteritis virus and duck tembusu virus cause in poultry, for example, the duck viral enteritis being caused by duck viral enteritis virus DEV, the duck tembusu virus disease being caused by duck tembusu virus etc.
In one embodiment of the invention, the invention provides a kind of vaccine, it comprises the restructuring duck viral enteritis virus vaccine strain CCTCC V201215 of expression-secretion type duck tembusu virus M/E albumen of the present invention, and medicinal adjuvant, vehicle etc.Those skilled in the art according to the application purpose of described vaccine, carry out the immune factors such as bird, can easily select applicable medicinal adjuvant, vehicle etc.
In a preferred embodiment of the invention, described vaccine can be effective to the transmissible disease that prevention is caused in poultry by duck viral enteritis virus and duck tembusu virus, for example, be effective to the duck viral enteritis that prevention is caused by duck viral enteritis virus DEV, the duck tembusu virus disease being caused by duck tembusu virus etc.
Therefore, the invention provides following:
1. the restructuring duck viral enteritis virus vaccine strain of expression-secretion type duck tembusu virus M/E albumen, its deposit number is CCTCC V201215, called after rDEV-TME-tPAS, described vaccine strain expression-secretion type duck tembusu virus M albumen and E albumen.
2. according to the restructuring duck viral enteritis virus vaccine strain of the expression-secretion type duck tembusu virus M/E albumen described in the 1st, wherein in the transcribed spacer between the genomic US7 of duck enteritis virus DEV and US8 gene, insert the gene fragment (SEQ ID NO:1) that comprises duck tembusu virus M and E albumen and SV40 promoter sequence.
3. according to the restructuring duck viral enteritis virus vaccine strain of the expression-secretion type duck tembusu virus M/E albumen described in the 1st or the 2nd, wherein said duck tembusu virus M and E albumen are obtained by duck tembusu virus PTD2010 strain amplification.
4. according to the restructuring duck viral enteritis virus vaccine strain of the expression-secretion type duck tembusu virus M/E albumen described in the 2nd, wherein said SV40 promoter sequence derives from the plasmid that comprises SV40 promotor.
5. according to the restructuring duck viral enteritis virus vaccine strain of the expression-secretion type duck tembusu virus M/E albumen described in the 4th, the plasmid of the wherein said SV40 of comprising promotor comprises pSI plasmid.
6. the method for the restructuring duck viral enteritis virus vaccine strain of the expression-secretion type duck tembusu virus M/E albumen described in building the 1st, described method comprises the steps:
(1) build the duck viral enteritis virus genomic Fosmid of DEV library, and therefrom select for saving 5 clay combined systems of duck viral enteritis virus, they are distinguished to called after pFOS1, pFOS2, pFOS3, pFOS4, pFOS5, and wherein pFOS5 clay comprises US7 and US8 gene and the transcribed spacer between them in duck viral enteritis viral genome;
(2) utilize obtain in step (1) comprise US7 in duck viral enteritis viral genome and the clay pFOS5 of US8 gene and the transcribed spacer between them, in the US7 of this clay and the transcribed spacer between US8 gene, insert the gene fragment (SEQ ID NO:1) that comprises duck tembusu virus M and E albumen and SV40 promoter sequence, build recombination mutation clay; With
(3) utilize the recombination mutation clay and pFOS1, pFOS2, pFOS3 and the pFOS4 cotransfection in the middle 5 clay combined systems that obtain of step (1) that in step (2), obtain inferior to chick embryo fibroblast CEF, save out recombinant virus CCTCC V201215, by its called after rDEV-TME-tPAS.
7. according to the method described in the 6th, Fse I-Sbf I-Pme I joint is contained at the DEV DNA fragmentation two ends that each clay clone in the 5 clay combined systems that wherein obtain in step (1) comprises, overlapped, and splicing covers the full genome of duck viral enteritis virus.
8. the application of the restructuring duck viral enteritis virus vaccine strain of the expression-secretion type duck tembusu virus M/E albumen described in the 1st, its vaccine viral for the preparation of preventing duck viral enteritis and the transmissible disease that duck tembusu virus causes.
9. according to the application described in the 8th, the transmissible disease that wherein said duck viral enteritis virus and duck tembusu virus cause comprises duck viral enteritis and duck tembusu virus disease.
10. a vaccine, it comprises the restructuring duck viral enteritis virus vaccine strain CCTCC V201215 of the expression-secretion type duck tembusu virus M/E albumen described in the 1st, and medicinal adjuvant, vehicle.
Accompanying drawing explanation
In detailed description below in conjunction with 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 pulse electrophoresis collection of illustrative plates after the viral dDEV of rescue and parent DEV vaccine strain viral genome are cut with BamH I, EcoR I, BbvC I enzyme respectively, DEV wherein: the parent DEV vaccine strain parental virus vaccine strain of construction of recombinant virus (for), dDEV: three strain virus that the 5 clay systems (referring to embodiment 1-3) that built and screened by the inventor are saved out, M1: low scope PFG molecule marker (Low Range PFG Marker); M2:DL15000 molecule marker; M3: λ-HindIII digests molecule marker (λ-Hind III digest Marker);
Fig. 3: pUC ccdB kan plasmid map;
Fig. 4: pFOS5 us78 Kan ccdB clay collection of illustrative plates;
Fig. 5: pENTR MCS plasmid map;
Fig. 6: pSI TME plasmid map;
Fig. 7: pENTR sv40-TME plasmid map;
Fig. 8: pFOS5us78 SV40 TME clay collection of illustrative plates;
Fig. 9: duck viral enteritis virus infection clones rescue recombinant virus schematic diagram;
Figure 10: the expression immunofluorescence of recombinant virus E albumen in CEF detects (C) detected result figure of (A and B, the wherein negative contrast of B) and western blotting (western blot);
Figure 11: PCR detects the situation of heterogenous expression framework in rDEV-TME-tPAS;
Figure 12: SEQ ID NO:1:SV40-ME expresses framework, wherein Italic capitals thickened portion is M and E gene; Small letter thickened portion is tPA signal peptide sequence.
