CN103667351A - Application of recombinant virus vaccine in galliformes poultry by adopting duck virus enteritis virus vaccine strain as vector - Google Patents

Abstract

The invention provides an application of living virus vector duck virus enteritis virus (DEV) attenuated vaccine vector in galliformes poultry. The DEV attenuated vaccine vector can be used for expressing relevant genes of the galliformes poultry pathogeny and can carry exogenous genes to be expressed in the galliformes poultry, and the protection for the pathogeny can be well induced in an immune animal body. Through the application of the recombinant duck virus enteritis virus vaccine strain (preservation serial number is CCTCC V201125, named as rDEVus78Ha-Re6) for expressing the avian influenza virus haemagglutinin (HA) genes and the recombinant duck virus enteritis virus vaccine strain (preservation serial number is CCTCC V201220, and named as rDEV F) for expressing newcastle disease virus gene F in chicken, the characteristics of the DEV attenuated vaccine vector can be proved.

Description

The application of the recombinant viral vaccine that the duck viral enteritis virus vaccine strain of take is carrier in Galliformes bird

Technical field

The invention belongs to recombinant viral vaccine field, more specifically belong to restructuring duck viral enteritis virus vaccines field.The invention provides a kind of live vector that can use in Galliformes bird; it is duck viral enteritis virus (DEV) attenuated vaccine strain vector; it can express the genes involved of Galliformes bird cause of disease; and can carry this foreign gene and express in Galliformes bird, in immune animal body, induce the good protection for this cause of disease.This research be take respectively and is expressed the restructuring duck viral enteritis virus vaccine strain of avian flu virus hemagglutinin (HA) gene (its deposit number is CCTCC V201125, called after rDEVus78Ha-Re6) and express the restructuring duck viral enteritis virus vaccine strain of F gene of NDV strain (its deposit number is CCTCC V201220, called after rDEV F) be model virus, proved the above characteristic of DEV attenuated vaccine strain.

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 with respect to other simplexvirus, compare less for the research of DEV.Therefore the international virusology classification of Ba Ci committee report is categorized as simplexvirus ^[1], and fail further to classify.2009, Li etc. 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.

At present, the DEV attenuated vaccine strain that China is used is in the contact of chicken embryo, to go down to posterity and succeed in developing the sixties in last century.Only for Anseriformes bird DEV such as preventing ducks.Use extensively, safe and effective ^[3].So far there are no report, it can be used as live vector and expresses the Galliformes bird transmissible disease pathogeny protective antigens such as chicken, and can be used for preventing Galliformes bird transmissible disease.

Since successfully utilizing the TK gene that vaccinia virus 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, existing a large amount of correlative study report.In common livestock and poultry simplexvirus disease, as to take Pseudorabies virus (PRV) be carrier, inserts the foreign genes such as E2 of CSF respectively, and use its immune swine in the genes such as gD, gE, gG and TK, obtained good immune effect ^[5-11].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 ^[12-14].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% ^[15-17]; 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 ^[18,19]; 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 ^[20].

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 current China between the genomic US7 of DEV and US8 gene for vaccine strain hemagglutinin (HA) gene of bird flu prevention, with its immune specific pathogen free duck (SPF duck), can make SPF duck produce the good antibody to anti influenza ^[21].Further research is found, with its immune chicken, can induce good HI antibody.Challenge test shows, latter two weeks of immunity, and immune chicken is protection completely all.Proof DEV attenuated vaccine strain can carry out a property crossed in chicken body to be copied, and can also express by foreign gene-carrying, makes foreign protein induce good antibody.

Summary of the invention

The invention provides a kind of live vector that can use in Galliformes bird; it is DEV attenuated vaccine strain vector; it can express the genes involved of Galliformes bird cause of disease; and can carry this foreign gene and express in Galliformes bird, in immune animal body, induce the good protection for this cause of disease.The present invention is respectively by expressing restructuring duck viral enteritis virus vaccine strain (its deposit number above characteristic of DEV attenuated vaccine strain vector that has been the restructuring duck viral enteritis virus vaccine strain (its deposit number is CCTCC V201220, called after rDEV F) of CCTCC V201125 (called after rDEVus78Ha-Re6) and the expression F gene of NDV strain application attestation in chicken of avian flu virus hemagglutinin (HA) gene.

