CN102533674B - Recombinant duck virus enteritis virus vaccine strain (rDEVul41HA) for expressing avian influenza virus hemagglutinin (HA) genes and construction method as well as application thereof - Google Patents
Recombinant duck virus enteritis virus vaccine strain (rDEVul41HA) for expressing avian influenza virus hemagglutinin (HA) genes and construction method as well as application thereof Download PDFInfo
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Abstract
The invention provides a recombinant duck virus enteritis virus vaccine strain CCTCC No.V201026, named as rDEVul41HA, for expressing avian influenza virus hemagglutinin (HA) genes and a construction method as well as application thereof. Specifically, gene segments SV40-HA containing avian influenza virus hemagglutinin HA genes and a SV40 promoter sequence are inserted into UL41 genes of duck virus enteritis virus to construct cosmids with SV40-HA expression cassette inserted into UL41 genes; and by the cosmids, the recombinant duck virus enteritis virus vaccine strain CCTCC V201026 for expressing the avian influenza virus hemagglutinin (HA) genes is saved and obtained. The invention also relates to a method for constructing the recombinant duck virus enteritis virus vaccine strain and application of the recombinant duck virus enteritis virus vaccine strain to preparing vaccines for preventing duck virus enteritis and avian influenza.
Description
Technical field
The present invention relates to recombinant viral vaccine field, duck viral enteritis virus vaccines field relates more specifically to recombinate.The invention provides a kind of restructuring duck viral enteritis virus vaccine strain CCTCC V201026 that expresses avian flu virus hemagglutinin (HA) gene, called after rDEVul41HA, 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.But compare less for the research of DEV with respect to other simplexvirus.Therefore the international virusology classification of Ba Ci committee report is categorized as simplexvirus
[1], and fail further to classify.Up to date, its complete sequence is just all surveyed logical
[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, the work of DEV vaccine strain genome sequencing is completed.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 vaccinia virus is vector expression hsv the eighties in last century, it is carrier that people start the various different DNA virus of trial, 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, existing a large amount of correlative study report.In common livestock and poultry simplexvirus disease, as take Pseudorabies virus (PRV) as carrier, in the genes such as gD, gE, gG and TK, insert respectively the foreign genes such as the E2 of CSF, and use its immune swine, obtain 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, attack poison with vMDV, vvMDV, 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]; Successful construction of recombinant virus between the UL45 of the promotor insertion herpes turkey virus (HVT) of Tsukamoto K etc. (2002) (IBDV) VP2 gene using Pec as infectious bursal disease virus and UL46 gene, the attack that is enough to resist the strong poison of IBDV with its immune SPF chicken
[19].Now, 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.But the genomic constitution of DEV and structure and MDV etc. have larger difference, 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 and insert the site of foreign gene and be not necessarily applicable to DEV.
Because the research to DEV relatively lags behind, up to now, be still blank about the research report of Nonessencial region in DEV gene both at home and abroad.The method that is usually used in building simplexvirus recombinant virus has three kinds.One is homologous recombination; The second is that viral genome is inserted in BAC, then on BAC, builds sudden change, goes out recombinant virus with the corresponding cellular rescue of its transfection; 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.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 this platform construction success, 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 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 genomic UL41 gene of DEV by current China for vaccine strain hemagglutinin (HA) gene of bird flu prevention, can make SPF duck produce the good antibody to anti influenza with its immune specific pathogen free duck (SPF duck), also not affect the immune effect of DEV simultaneously.