Embodiment
Carry out by the following examples further to illustrate the present invention.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
Press EPICENTRE company " CopyControl Fosmid Library Production Kit " test kit specification sheets and build the genomic Fosmid of DEV library.
Method is as follows: by DEV vaccine strain virus (CVCC AV1222) (GeneBank EU082088) (Chinese veterinary microorganism culture presevation administrative center, catalog number AV1222; Purchased from China Veterinery Drug Inspection Office) DNA use No. 25 syringe needles (purchased from Shanghai Zhi Yu Medical Devices Co., Ltd.) suctions repeatedly to cut off processing with physical method, with T4 archaeal dna polymerase (T4 DNA Polymerase, purchased from New England Biolabs) and alkaline phosphatase (Alkaline Phosphatase, purchased from New England Biolabs) DNA fragmentation is carried out to end smoothing and dephosphorylation processing, pulse electrophoresis is (with the CHEF of Bio-Rad company
xA Pulsed Field system is carried 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.DEV DNA segment two ends after reclaiming are connected to upper Fse I-Sbf I-Pme I joint with T4 ligase enzyme, after refining, be connected on pCC1Fos (purchased from EPICENTRE, Fig. 1 is shown in by collection of illustrative plates) carrier 4 ℃ of connections of spending the night.Mixed liquid is packed to transfection Escherichia coli EPI300-T1 (purchased from EPICENTRE).Library titre is confirmed, its process of the test is as follows: packaged mixed liquid is carried out to 10 times of gradient dilutions, get respectively 10
-2, 10
-4, 10
-5, 10
-6four dilution diluent 10 μ l infect 100 μ l EPI300-T1 cells, and this bacterium is applied to the LB flat board containing 12.5 μ g/ml paraxin, 37 ℃ of incubated overnight, and statistics colony counts, and calculate its titre, result is 3.8x10
5cfu/lib.Successfully build the fosmid library of DEV.
Embodiment 2. is for saving the selection of DEV virus clay
After library construction success, 286 clones of picking extract clay, use alkaline lysis
[5]extract clay, send the precious biotech firm in Dalian to check order to the DEV DNA fragmentation end inserting in pCC1 Fos, sequencing primer sequence is as follows:
Primer 1：5’-TAATACGACTCACTATAGGG-3’
Primer 2：5’-GCCAAGCTATTTAGGTGAGA-3’
Through the analysis of end sequencing, obtain altogether 250 of the clones that Insert Fragment two ends are all connected with complete Fse I-SbfI-Pme I joint.From these 250 clones, choose many groups for saving the 5 clay combinations of DEV.Fse I-Sbf I-PmeI joint is all contained at the DEV DNA fragmentation two ends of wherein cloning in every group, can be overlapped, and can splice the full DEV genome of covering.
Embodiment 3. virus rescues
By the middle amount of Qiagen company, extract the DNA that test kit extracts selected clay.With Fse I, Sbf I or Pme I restriction endonuclease (all purchased from New England Biolabs), selected clay is carried out to linearization process, reaction conditions is as follows: Sbf I restriction endonuclease 20U (also can use Fse I or Pme I restriction endonuclease), clay 10 μ g, 37 ℃ act on 1 hour, the extracting of phenol/chloroform, ethanol precipitation transfection DEV DNA.
Calcium phosphate method with reference to Reddy SM (2002) is inferior to chick embryo fibroblast (CEF) by 5 sections of DEV DNA cotransfections
[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.Gather in the crops the virus that this organizes 5 clay cotransfection rescues, called after dDEV, inoculates respectively inferior to CEF by this dDEV and parent DEV virus (for building the parental virus of this infections clone).
The preparation method of CEF is as follows: get 9-10 age in days SPF chicken embryo, with 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 that fills Hank ' s liquid (purchased from HyClone) and wash, and remove head, four limbs and internal organ, with scissors, shred.Pancreatin with 0.25% (4mL/ embryo) digests 4-5 minute in 37 ℃ of water-baths, discards pancreatin, with Hank ' s liquid washing 2 times.Add the appropriate M199 nutritive medium (purchased from HyClone) containing serum and dual anti-(penicillin 100u/mL, Streptomycin sulphate 100mg/mL), piping and druming disperses cell, with making 10 after four layers of filtered through gauze
6-10
7the cell suspension of cells/ml, is finally sub-packed in and cultivates 37 ℃ 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 ℃ of 6000g, remove cell debris; Get centrifugal 2 hours enrichment virus of supernatant 50000g; Then through 20% and 60% sucrose density gradient centrifugation, centrifugal 2 hours of 50000g, reclaims 20% and 60% middle layer; Through 50000g ultracentrifugation desugar in centrifugal 2 hours, process and obtain the good virus of purifying afterwards.Extract viral complete genome DNA
[29], use respectively BamH I, EcoR I and BbvC I (all purchased from New England Biolabs) to carry out enzyme to former vaccine strain DEV and dDEV and cut.Reaction conditions is as follows: get respectively BamHI, EcoR I and each 20U of BbvC I, mixed with DEV genomic dna 8 μ g respectively, in 50 μ l systems, 37 ℃ act on 1 hour.With the CHEF of Bio-Rad company
xA Pulsed Field system is carried out pulse electrophoresis, and the condition of pulse electrophoresis is: electrophoretic buffer position 0.5xTBE, and agarose gel concentration is 1%, program is 2K-70K.