Particularly, the inventor utilizes recombinant clone technology, the gene fragment SV40-HA (SEQ ID NO:1) that will comprise respectively avian flu virus hemagglutinin (HA) gene and SV40 promoter sequence, and the gene fragment SV40-F that comprises F gene of NDV strain and SV40 promoter sequence (SEQ ID NO:2) is inserted in the US7 and the transcribed spacer between US8 gene (nucleotide sequence of the transcribed spacer between US7 and US8 gene is shown in SEQ ID NO:6) of DEV, build respectively and obtain the clay pFOS5us78 SV40 HA that inserts SV40-HA expression cassette between US7 and US8 gene, with the clay pFOS5us78 SV40 F that inserts SV40-F expression framework.And rescue obtains the restructuring duck viral enteritis virus vaccine strain CCTCC V201125 that expresses avian flu virus hemagglutinin (HA) gene, (this restructuring duck viral enteritis virus vaccine strain was preserved in Chinese Typical Representative culture collection center (CCTCC to called after rDEVus78Ha-Re6 on July 15th, 2011, Wuhan, China, Wuhan University)); With the restructuring duck viral enteritis virus vaccine strain CCTCC V201220 that expresses F gene of NDV strain, called after rDEV F (is preserved in Chinese Typical Representative culture collection center (CCTCC on May 24th, 2012, Wuhan, China, Wuhan University, postcode: 430072)).The restructuring duck viral enteritis virus vaccine strain CCTCC V201125 that expresses avian flu virus hemagglutinin (HA) gene applies for a patent on September 26th, 2011, application number is 201110286743.6, which describes in detail the construction process of the restructuring duck viral enteritis virus vaccine strain of expressing avian flu virus hemagglutinin (HA) gene, and proved that this recombinant vaccine strain can prevent the transmissible disease being caused in duck, goose and other Anseriformes bird by duck viral enteritis virus and avian influenza virus effectively.The construction process of the restructuring duck viral enteritis virus vaccine strain CCTCC V201220 of expression F gene of NDV strain is referring to following embodiment 4.

By the understanding of state of the art, DEV attenuated vaccine strain vector is the virus vector that is structured in the widespread use of the recombinant viral vaccine strain that has prophylactic activity in the Anseriformes bird that comprises duck goose, due to the species difference between Anseriformes and Galliformes, also nobody is used for being structured in by DEV attenuated vaccine strain vector the recombinant viral vaccine strain that Galliformes has prophylactic activity at present.Yet the inventor finds that surprisingly the restructuring duck viral enteritis virus vaccine strain of above-mentioned expression avian flu virus hemagglutinin (HA) gene by DEV attenuated vaccine strain vector construction also has effective active in Galliformes bird in the follow-up study of the restructuring duck viral enteritis virus vaccine strain about expression avian flu virus hemagglutinin (HA) gene.On this basis, the inventor further studies and finds that duck enteritis virus (DEV) attenuated vaccine strain vector also can prevent the recombinant viral vaccine strain of Galliformes poultry disease for building, and then has completed the present invention.Simultaneously, contriver has further built the restructuring duck viral enteritis virus vaccine strain rDEV F that expresses F gene of NDV strain, and in chicken, carried out system evaluation, further prove that DEV attenuated vaccine strain vector can be used for being structured in and in Galliformes, has the strain of the recombinant viral vaccine of prophylactic activity.

In one embodiment of the invention; the invention provides duck viral enteritis virus (DEV) attenuated vaccine strain vector for building the application of the recombinant viral vaccine strain that can prevent Galliformes poultry disease; the genes involved that this duck viral enteritis virus (DEV) attenuated vaccine strain vector can be expressed Galliformes bird cause of disease; and can in Galliformes bird, express by foreign gene-carrying, in immune animal body, induce the good protection for this cause of disease.Wherein said duck viral enteritis virus attenuated vaccine strain can be CVCC AV1222.External source Galliformes pathogenic genes is generally inserted into the US7 of duck viral enteritis virus (DEV) attenuated vaccine strain vector and the transcribed spacer between US8 gene, those skilled in the art can select suitable insertion point according to prior art and experience, utilize the method for duck viral enteritis virus attenuated vaccine strain construction of recombinant virus can be referring to the construction process of CCTCC V201125 (can referring to patent application 201110286743.6).