Summary of the invention
The invention provides a kind of restructuring duck viral enteritis virus vaccine strain of expressing avian flu virus hemagglutinin (HA) gene, its deposit number is CCTCC V201026, called after rDEVul41Ha, and construction process and application.Particularly, the present invention utilizes recombinant clone technology, the gene fragment SV40-HA that comprises avian flu virus hemagglutinin (HA) gene and SV40 promoter sequence (nucleotides sequence of described gene fragment is classified SEQ ID NO:1 as) is inserted into duck enteritis virus (duck enteritis virus, DEV) in UL41 gene, build and obtain the clay pFOSlu141SV40HA that inserts SV40-HA expression cassette in UL41 gene, obtain by its rescue the restructuring duck viral enteritis virus vaccine strain CCTCC V201026 that expresses avian flu virus hemagglutinin (HA) gene, called after rDEVul41HA.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 the vaccine of preventing duck viral enteritis and bird flu.
In one embodiment of the invention, the invention provides a kind of restructuring duck viral enteritis virus vaccine strain of expressing avian flu virus hemagglutinin (HA) gene, its deposit number is CCTCCV201026, called after rDEVul41HA, it is preserved in Chinese Typical Representative culture collection center (CCTCC on October 26th, 2010, Wuhan, China, Wuhan University).The restructuring duck viral enteritis virus vaccine strain CCTCC V201026 of described expression avian flu virus hemagglutinin (HA) gene inserts the gene fragment SV40-HA (SEQ ID NO:1) that comprises avian flu virus hemagglutinin HA gene and SV40 promoter sequence in the duck viral enteritis virus genomic UL41 gene of DEV (SEQ ID NO:5).
In one embodiment of the invention, the invention provides the method for the restructuring duck viral enteritis virus vaccine strain CCTCC V201026 of construction expression avian flu virus hemagglutinin HA gene, described method comprises the steps:
(1) build the genomic Fosmid of duck viral enteritis virus (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 pFOS1 clay comprises the UL41 gene in duck viral enteritis viral genome;
(2) utilize the clay pFOS1 that comprises the UL41 gene in DEV genome obtaining in step (1), in the UL41 of this clay gene, insert the gene fragment SV40-HA (SEQ ID NO:1) that comprises avian flu virus hemagglutinin HA gene and SV40 promoter sequence, build recombination mutation clay; With
(3) utilize pFOS2, pFOS3, pFOS4 and pFOS5 cotransfection in recombination mutation clay and the middle 5 clay combined systems that obtain of step (1) that obtain in step (2) inferior to chick embryo fibroblast CEF, save out recombinant virus CCTCC V201026, by its called after rDEVul41HA.
In preferred embodiments, 5 clays that obtain in above-mentioned steps (1) are cloned the duck viral enteritis viral dna fragment two ends that comprise and are all contained Fse I-SbfI-Pme I joint, 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, avian flu virus hemagglutinin HA gene in above-mentioned steps (2) is the HA gene that has lacked alkaline bleach liquor cleavage site, by the H5NI type bird flu strain from Anhui separation in 2006, (its detailed name was called A/duck/Anhui/106/2006 (H5N1) for it, national bird flu reference laboratory by inventor place is preserved, and this laboratory is the mechanism of domestic legal preservation avian influenza virus) increasing obtains; 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 HA gene does not affect the immune effect (data do not show) of constructed recombinant vaccine strain to duck viral enteritis virus.But HA gene inserts other positions, whether can affect its protection effect to duck viral enteritis virus and 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 V201026 of described expression avian flu virus hemagglutinin HA gene, its vaccine viral for the preparation of preventing duck viral enteritis and the transmissible disease that avian influenza virus causes.
In a preferred embodiment of the invention, the transmissible disease that described duck viral enteritis virus and avian influenza virus cause comprises the transmissible disease that duck viral enteritis virus and avian influenza virus cause in duck, goose and other Anseriformes bird, for example, the duck viral enteritis being caused by duck viral enteritis virus DEV, the bird flu being caused by avian influenza virus A/duck/LN/51/2005 (H5N1) and A/duck/HuB/49/2005 (H5N1) etc. 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 V201026 of expression avian flu virus hemagglutinin HA gene 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 factors such as immune 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 duck, goose and other Anseriformes bird by duck viral enteritis virus and avian influenza virus, for example, be effective to the duck viral enteritis that prevention is caused by duck viral enteritis virus DEV, the bird flu being caused by avian influenza virus A/duck/LN/51/2005 (H5N1) and A/duck/HuB/49/2005 (H5N1) etc. etc.