Save the viral restriction enzyme mapping obtaining identical with parental virus, as shown in Figure 2.Illustrate that selected 5 clays form merit rescue DEV virus.The inventor is by selected 5 clay group memberships difference called after pFOS1, pFOS2, pFOS3, pFOS4, pFOS5, Fse I-Sbf I-Pme I joint is all contained at the DEVDNA fragment two ends that this 5 clay clone comprises, can be overlapped, and can splice and cover full DEV genome (their overlapping and replace mode can referring to Fig. 9), and wherein pFOS5 comprises the genomic US7 of DEV and US8 gene and the transcribed spacer between them (nucleotide sequence of the transcribed spacer between US7 and US8 gene is referring to SEQ ID NO:5).About this 5 clay system, the inventor applies for a patent, and application number is 201010207207.8, and exercise question is " duck viral enteritis virus vaccine strain infections clone system and construction process and application ", and the date of application is on June 13rd, 2010.
In the transcribed spacer of embodiment 4. between the genomic US7 of DEV, US8 gene, insert the structure that SV40-TME expresses the recombination mutation clay of framework (SEQ ID NO:1)
Based on above-described embodiment 1-3 result, in transcribed spacer (SEQ ID NO:5) in selected 5 clay group membership pFOS5 between the genomic US7 of DEV and US8 gene, particularly, transcribed spacer between US7 and US8 gene is 223bp altogether, in this research, this transcribed spacer has lacked wherein four Nucleotide of the 108th to 111, (SV40-TME expresses the nucleotides sequence of framework and classifies SEQ ID NO:1 as to replace insertion SV40-TME expression framework, wherein comprise SV40 promotor, referring to Figure 12, wherein italic thickened portion is E gene (SEQ ID NO:4)), build 1 recombination mutation clay, (collection of illustrative plates of this sudden change clay as shown in Figure 8 for pFOS5us78 SV40 TME, its structure pattern can be referring to Fig. 9).In this research, inventor's discovery, the on position of M and E gene does not affect its immune effect to duck viral enteritis virus.But M and E gene insert other positions, whether can affect its protection effect to duck viral enteritis virus and need to test to prove.The building process of pFOS5us78 SV40 TME clay is summarized as follows:
The structure of 4.1pUC ccdB kan:
With the three pairs of primers (being synthesized by TaKaRa company) shown in table 1, respectively " RfA " (wherein gene is aatR1-paraxin-ccdB-aatR2) gene (SEQ ID NO:2) providing in the Gateway Vector Conversion System with One Shot ccdB Survival of Invitrogen company 2 T1Competent Cells test kits is carried out to multiplex PCR amplification.
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 respectively tR1 and tR2, and ccdB1 and these two pairs of primers of ccdB2 increase and obtain aatR1 fragment and ccdB-aatR2 fragment from Reading Frame Cassette A, its reaction conditions is :-72 ℃ of 10min of 95 ℃ 5min-35 circulation (94 ℃ 45s-54 ℃ 45s-72 ℃ of 45s).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 :-72 ℃ of 10min of 95 ℃ of 5min-35* (94 ℃ 45s-54 ℃ 45s-72 ℃ of 45s).These three sheet segment DNAs of difference purifying, and using these three fragments jointly as template, using tR1 and ccdB2 as primer, amplification obtains RfKan gene (SEQ ID NO:3), be that its gene is aatR1-kantlex-ccdB-aatR2, its reaction conditions is :-72 ℃ of 10min of 95 ℃ 5min-35 circulation (94 ℃ 45s-54 ℃ 45s-72 ℃ of 1.5min).
And utilize XbaI and HindIII to be cloned in pUC18 carrier (purchased from TaKaRa company) " RfKan " fragment obtaining, obtain pUC ccdB kan, as shown in Figure 3.
The structure of 4.2pFOS5 us78 Kan ccdB clay:
With the primer US78ccd1 shown in table 2 and US78ccd2 (being synthesized by TaKaRa company), increase with the ccdB gene of restructuring arm from the pUC ccdB kan of above-mentioned structure, its PCR reaction conditions is :-72 ℃ of 10min of 95 ℃ 5min-35 circulation (94 ℃ 45s-54 ℃ 45s-72 ℃ of 2min).With the Counter-Selection BAC Modification Kit test kit of Gene Bridges company, increased fragment is cloned in pFOS5 clay, obtain pFOS5 us78Kan ccdB clay, between the US7 of pFOS5 clay and US8 gene, insert ccdB and kalamycin resistance gene, as shown in Figure 4.