By detection, express the restructuring duck viral enteritis virus vaccine strain of avian flu virus hemagglutinin (HA) gene and the prophylactic activity proof duck viral enteritis virus vaccine strain above feature of the restructuring duck viral enteritis virus vaccine strain of expression F gene of NDV strain in Galliformes bird.The restructuring duck viral enteritis virus vaccine strain deposit number of expressing avian flu virus hemagglutinin (HA) gene is CCTCC V201125, called after rDEVus78Ha-Re6, it is preserved in Chinese Typical Representative culture collection center (CCTCC on July 15th, 2011, Wuhan, China, Wuhan University); The restructuring duck viral enteritis virus vaccine strain deposit number of expressing F gene of NDV strain is CCTCC V201220, called after rDEV F (is preserved in Chinese Typical Representative culture collection center (CCTCC on May 24th, 2012, Wuhan, China, Wuhan University, postcode: 430072)).In the transcribed spacer (SEQ ID NO:6) of the restructuring duck viral enteritis virus vaccine strain CCTCC V201125 of described expression avian flu virus hemagglutinin (HA) gene between the genomic US7 of duck enteritis virus DEV and US8 gene, insert the gene fragment SV40-HA (SEQ ID NO:1) that comprises avian flu virus hemagglutinin HA gene and SV40 promoter sequence.In the transcribed spacer (SEQ ID NO:6) of the restructuring duck viral enteritis virus vaccine strain CCTCC V201220 of described expression F gene of NDV strain between the genomic US7 of duck enteritis virus DEV and US8 gene, insert the gene fragment SV40-F (SEQ ID NO:2) that comprises F gene of NDV strain and SV40 promoter sequence.

Therefore, in another embodiment of the invention, the invention provides the application of restructuring duck viral enteritis virus vaccine strain CCTCC V201125 in the recombinant viral vaccine of the bird flu disease for the preparation of in prevention Galliformes bird of expressing avian flu virus hemagglutinin HA gene.The present invention also provides the application of the restructuring duck viral enteritis virus vaccine strain CCTCC V201220 that expresses F gene of NDV strain in the recombinant viral vaccine of the newcastle disease disease for the preparation of in prevention Galliformes bird.

In a preferred embodiment of the invention, except avian influenza virus gene, newcastle disease virus gene, other cause of disease of described Galliformes bird, other can infect the various diseases cause of disease of Galliformes bird to comprise bursa of Fabricius in poultry virus, avian infectious bronchitis virus etc.For example, can be avian flu virus hemagglutinin HA gene, the F gene of Avian pneumo-encephalitis virus or HN gene, the VP2 gene of bursal disease virus etc.

In one embodiment of the invention; the invention provides a kind of live vector that can use in Galliformes bird; DEV attenuated vaccine strain vector; it can express the genes involved of Galliformes bird cause of disease; and can carry this foreign gene and express in Galliformes bird, in immune animal body, induce the good protection for this cause of disease.By expressing the restructuring duck viral enteritis virus vaccine strain of avian flu virus hemagglutinin (HA) gene and the above feature of restructuring duck viral enteritis virus vaccine strain proof duck viral enteritis virus vaccine strain of expression F gene of NDV strain.And develop the new vaccine using with this carrier in Galliformes bird.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 DEV attenuated vaccine strain, can be used as carrier development of new vaccine applies in Galliformes bird, be effective to the transmissible disease that prevention is caused by the relevant cause of disease of Galliformes bird, for example, be effective to newcastle disease disease that the fabricius bursa that prevents bursal disease virus to cause, the bird flu being caused by avian influenza virus etc. etc., Avian pneumo-encephalitis virus cause etc.Therefore, the invention provides following:

1. duck viral enteritis virus attenuated vaccine strain is for the preparation of the application of the recombinant viral vaccine strain of disease in prevention Galliformes bird, and wherein said duck viral enteritis virus attenuated vaccine strain is as carrying the expression vector that exogenous Galliformes bird pathogenic genes is expressed in Galliformes bird.

2. the application of the 1st, wherein said duck viral enteritis virus attenuated vaccine strain is CVCC AV1222.

3. the application of the 1st, wherein said exogenous Galliformes bird pathogenic genes is selected from the gene of the Galliformes bird common transmittable disease pathogens such as avian influenza virus, Avian pneumo-encephalitis virus, bursa of Fabricius in poultry virus or avian infectious bronchitis virus.

4. the application of the 3rd, wherein said exogenous Galliformes bird pathogenic genes is selected from the F gene of avian flu virus hemagglutinin HA gene, Avian pneumo-encephalitis virus or the VP2 gene of HN gene or bursal disease virus etc.

5. the application of the 1st, the disease in wherein said Galliformes bird is selected infectious bronchitis that bird flu, the fabricius bursa being caused by bursal disease virus, the Newcastle disease being caused by Avian pneumo-encephalitis virus, avian infectious bronchitis virus that free avian influenza virus causes cause etc.

6. a restructuring duck viral enteritis virus vaccine strain of expressing F gene of NDV strain, its deposit number is CCTCC V201220.

7. the application of the restructuring duck viral enteritis virus vaccine strain CCTCC V201125 of expression avian flu virus hemagglutinin HA gene in the recombinant viral vaccine of the bird flu disease for the preparation of in prevention Galliformes bird.

8. the application of the restructuring duck viral enteritis virus vaccine strain CCTCC V201220 of expression F gene of NDV strain in the recombinant viral vaccine of the newcastle disease disease for the preparation of in prevention Galliformes bird.