Therefore, the invention provides following:
1. the restructuring duck viral enteritis virus vaccine strain of expressing avian flu virus hemagglutinin HA gene, its deposit number is CCTCC V201026, called after rDEVul41HA.
2. according to the restructuring duck viral enteritis virus vaccine strain of the expression avian flu virus hemagglutinin HA gene described in the 1st, wherein in the genomic UL41 gene of duck viral enteritis virus DEV, insert the gene fragment SV40-HA that comprises avian flu virus hemagglutinin HA gene and SV40 promoter sequence, the nucleotides sequence of described gene fragment is classified SEQ ID NO:1 as.
3. according to the restructuring duck viral enteritis virus vaccine strain of the expression avian flu virus hemagglutinin HA gene described in the 1st or the 2nd, wherein said avian flu virus hemagglutinin HA gene is the hemagglutinin gene that has lacked alkaline bleach liquor cleavage site, and it is obtained by H5N1 type bird flu strain A/duck/Anhui/106/2006 (H5N1) amplification.
4. the restructuring duck viral enteritis virus vaccine strain of expression avian flu virus hemagglutinin HA gene claimed in claim 2, the wherein said gene fragment that comprises avian flu virus hemagglutinin HA gene and SV40 promoter sequence is replaced the nucleotide fragments of the 506th to 891 of the virus genomic UL gene of described duck viral enteritis.
5. according to the restructuring duck viral enteritis virus vaccine strain of the expression avian flu virus hemagglutinin HA gene described in the 2nd, wherein said SV40 promoter sequence derives from the plasmid that comprises SV40 promotor, for example pSI plasmid.
6. the method for the restructuring duck viral enteritis virus vaccine strain of the expression avian flu virus hemagglutinin HA gene 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 pFOS1 clay comprises the UL41 gene in duck viral enteritis viral genome;
(2) utilize the clay pFOS1 that comprises the UL41 gene in duck viral enteritis viral genome obtaining in step (1), in the UL41 of this clay gene, insert the gene fragment SV40-HA (SEQ ID NO:1) that comprises avian flu virus hemagglutinin HA gene and SV40 promoter sequence, build recombination mutation clay; With
(3) utilize pFOS2, pFOS3, pFOS4 and pFOS5 cotransfection in recombination mutation clay and the middle 5 clay combined systems that obtain of step (1) that obtain in step (2) inferior to chick embryo fibroblast CEF, save out recombinant virus CCTCC V201026, by its called after rDEVul41HA.
7. according to the method described in the 6th, each clay in the 5 clay combined systems that wherein obtain in step (1) is cloned the DEV DNA fragmentation two ends that comprise and is contained Fse I-SbfI-Pme I joint, can be overlapped, and can splice the full genome of covering duck viral enteritis virus.
8. the application of the restructuring duck viral enteritis virus vaccine strain of the expression avian flu virus hemagglutinin HA gene described in the 1st, its vaccine viral for the preparation of preventing duck viral enteritis and the transmissible disease that avian influenza virus causes.
9. according to the application described in the 8th, the transmissible disease that wherein said duck viral enteritis virus and avian influenza virus cause comprises the transmissible disease that duck viral enteritis virus and avian influenza virus cause in duck, goose and other Anseriformes bird.
10. a vaccine, it comprises the restructuring duck viral enteritis virus vaccine strain of the expression avian flu virus hemagglutinin HA gene described in the 1st, and medicinal adjuvant, vehicle etc.