Table 2: for the primer with the ccdB gene of restructuring arm from pUC ccdB kan amplification
The structure of 4.3pENTR MCS plasmid:
For convenience of follow-up test, this research has been done following transformation by the pENTR-gus plasmid (purchased from Invitrogen company) providing in the Gateway Vector Conversion System with One Shot ccdB Survival 2T1 Competent Cells of Invitrogen company test kit: by the gus gene elmination in pENTR-gus, and added BamHI, BglII, EcoRI, EcoRV, SacI, SalI, KpnI and eight restriction enzyme sites of XbaI.With two primer MCS1 shown in table 3 and MCS2, with point mutation, clone test kit (purchased from Invitrogen company) pENTR-gus is transformed into pENTR MCS, as shown in Figure 5.
Table 3: for pENTR-gus being transformed into the primer of pENTR MCS
The structure of 4.4pSI TME plasmid:
With primer pTMEf and the pTMEr shown in table 4, amplification M and E gene (SEQ ID NO:4), this M and E gene source are in duck tembusu virus PTD2010 strain, it is separated in 2010 by the inventor from Putian, fujian Province duck group, by Vet Biotechnology National Key Laboratory and animal influenza key lab of the Ministry of Agriculture, is preserved.With BamH I and EcoR V (purchased from New England Biolabs) enzyme, cut M and E gene are connected in pSI plasmid (purchased from Promega company), successfully build pSI M and E, as shown in Figure 6.Wherein pSI plasmid comprises SV40 promotor (seeing Fig. 6).
Table 4: for building the primer of pSI TME
The structure of 4.5pENTR sv40-TME plasmid:
With primer entry1 and the entry2 shown in table 5, the sv40-TME gene that increases from the above-mentioned pSI TME successfully constructing, and be cloned in the pENTR MCS having built, obtain pENTRsv40-TME, as shown in Figure 7.
Table 5: for building the primer of pENTR sv40-TME
| Primer title | Sequence |
| entry1 | 5’-TGA GGA TCC GGG CGG AGC CTA TGG AAA A-3’ |
| entry2 | 5’-CGA GGA TCC CAG CCC GGA TCC TTA TCG A-3’ |
4.6pFOS5us78 the structure of SV40 TME clay:
PFOS5us78 Kan ccdB and pENTR sv40-E are utilized to the effect of the Gateway Vector Conversion System with One Shot ccdB Survival 2T1 Competent Cells of Invitrogen company test kit, make the sv40-TME in pENTR sv40-TME express the kan ccdB gene in framework replacement pFOS5us78 Kan ccdB, thereby obtain the clay pFOS5us78 SV40 TME that inserts sv40-TME expression cassette in the transcribed spacer between US7 and US8, as shown in Figure 8.Particularly, described sv40-TME expresses the tetranucleotide fragment of the 108th to 111 that framework is replaced the transcribed spacer (SEQ ID NO:5) between described DEV genome US7 and US8.
The rescue of embodiment 5. recombinant viruses
With the middle extraction reagent kit of Qiagen company, extract pFOS1, pFOS2, pFOS3, pFOS4 (by embodiment 1-3, build and screen) and five cosmid DNAs of pFOS5us78 SV40 E (being built by embodiment 4).With Fse I or Sbf I restriction endonuclease (purchased from New England Biolabs company) by clay linearization process used, its reaction conditions is as follows: Sbf I restriction endonuclease 20U (also can use Fse I or Pme I restriction endonuclease), clay 10 μ g, 37 ℃ act on 1 hour, the extracting of phenol/chloroform, ethanol precipitation, preparation transfection DEV DNA.Method with reference to Reddy SM (2002) is inferior to chick embryo fibroblast CEF by five clay cotransfections respectively
[28].PFOS1, pFOS2, pFOS3, pFOS4 and pFOS5us78 SV40 E and the genomic relation of 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, with 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 that fills Hank ' s liquid (purchased from HyClone) and wash, and remove head, four limbs and internal organ, with scissors, shred.Pancreatin with 0.25% (4mL/ embryo) digests 4-5min in 37 ℃ of water-baths, discards pancreatin, with Hank ' s liquid washing 2 times.Add the appropriate M199 nutritive medium (purchased from HyClone) containing serum and twin antibiotic (the two is purchased from Sigma company for penicillin 100u/mL, Streptomycin sulphate 100mg/mL), piping and druming disperses cell, with making 10 after four layers of filtered through gauze
6-10
7the cell suspension of cells/ml, is finally sub-packed in 37 ℃ of cultivations in culturing bottle.Then, respectively that above-mentioned five clay cotransfections are inferior to CEF with reference to the method for Reddy SM (2002)
[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-TME-tPAS, this restructuring duck viral enteritis virus vaccine strain is preserved in Chinese Typical Representative culture collection center (CCTCC, Wuhan, China on April 16th, 2012, Wuhan University), deposit number is CCTCC V201215.
Embodiment 6. recombinant virus E protein expression western blottings (western blot) are identified
The recombinant virus rDEV-TME-tPAS of rescue and parental virus DEV are inoculated respectively inferior to CEF.When appearring in 80% cell, pathology detects the expression of E albumen by western blotting (western blot) detection method.
Western blotting step is summarized as follows: collect respectively 80% cell occur pathology infection recombinant virus rDEV-TME-tPAS and TMUV time for CEF.Carry out SDS-page electrophoresis, use transferring film immersion bubble nylon membrane (purchased from Sartorius company) and filter paper 10 minutes, wet and turn, transferring film condition is voltage 20mA/cm2, and 4 ℃ are spent the night.With PBS, wash film 2 times, each 5 minutes.Nylon membrane is placed in plate, adds 5% skimming milk sealing immersion film, 37 ℃ are swayed 1 hour.Add with 1%BSA confining liquid by the anti-E protein antibodies of the rabbit of the dilution proportion of 1: 100 (Vet Biotechnology National Key Laboratory and animal influenza key lab of the Ministry of Agriculture by inventor place prepare and preserve) (as primary antibodie), in every square centimeter of ratio that adds 0.1ml, add primary antibodie liquid, room temperature is swayed 1 hour.With PBST, wash film 3 times, each 10 minutes.Add and use 1%BSA confining liquid by the anti-rabbit igg antibody (purchased from Sigma company) of the Infrared fluorescence mark of the dilution proportion of 1: 5000 (anti-as two), in every square centimeter of ratio that adds 0.1ml, add two anti-liquid, room temperature is swayed 1 hour.With PBST, wash film 3 times, each 10 minutes.Finally in infrared scanner, take a picture.
Embodiment 7. experimentation on animalies
To recombinate rDEV-TME-tPAS and parent DEV respectively by 10
6tCID
508 of the SPF ducks (being provided by Harbin veterinary institute Animal House) in 16 week age are provided.After 3 weeks, use respectively 100DLD
50the strong poison of DEV (CVCC AV1222, purchased from China Veterinery Drug Inspection Office); Strain (is separated in 2010 by the inventor from Putian, fujian Province duck group with duck tembusu virus PTD2010; by Vet Biotechnology National Key Laboratory and animal influenza key lab of the Ministry of Agriculture, preserved) attack, observe the protection effect of recombinant virus to the strong poison of DEV and duck tembusu virus.
Result
1.E protein expression detects
With the 8th generation recombinant virus rDEV-TME-tPAS, infect after CEF, when pathology appears in 80% cell, by western blotting detection method, detect the expression of E albumen.Its result as shown in figure 10.Recombinant virus can be good in CEF expression M and E albumen.
2. genetic stability detects
Recombinant virus is passed to 20 generations continuously, extract the DNA of parent DEV virus and recombinant virus, by PCR method, identify.As shown in figure 11.And it is carried out to sequencing analysis (sequencing result does not show), have no the generation of disappearance or sudden change.
3. results of animal
To recombinate rDEV-TME-tPAS and parent DEV respectively by 10
6tCID
508 of the SPF ducks (being provided by Harbin veterinary institute Animal House) in 16 week age are provided.After 3 weeks, use respectively 100DLD
50the strong poison of DEV (CVCC AV1222, purchased from China Veterinery Drug Inspection Office); Attack with duck tembusu virus PTD2010 strain.No matter result is for duck viral enteritis or for duck tembusu virus, with the duck of recombinant virus rDEV-TME-tPAS immunity, obtain 100% protection.Its result is as shown in table 6.
Table 6: results of animal table
Note: TMUV:PTD2010 wherein; DEV is poison by force: CVCC AV1222; The expression mode (*/8) of protection result is the number that obtains the SPF duck of protection in 8 tested SPF ducks; Wherein PBS is as negative control.
Discuss
The platform of this research by utilizing duck viral enteritis virus infection clones (, the DEV virus 5 clay group that the inventor builds, referring to embodiment 1-3, the application for a patent for invention that can be also 201010207207.8 referring to the inventor's application number), success obtains the duck viral enteritis virus of restructuring duck tembusu virus M and E gene, at home and abroad still belongs to the first time.
By to recombinant virus rDEV-TME-tPAS in vivo with the detection of growth characteristics of cultured, this research proves first that the transcribed spacer between the genomic US7 of DEV and US8 can stablize to insert and take duck tembusu virus M and the E gene expression construct that SV40 is promotor.This recombinant virus is good representation M and E albumen in vitro.With it, duck is carried out not only providing the immune effect suitable with former vaccine strain DEV after primary immune response, and M and E albumen can obtain good expression in SPF duck body, can induce good immune effect, the attack that can resist the strong poison of duck tembusu virus after immune duck completely.
As can be seen here, the vaccine of the transmissible disease that the restructuring duck viral enteritis virus vaccine strain CCTCC V201215 (its called after rDEV-TME-tPAS) of the expression-secretion type duck tembusu virus M/E albumen that the present invention obtains can cause for the preparation of preventing duck viral enteritis virus and duck tembusu virus, and transmissible disease that duck tembusu virus poultry cause viral for preventing duck viral enteritis.