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, wherein DEV: 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 digestion molecule marker (λ-Hind III digest Marker);
Fig. 3: pUC ccdB kan plasmid map;
Fig. 4: pFOS1u141Kan ccdB clay collection of illustrative plates;
Fig. 5: pENTR MCS plasmid map;
Fig. 6: pSI HA plasmid map;
Fig. 7: pENTR sv40-ha plasmid map;
Fig. 8: pFOSlu141SV40HA clay collection of illustrative plates;
Fig. 9: duck viral enteritis virus infection clones rescue recombinant virus schematic diagram;
Figure 10: the expression immunofluorescence detected result figure of recombinant virus HA gene in CEF;
Figure 11: PCR detects the situation of heterogenous expression framework in rDEVul41Ha;
Figure 12: after immune recombinant virus, induce the situation of HI antibody in SPF duck body.
Figure 13: SEQ ID NO:1:SV40-HA expresses framework, wherein italic thickened portion is avian influenza hemagglutinin HA gene.
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 (CVCC), 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 T4DNA polysaccharase (T4DNAPolymerase, 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 T4 ligase enzyme after reclaiming is connected to upper Fse I-SbfI-Pme I joint, after refining, be connected on pCCl Fos (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 EPl300-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 lEPI300-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.
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 pCCl Fos, sequencing primer sequence is as follows:
Primer1：5’-TAATACGACTCACTATAGGG-3’
Primer2：5’-GCCAAGCTATTTAGGTGAGA-3’
Through the analysis of end sequencing, obtain altogether Insert Fragment two ends and be all connected with 250 of the clones of complete Fse I-Sbf I-Pme I joint.From these 250 clones, choose many groups for saving the 5 clay combinations of DEV.Fse I-SbfI-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.
Extract the DNA of selected clay with the middle amount extraction test kit of Qiagen company.Selected clay is carried out to linearization process with Fse I, SbfI or Pme I restriction endonuclease (all purchased from New England Biolabs), reaction conditions is as follows: SbfI 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.
With reference to the calcium phosphate method of Reddy SM (2002) by 5 sections of DEV DNA 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.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, shred with scissors.Pancreatin (4mL/ embryo) with 0.25% digests 4-5 minute in 37 ℃ of water-baths, discards pancreatin, with Hank ' s liquid washing 2 times.Add the appropriate MI99 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 be 1:1000 by virus inoculation in CEF.In the time that 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; Process and obtain the good virus of purifying through 50000g ultracentrifugation desugar in centrifugal 2 hours 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 BamH I, EcoR I and the 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 0.5x TBE, 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.Selected 5 clay composition merit rescue DEV viruses are described.The inventor is by selected 5 clay group memberships called after pFOS1, pFOS2, pFOS3, pFOS4, pFOS5 respectively, this 5 clay is cloned the DEVDNA fragment two ends that comprise and is all contained Fse I-Sbf I-Pme I joint, can be overlapped, and can splice and cover full DEV genome (their overlapping and replace mode can referring to Fig. 9), and wherein pFOS1 comprises the genomic UL41 gene of DEV (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.
Embodiment 4. inserts the structure of the recombination mutation clay of SV40-HA expression framework (SEQ ID NO:1) in the genomic UL41 gene of DEV
Based on above-described embodiment 1-3 result, in selected 5 clay group membership pFOS1 in the genomic UL41 gene of DEV, be specially the 506th to 891 Nucleotide of disappearance UL41 gene, and replace insert SV40-HA express framework, be that SV40-HA expresses framework and replaces the nucleotide fragments of the 506th to 891 of described UL gene (SEQ ID NO:5) (wherein SV40-HA expresses the nucleotides sequence of framework and classifies SEQ ID NO:1 as, wherein comprise SV40 promotor, also referring to Figure 13, wherein italic thickened portion is HA 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 pFOSlul41SV40HA, its structure pattern can be referring to Fig. 9).In this research, inventor's discovery, the on position of HA gene does not affect its immune effect to duck viral enteritis virus.But HA gene inserts other positions, whether can affect its protection effect to duck viral enteritis virus and need to test to prove.