Therefore, the present invention also provides the vaccine that comprises the restructuring duck viral enteritis virus vaccine strain CCTCC V201215 (that is, rDEV-TME-tPAS of the present invention) of expression-secretion type duck tembusu virus M/E albumen and medicinal adjuvant, vehicle etc.Those skilled in the art according to the application purpose of described vaccine, carry out the immune factors such as bird, can easily select applicable medicinal adjuvant, vehicle etc.。
Spring in 2010 there is suddenly a kind of acute infectious disease that to decline to a great extent as feature, ovarian hemorrhage be cardinal symptom of laying eggs of take in the minority duck group in China East China, South China, and rapid spread is to national most areas afterwards.Through domestic a plurality of laboratory diagnosiss, this disease is caused by a kind of flavivirus; Initial stage is called duck hemorrhagic ovaritis, egg drop syndrome, Duckling flavivirus disease etc., renames duck tembusu virus as sick along with the development later stage to the parsing of the full gene of cause of disease and this disease course of disease is unified.This disease can betide the egg duck of a plurality of kinds, also can betide meat duck and wild duck.Sick duck starts to occur that food consumption and egg productivity sharply decline, and heating, One's spirits are drooping subsequently, 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 stage strain isolated can cause duck and occur nervous symptoms, both legs paralysis, astasia, some strain also can cause that heart, liver are hemorrhage in addition, mortality ratio 5%-30% is not etc.According to incompletely statistics, in egg duck and meat duck main producing region, approximately have 1.2 hundred million egg ducks and more than 1,500 ten thousand meat duck morbidities, accumulative total financial loss is over 5,000,000,000 yuan.Because the morbidity of this disease is unexpected, also not effectively prevention and treatment measure at present, the recombinant virus of the present invention utilizes ripe duck enteritis virus attenuated vaccine to be vector construction express duck tembusu virus M and E albumen, this recombinant virus, for the generation of preventing duck tembusu virus disease, has good application prospect and using value.
Should be appreciated that, although with reference to its exemplary embodiment, the present invention is shown particularly and described, but will be understood by those skilled in the art that, under the condition not deviating from by the defined the spirit and scope of the present invention of accompanying claim, the variation of various forms and details can be carried out therein, the arbitrary combination of various embodiments can be carried out.
Reference
1.Fauquet CM，Mayo MA，Maniloff J，et al.Virus Taxonomy：Eighth Report of the International Committee on Taxonomy of Viruses(Elsevier Academic Press，California，2005)，p.208
2.Li Y.，Huang B.b，Ma X.，Wu J.，et al.Molecular characterization of the genome of duck enteritis virus.Virology，2009，391(2)：151-161.
3.Hooft van Iddekinge BJ.De wind N.，Wensvoort G.Comparison of the protective efficacy of recombinant pseudorabies viruses against pseudorabies and classical swi ne fever in pigs’influence of different promoters on gene expression and on protection.Vaccine，1996.14：6-12.
4.Peeters B.，Bienkowska Szewczyk K.，Hulst M.，et al.Biologically safe，non-transmissible pseudorabies virus vector vaccine protects pigs against both Aujeszky’s disease and classical swine fever.J Gen Virol.，1997，78：3311-3315.
5.Mulder WAM.Virulence and pathogenesis of non-virulent and virulent strains of pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus.Journal of General Virological，1994，78：117-124.
6.Knapp A C.，Enquist LW.Pseudorabies virus recombinants exp ressing functional virulence determinants gE and gI from bovine herpesvirus I.J Virol.，1997，71：2731-2739.
7.Xu G.，Xu X.，Chen HC.Construction of recombinant pseudorabies virus expressing NS1 protein of Japanese encephalitis(SAI4-14-2)virus and its safety and immunogenicity.Vaccine，2004，22(15-16)：1846-1853.
8.Qian P.，Li XM.，Jin ML.，et al.An approach to a FMD vaccine based on genetic engineered attenuated pseudorabies virus l one expeHment using VP1 gene alone generates fin an tibody responds on FIvlD and pseudorabies in swine.Vaccime，2004，22(17-18)：2129-2136.
9.Van Zijl M.，Wensvoort G.，De Kluyver E.，et al.Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protects swine against both pseudorabies and hog cholera.J Virol.，1991.65(5)：2761-2765.
10.Metteuleiter TC.A glycoprotein gX-19-galactosidase fusion gene as insertional marker for rapid identification of pseudorabies vius mutants.J Virol.，1990，30：55-66.
11.Luschow D.，Werner O.Mettenleiter TC.，FuchsW.Protection of chickens from lethal avian influenza A virus infection by live-virus vaccination with infectious laryngotracheitis virus recombinants expressing the hemagglutinin(H5)gene.Vaccine，2001，19(30)：4249-59.
12.Veits J.，Luschow D.，Kindermann K.，et al.Deletion of the non-essentialUL0 gene of infectious laryngotracheitis(ILT)virus leads to attenuation in chickens，and UL0 mutants expressing influenza virus haemagglutinin(H7)protect against ILT and fowl plague.J Gen Virol.，2003，84(12)：3343-52
13.Pavlova SP.，Veits J.，Keil GM.et al.Protection of chickens against H5N1highly pathogenic avian influenza virus infection by live vaccination with infectious laryngotracheitis virus recombinants expressing H5 hemagglutinin and N1 neuraminidase.Vaccine，2009，27(37)：5085-5090.
14.Sakaguchi M.，Urakawa T.，Hirayama Y.，et al.Marek′s disease virus protein kinase gene identified within the shote unique region of the viral genome is not essential for viral replication in cell culture and vaccine-induced immunity in chickens.Virology，1993，195：140-148.
15.Sakaguchi M.，Hirayama Y.，Maeda H.，et al.Construction of recombinant Marek′s disease virus type 1(MDV 1)expressing the E coli LacZ gene as a possible live vaccine vector：the US10 gene of MDVl as a stable insertion site.Vaccine，1994，12：953-957.
16.Sonoda k.，Sakaguchi M.，Matsuo G.，et al.Asymmetric deletion of the junction between the short unique region and the inverted repeat does not affect virua growth in culture and vaccine-induced immunity in chickens.Vaccine，1996，14：277-284.
17.Sakaguchi M.，Nakamura H.，Sonoda K.，et al.protection of chickens with or without maternal antibodies against both Marek′s disease virus and Newcastle disease by one time vaccination with recombinant vaccine of Marek′s disease virus type 1.Vaccine，1998，16：472-479.
18.Tsukamoto K.，Kojima C.，Komori Y.，et al.Protection of chickens against very virulent infectious bursal disease virus and Marek′s disease virus with a recombinant MDV expressing IBDV VP2.Virology，1999，257：352-362.
19.Tsukamoto K.，Saito S.，Saeki S.et al.Complete，Long-Lasting Protection against Lethal Infectious Bursal Disease Virus Challenge by a Single Vaccination with an Avian Herpesvirus Vector Expressing VP2 Antigens.J.Virol.，2002，76(11)：5637-5645.
20.Van Zijl M.，Quint W.，Briair e J.，et al.Regeneration of herpesviruses from molecularly cloned subgenomic fragments.J.Virol.，1988，62：2191-2195.
21.Tomkinson B.，Robertson E.，Yalamanchili R.，Longnecker R.，et al.Epstein-Barr virus recombinants from overlapping cosmid fragments.J.Virol.，1993，67：7298-7306.
22.Cohen JI.，Seidel KE.Generation of varicella-zoster virus(VZV)and viral mutants from cosmid DNAs：VZV thymidylate synthetaseis not essential for replication in vitro.Proc.Natl.Acad.Sci.，1993.90：7376-7380.
23.Cunningham C.，Davison AJ.A cosmid-based system for constructing mutants of herpes simplex virus type 1.Virology，1993，197：116-124.
24.Kemble G.，Duke G.，Winter R.，et al.Defined large-scale alterations of the human cytomegalovirus genome constructed by cotransfection of overlapping cosmids.J.Virol.，1996，70：2044-2048.
25.Register RB.，Shafer JA.A facile system for construction of HSV-1 variants：site-directed mutation of the UL26 protease gene in HSV-1.J.Virol.Methods，1996，57：181-193.
26.Reddy SM.，Lupiani B.，Gimeno IM.，et al.Rescue of a patho genic Marek’s disease virus with overlapping cosmid DNAs：use of a pp38 mutant to validate the technology for the study of gene function.Proc.Natl.Acad.Sci.，2002，99：7054-7059.
27.Gray WL.，Mahalingam R.A cosmid-based system for inserting mutations and foreign genes into the simian varicella virus genome.J.Virol.Methods，2005，130：89-94
28.Reddy SM.，Lupiani B.，Gimeno IM.，et al.Rescue of a patho genic Marek’s disease virus with overlapping cosmid DNAs：use of a pp38 mutant to validate the technology for the study of gene function.Proc.Natl.Acad. Sci.，2002，99：7054-7059.
29.Morgan RB，John L.Cantello；Caroline H.McDermott.1990.Transfection of Chicken Embryo Fibroblasts with Marek′s Disease Virus DNA.Avian Diseases，Vol.34，No.2.(Apr.-Jun.，1990)，pp.345-351.
Claims (10)
1. the restructuring duck viral enteritis virus vaccine strain of expression-secretion type duck tembusu virus M/E albumen, its deposit number is CCTCC V201215, described vaccine strain expression-secretion type duck tembusu virus M albumen and E albumen.
2. the restructuring duck viral enteritis virus vaccine strain of expression-secretion type duck tembusu virus M/E albumen according to claim 1, wherein in the transcribed spacer between the duck viral enteritis virus genomic US7 of DEV and US8 gene, insert the gene fragment that comprises duck tembusu virus M and E gene and SV40 promoter sequence, the nucleotides sequence of described gene fragment is classified SEQ ID NO:1 as.
3. the restructuring duck viral enteritis virus vaccine strain of expression-secretion type duck tembusu virus M/E albumen according to claim 1 and 2, wherein said duck tembusu virus M and E gene source are in PTD2010 strain virus.
4. the restructuring duck viral enteritis virus vaccine strain of expression-secretion type duck tembusu virus M/E albumen according to claim 2, the wherein said gene fragment that comprises duck tembusu virus M and E gene and SV40 promoter sequence is replaced the tetranucleotide fragment of the 108th to 111 of the transcribed spacer between the virus genomic US7 of described duck viral enteritis and US8 gene.
5. the restructuring duck viral enteritis virus vaccine strain of expression-secretion type duck tembusu virus M/E albumen according to claim 2, wherein said SV40 promoter sequence derives from the plasmid that comprises SV40 promotor, for example pSI plasmid.
6. build the method for the restructuring duck viral enteritis virus vaccine strain of expression-secretion type duck tembusu virus M/E albumen claimed in claim 1, described method comprises the steps:
(1) build the duck viral enteritis virus genomic Fosmid of DEV library, and therefrom select for saving 5 clay combined systems of duck viral enteritis virus, they are distinguished to called after pFOS1, pFOS2, pFOS3, pFOS4, pFOS5, and wherein pFOS5 clay comprises US7 and US8 gene and the transcribed spacer between them in duck viral enteritis viral genome;
(2) utilize obtain in step (1) comprise US7 in duck viral enteritis viral genome and the clay pFOS5 of US8 gene and the transcribed spacer between them, in the US7 of this clay and the transcribed spacer between US8 gene, insert the gene fragment SEQ ID NO:1 that comprises duck tembusu virus TME gene and SV40 promoter sequence, build recombination mutation clay; With
(3) utilize the recombination mutation clay and pFOS1, pFOS2, pFOS3 and the pFOS4 cotransfection in the middle 5 clay combined systems that obtain of step (1) that in step (2), obtain inferior to chick embryo fibroblast CEF, save out recombinant virus CCTCC V201215, by its called after rDEV-TME-tPAS.