The building process of pFOSlul41SV40HA clay is summarized as follows:
The structure of 4.1pUC ccdB kan:
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 Survival2T1Competent Cells of Invitrogen company test kit is carried out to multiplex PCR amplification with the three pairs of primers (being synthesized by TaKaRa company) shown in table 1.
Table 1: for the PCR primer of clone " Rfkan " (SEQ ID NO:3, 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 to aatR1 gene and ccdB-aatR2 gene from Reading Frame Cassette A, its reaction conditions is :-72 ℃ of 10min of 95 ℃ of 5min-35* (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 purifying respectively, 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 ℃ of 5min-35* (94 ℃ 45s-54 ℃ 45s-72 ℃ of 1.5min).
And utilize XbaI and HindIII to be cloned in pUC18 carrier (purchased from TaKaRa company) by " RfKan " that obtain, obtain pUC ccdB kan, as shown in Figure 3.
The structure of 4.2pFOS1u141Kan ccdB clay:
With the primer UL41ccd1 shown in table 2 and UL41ccd2 from the pUC ccdB kan of above-mentioned structure, increase with restructuring arm ccdB gene, its reaction conditions is :-72 ℃ of 10min of 95 ℃ of 5min-35* (94 ℃ 45s-54 ℃ 45s-72 ℃ of 2min).Increased fragment is cloned in pFOS1 clay with the Counter-Selection BAC Modification Kit test kit of Gene Bridges company, obtain pFOS1u141Kan ccdB clay, in the UL41 of pFOS1 clay 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, the pENTR-gus plasmid (purchased from Invitrogen company) providing in the Gateway Vector Conversion System with One Shot ccdB Survival2T1Competent Cells of invitrogen company test kit has been done following transformation by this research: 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, clone test kit (purchased from Invitrogen company) with point mutation 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 HA plasmid:
With the primer pHa031 shown in table 4 and pHa ORF2, amplification has lacked the avian influenza hemagglutinin HA gene (SEQ ID NO:4) in alkaline bleach liquor cleavage site, (its detailed name is called A/duck/Anhui/106/2006 (H5N1) to the H5N1 type bird flu strain that this HA gene source separated from Anhui in 2006, national bird flu reference laboratory by inventor place is preserved, this laboratory is the mechanism of domestic legal preservation avian influenza virus),, be namely the malicious HA gene of kind of seedling for 5 of avian influenza prevention at present.And cut and be connected in pSI plasmid (purchased from Promega company) with Not I and Nhe I (purchased from New England Biolabs) enzyme, successfully build pSI HA, as shown in Figure 6.Wherein pSI plasmid comprises SV40 promotor (seeing Fig. 6).
Table 4: for building the primer of pSI HA
| Primer title | Sequence |
| pHaO31 | 5′-AAT?GCT?AGC?CGC?CAC?CAT?GGAGAAAAT?AGT?GCT?TCT-3′ |
| pHa?ORF2 | 5′-GAT?GGC?GGC?CGC?TTAAAT?GCAAAT?TCT?GCATT-3′ |
The structure of 4.5pENTR sv40-ha plasmid:
With primer entryha1 and entryha2 shown in table 5, HA gene increases from the above-mentioned pSI HA successfully constructing, and be cloned into (BamHI enzyme is purchased from New England Biolabs company) in the pENTR MCS having built with BamHI restriction enzyme site, obtain pENTR sv40-ha, as shown in Figure 7.