7. method according to claim 6, Fse I-Sbf I-Pme I joint is contained at the duck viral enteritis viral dna fragment two ends that each clay clone in the 5 clay combined systems that wherein obtain in step (1) comprises, overlapped, and splicing covers the full genome of duck viral enteritis virus.
8. the application of the restructuring duck viral enteritis virus vaccine strain of expression-secretion type duck tembusu virus M/E albumen claimed in claim 1, its vaccine viral for the preparation of preventing duck viral enteritis and the transmissible disease that duck tembusu virus causes.
9. application according to claim 8, the transmissible disease that wherein said duck viral enteritis virus and duck tembusu virus cause comprises duck viral enteritis and duck tembusu virus disease.
10. a vaccine, it comprises the restructuring duck viral enteritis virus vaccine strain CCTCC V201215 of expression-secretion type duck tembusu virus M/E albumen claimed in claim 1, and medicinal adjuvant, vehicle.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039268A (en) * | 2015-06-01 | 2015-11-11 | 浙江省农业科学院 | Recombinant duck plague virus of expressing duck tembusu virus E protein as well as construction method and application of recombinant duck plague virus |
| CN105039267A (en) * | 2015-06-01 | 2015-11-11 | 浙江省农业科学院 | Recombinant duck plague virus for expressing gosling plague virus VP2 and its constructing method and application |
| CN106520710A (en) * | 2016-11-28 | 2017-03-22 | 广东省农业科学院动物卫生研究所 | Preparation method and application of live vector vaccine for expressing duck Tembusu virus (DTMUV) prm and E protein recombinant Newcastle disease virus (NDV) |
| CN110904058A (en) * | 2018-09-14 | 2020-03-24 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Recombinant duck plague virus vaccine and construction method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277368A (en) * | 2010-06-13 | 2011-12-14 | 中国农业科学院哈尔滨兽医研究所 | Duck enteritis virus infectious recombinant cloning system and construction method and application thereof |
| CN102373180A (en) * | 2011-09-26 | 2012-03-14 | 中国农业科学院哈尔滨兽医研究所 | Recombinant duck enteritis virus (DEV) vaccine strain for expressing avian influenza virus haemagglutinin (HA) gene and constructing method and application thereof |
| CN102533674A (en) * | 2010-12-08 | 2012-07-04 | 中国农业科学院哈尔滨兽医研究所 | Recombinant duck virus enteritis virus vaccine strain (rDEVul41HA) for expressing avian influenza virus hemagglutinin (HA) genes and construction method as well as application thereof |
-
2012
- 2012-09-10 CN CN201210331133.8A patent/CN103667197B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277368A (en) * | 2010-06-13 | 2011-12-14 | 中国农业科学院哈尔滨兽医研究所 | Duck enteritis virus infectious recombinant cloning system and construction method and application thereof |
| CN102533674A (en) * | 2010-12-08 | 2012-07-04 | 中国农业科学院哈尔滨兽医研究所 | Recombinant duck virus enteritis virus vaccine strain (rDEVul41HA) for expressing avian influenza virus hemagglutinin (HA) genes and construction method as well as application thereof |
| CN102373180A (en) * | 2011-09-26 | 2012-03-14 | 中国农业科学院哈尔滨兽医研究所 | Recombinant duck enteritis virus (DEV) vaccine strain for expressing avian influenza virus haemagglutinin (HA) gene and constructing method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 徐大伟等: "鸭坦布苏病毒E基因DNA疫苗构建及免疫原性的初步研究", 《中国预防兽医学报》, vol. 34, no. 4, 30 April 2012 (2012-04-30) * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039268A (en) * | 2015-06-01 | 2015-11-11 | 浙江省农业科学院 | Recombinant duck plague virus of expressing duck tembusu virus E protein as well as construction method and application of recombinant duck plague virus |
| CN105039267A (en) * | 2015-06-01 | 2015-11-11 | 浙江省农业科学院 | Recombinant duck plague virus for expressing gosling plague virus VP2 and its constructing method and application |
| CN106520710A (en) * | 2016-11-28 | 2017-03-22 | 广东省农业科学院动物卫生研究所 | Preparation method and application of live vector vaccine for expressing duck Tembusu virus (DTMUV) prm and E protein recombinant Newcastle disease virus (NDV) |
| CN106520710B (en) * | 2016-11-28 | 2020-01-14 | 广东省农业科学院动物卫生研究所 | Preparation and application of recombinant Newcastle disease virus live vector vaccine expressing duck tembusu virus prm and E proteins |
| CN110904058A (en) * | 2018-09-14 | 2020-03-24 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Recombinant duck plague virus vaccine and construction method and application thereof |
| CN110904058B (en) * | 2018-09-14 | 2023-03-07 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Recombinant duck plague virus vaccine and construction method and application thereof |
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