Table 5: for building the primer of pENTR sv40-ha
| Primer title | Sequence |
| entryha1 | 5′-TGA?GGA?TCC?GGG?CGG?AGC?CTA?TGG?AAAA-3′ |
| entryha2 | 5′-CGA?GGA?TCC?CAG?CCC?GGA?TCC?TTA?TCGA-3′ |
The structure of 4.6pFOS1ul41SV40HA clay:
By the effect of pFOS1u141Kan ccdB and the pENTR sv40-ha use GatewayVector Conversion System with One Shot ccdB Survival2T1Competent Cells of Invitrogen company test kit, make the sv40-ha in pENTR sv40-ha express the kan ccdB gene in framework replacement pFOS1u141Kan ccdB, thereby obtain the clay pFOS1ul41SV40HA that inserts sv40-ha expression cassette in UL41 gene, as shown in Figure 8.Particularly, described sv40-ha expression framework is replaced the nucleotide fragments of the 506th to 891 of described DEV genome UL gene.
The rescue of embodiment 5. recombinant viruses
With middle extraction reagent kit extraction pFOS1ul41SV40HA (being built by embodiment 4) and pFOS2, pFOS3, five cosmid DNAs of pFOS4, pFOS5 (build and screen by embodiment 1-3) of Qiagen company.With Fse I or SbfI restriction endonuclease (purchased from New England Biolabs company) by clay linearization process used, its reaction conditions is as follows: SbfI 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.With reference to the method for Reddy SM (2002) respectively by five clay cotransfections time for chick embryo fibroblast CEF
[3].The genomic relation of pFOSlul41SV40HA and pFOS2, pFOS3, pFOS4, pFOS5 and DEV 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, shred with scissors.Pancreatin (4mL/ embryo) with 0.25% 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, with reference to the method for Reddy SM (2002) respectively by above-mentioned five clay cotransfections time for CEF
[3], after transfection 6-9 days, can be observed the appearance of the typical cytopathics such as cell circle contracting.Save out recombinant virus, called after rDEVul41HA, this restructuring duck viral enteritis virus vaccine strain is preserved in Chinese Typical Representative culture collection center (CCTCC, Wuhan, China on October 26th, 2010, Wuhan University), deposit number is CCTCC V201026.
Embodiment 6. recombinant virus HA express identified by immunofluorescence
The recombinant virus rDEVul41HA of rescue and parental virus DEV are inoculated respectively inferior to CEF.In the time that appearring in 80% cell, pathology detects the expression of HA gene by indirect immunofluorescene assay method.
Wherein indirect immunofluorescence step is summarized as follows: waited to infect the inferior of rDEVul41HA and DEV and occurred pathology for CEF80% cell, with 4% paraformaldehyde fixed cell 30 minutes, PBS washes 3 times, add with 1%BSA confining liquid by the anti-HA antibody of the chicken of the dilution proportion of 1:100 (the national bird flu reference laboratory by inventor place is prepared according to a conventional method and preserves) 37 ℃ of effects 1 hour.Wash 3 times each 10 minutes with PBST.With the goat-anti chicken igg antibody (purchased from Sigma company) of green fluorescent protein (GFP) mark 37 ℃ of effects 1 hour.Wash 3 times with PBS, observe and take pictures.
Embodiment 7. genetic stabilities detect
Recombinant virus rDEVul41HA is passed continuously in CEF to 20 generations, extract the DNA of parent DEV virus and recombinant virus rDEVul41HA, identify by PCR method, the primer is U141d21:5 '-GGC TTG GTA GAT TTG AGG C-3 '; U141d22:5 '-TTC CAT CGC AAT GAG TAG G-3 '.And it is carried out to sequencing analysis.
Embodiment 8. blood clottings suppress experiment antibody titer (HI antibody) induction situation
With recombinating, rDEVul41HA and parent DEV press 10 respectively
5tCID
50infect 6 of the SPF ducks (each 3 of male and female, approximately 500 grams of body weight are provided by Harbin veterinary institute Animal House) in 4 week age, blood sampling weekly, separation of serum, surveys its hemagglutination inhibition antibody (HI antibody).
Step is as follows: doubling dilution serum in 96 hole blood-coagulation-boards, add afterwards 4 unit antigens (being avian influenza virus), described A/duck/Anhui/106/2006 (H5N1) strain (being preserved by inventor the country one belongs to bird flu reference laboratory) preparation for antigen, under room temperature, act on 30 minutes, add afterwards 25 microlitre chicken red blood cells (being prepared according to a conventional method by this research department).Observations.
Embodiment 9. experimentation on animalies
By parent DEV virus with recombinant virus rDEVul41HA respectively with 10
5tCID
50immunity SPF duck in 4 week age (each 3 of male and female, approximately 500 grams of body weight are provided by Harbin veterinary institute Animal House), 10 every group.After 3 weeks, use respectively 100DLD
50the strong poison of DEV (CVCC AV1222, purchased from China Veterinery Drug Inspection Office); And A/duck/LN/51/2005 (H5N1), A/duck/HuB/49/2005 (H5N1) two influenzae strain viruses (preserve by the national bird flu reference laboratory by inventor place; this laboratory is the mechanism of domestic legal preservation avian influenza virus) attack, observe the protection effect of recombinant virus to the strong poison of DEV and influenza.
Result
1.HA genetic expression detects
With after the 4th generation recombinant virus rDEVul41HA infection CEF, in the time that appearring in 80% cell, pathology detects the expression of HA gene by indirect immunofluorescene assay method.Its result as shown in figure 10.Recombinant virus can be good in CEF expression HA albumen.
2. genetic stability detects
Recombinant virus is passed continuously to 20 generations, extract the DNA of parent DEV virus and recombinant virus, identify by PCR method.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.
The experiment of 3.HI antibody extended period
With recombinating, rDEVul41HA and parent DEV press 10 respectively
5tCID
50infect 6 of the SPF ducks in 4 week age, its HI antibody is surveyed in blood sampling weekly.Its result as shown in figure 12.The HI antibody titer average out to 1 of first week, second and third, on average tiring of surrounding be respectively 2.67,2.33 and 2.The HI antibody with parent DEV infection SPF duck was in contrast for 0 (result does not show).
4. results of animal
By parent DEV virus with recombinant virus rDEVul41HA respectively with 10
5tCID
50immunity SPF duck in 4 week age, 10 every group.After 3 weeks, use respectively 100DLD
50dEV strong poison (CVCC AV1222) and two influenzae strain viruses (A/duck/LN/51/2005 (H5N1) and A/duck/HuB/49/2005 (H5N1)) attack, the protection effect of observation recombinant virus to the strong poison of DEV and influenza.No matter result is for duck viral enteritis or for bird flu, obtain 100% protection with the duck of recombinant virus rDEVu141Ha immunity.Its result is as shown in table 6.
Table 6: results of animal table
Note: wherein Aiv1:A/duck/LN/51/2005 (H5N1); Aiv2:A/duck/HuB/49/2005 (H5N1); DEV is poison by force: CVCC AV1222; The expression mode (*/10) of protection result is the number that obtains the SPF duck of protection in 10 tested SPF ducks; Wherein PBS is as negative control.
Discuss
The platform of this research by the duck viral enteritis virus infection clones that utilizes this research department and built (, 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 restructuring duck viral enteritis virus of expressing avian flu virus hemagglutinin HA gene, and this at home and abroad still belongs to the first time.
By to recombinant virus rDEVu141HA in vivo with the detection of growth characteristics of cultured, this research proves in DEV genome, in UL41 gene, to stablize the HA gene expression construct inserting take SV40 as promotor first, it is good representation HA gene 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 HA gene can obtain good expression in SPF duck body, can induce good immune effect, can resist the attack of the strong poison of bird flu.
As can be seen here, the vaccine of the transmissible disease that the restructuring duck viral enteritis virus vaccine strain CCTCC V201026 (its called after rDEVu141HA) of the expression Avian Influenza Virus HA Gene that the present invention obtains can cause for the preparation of preventing duck viral enteritis virus and avian influenza virus, and transmissible disease that avian influenza virus duck, goose and other Anseriformes bird 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 V201026 (its called after rDEVu141HA) of expression of influenza virus HA gene and medicinal adjuvant, vehicle etc.Those skilled in the art according to the application purpose of described vaccine, carry out the factors such as immune bird, can easily select applicable medicinal adjuvant, vehicle etc.
There are long edible duck and the history of duck's egg in China, and it occupies an important position in the food consumption of China.Meanwhile, China is maximum in the world duck producing country.According to Food and Argriculture OrganizationFAO (FAO) statistics, 6.61 hundred million of China's duck breeding stocks in 2002, account for respectively 69.7% and 78.3% of the world and the total breeding stock of Asia duck
[30].In recent years, the egg of duck, meat and suede also export to other countries, increase farmers' income and have played vital role for some areas.Although Shi Yang duck big country of China, raise competence is not high, and the research such as the disease control of duck and different times egg, fleshy duck fodder nutritional needs lags behind with respect to other domestic animals and fowls.The aquatic birds such as duck are the natural storage vaults of all subtype influenza viruses, and meanwhile, bird flu also can cause a large amount of duck death
[31,32].Duck plague and bird flu, duck hepatitis are to threaten at present the virus disease of supporting of paramount importance three kinds of duck industry.At present, the prevention of duck hepatitis mainly, by duckling injection of antibodies, does not have vaccine so far.For bird flu prevention is the deactivation vaccine identical with chicken.But the epidemiology survey result to aquatic bird every year from this research department, influenza immune state is unsatisfactory.And due to the high mortality of DEV, most of raiser can carry out to duck group the immunity of DEV, thereby the immune popularity rate of DEV will be higher than bird flu.This vaccine not only can provide the good immune effect to DEV to immune duck, and the good immune effect to bird flu can also be provided.Living vaccine wants cheap a lot of than the current inactivated vaccine price for duck preventing and controlling influenza, and can also will get well than inactivated vaccine at cellular immune level.Therefore, the development of this vaccine not only has most important theories and practice significance, also has very important economy and public health meaning simultaneously.
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.
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Claims (3)
1. the restructuring duck viral enteritis virus vaccine strain of expressing avian flu virus hemagglutinin HA gene, its deposit number is CCTCC NO:V201026.
2. the application of the restructuring duck viral enteritis virus vaccine strain of expression avian flu virus hemagglutinin HA gene claimed in claim 1, its vaccine viral for the preparation of preventing duck viral enteritis and the transmissible disease that H5N1 type avian influenza virus causes in duck.
3. a vaccine, it comprises the restructuring duck viral enteritis virus vaccine strain CCTCC NO:V201026 of expression avian flu virus hemagglutinin HA gene claimed in claim 1, and medicinal adjuvant or vehicle.
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| CN103667198B (en) * | 2012-09-10 | 2016-04-13 | 中国农业科学院哈尔滨兽医研究所 | The structure of the restructuring duck enteritis virus vaccine of expression-secretion type duck tembusu virus E protein and application |
| CN103497967A (en) * | 2013-08-26 | 2014-01-08 | 华中农业大学 | Construction method and use of duck enteritis virus bacterial artificial chromosome |
| BR112017028420A2 (en) * | 2015-06-30 | 2018-08-28 | Ceva Sante Animale | duck enteritis virus and its uses |
| CN105255841A (en) * | 2015-11-26 | 2016-01-20 | 中国兽医药品监察所 | Establishment method and application of H9 subtype avian influenza recombined duck enteritis virus rDEVdeltagE-H9 strain |
| CN109609549A (en) * | 2018-12-26 | 2019-04-12 | 四川农业大学 | A kind of seamless gene-deleted strain CHv-BAC-G- Δ UL41 of duck plague virus UL41 gene and its construction method |
| CN110184287B (en) * | 2019-05-24 | 2024-01-30 | 华南农业大学 | Method for preparing recombinant virus and application thereof |
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