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-Hind III 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 HA (A) and pSI F (B) plasmid map;

Fig. 7: pENTR sv40-ha (A) and pENTR sv40-F (B) plasmid map;

Fig. 8: pFOS5us78 SV40 HA (A) and pFOS5us78 SV40F (B) clay collection of illustrative plates;

Fig. 9: duck viral enteritis virus infection clones rescue recombinant virus schematic diagram;

Figure 10: recombinant virus HA gene and the F gene expression immunofluorescence detected result figure in CEF;

Figure 11: after immune recombinant virus, induce the situation of HI antibody in SPF chicken body; " ◆ ": rDEVus78Ha-Re6 immune group, " ◇ ": DEV immune group, " zero ": PBS control group.

Figure 12: SEQ ID NO:1:SV40-HA expresses framework, wherein italic underlines part for the avian influenza hemagglutinin HA gene from H5N1 type bird flu strain A/duck/Guangdong/S1322/2010 (H5N1).

Figure 13: SEQ ID NO:2:SV40-F expresses framework, wherein italic underlines part for from Avian pneumo-encephalitis virus LaSota strain F gene.

Sequence description

SEQ ID NO:1:SV40-HA expresses framework, comprises the gene fragment of A/duck/Guangdong/S1322/2010 (H5N1) avian flu virus hemagglutinin HA gene and SV40 promoter sequence;

SEQ ID NO:2:SV40-F expresses framework, and wherein italic underlines part for from Avian pneumo-encephalitis virus LaSota strain F gene.

SEQ ID NO:3: " RfA " gene, wherein gene is aatR1-paraxin-ccdB-aatR2;

SEQ ID NO:4:RfKan gene;

SEQ ID NO:5:A/duck/Guangdong/S1322/2010 (H5N1) avian flu virus hemagglutinin HA gene;

The nucleotide sequence of the transcribed spacer between SEQ ID NO:6:US7 and US8 gene;

SEQ ID NO:7: the F gene of NDV strain that derives from the LaSota strain of Avian pneumo-encephalitis virus.

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 pCC1 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 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-Sbf I-Pme I joint.From these 250 clones, choose many groups for saving the 5 clay combinations of DEV.Fse I-Sbf I-Pme I 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 ^[22], 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 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 ^[23], 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 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.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 DEV DNA fragmentation 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:6).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 respectively the structure that SV40-HA expresses the recombination mutation clay of framework (SEQ ID NO:1) and SV40-F expression framework (SEQ ID NO:2)

Based on above-described embodiment 1-3 result, in transcribed spacer (SEQ ID NO:6) 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, replace and insert respectively SV40-HA and SV40-F and express framework (nucleotide sequence that SV40-HA and SV40-F express framework is respectively SEQ ID NO:1 and SEQ ID NO:2, wherein comprise SV40 promotor, referring to Figure 12 and Figure 13, wherein italic underlines part and derives from respectively A/duck/Guangdong/S1322/2010 (H5N1) avian flu virus hemagglutinin HA gene (SEQ ID NO:5)) and derive from the F gene of NDV strain (SEQ ID NO:7) of the LaSota strain of Avian pneumo-encephalitis virus.Building 2 recombination mutation clays, is respectively pFOS5us78 SV40 HA and pFOS5us78 SV40 F (collection of illustrative plates of this sudden change clay as shown in Figure 8, its structure pattern can 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 FOS5us78 SV40 HA clay is summarized as follows:

4.1 the structure of pUC 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:3) providing in the Gateway Vector Conversion System with One Shot ccdB Survival of Invitrogen company 2 T1 Competent 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

Primer title	Sequence
		tR1	5’-GCG TCT AGA ATC ACA AGT TTG TAC AAA AAA GCT GAA CG-3’(XbaI)
tR2	5’-GTG TCC ACA CAT TAT ACG AGC CGG-3’
		P6K1	5’-CCG GCT CGT ATA ATG TGT GGA CAC ATC TCA ACC ATC ATC G-3’
P6K2	5’-CAG CGC GCA AAT ACG CAT ACC GAG CTC GAA TTC GAT GAA-3’
		ccdB1	5’-TCG GTA TGC GTA TTT GCG CGC TG-3’
ccdB2	5’-GCG AAG CTT ATC ACC ACT TTG TAC AAG AAA G-3’(HindIII)

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 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 difference purifying, and using these three fragments jointly as template, using tR1 and ccdB2 as primer, amplification obtains RfKan gene (SEQ ID NO:4), 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) " RfKan " fragment obtaining, obtain pUC ccdB kan, as shown in Figure 3.

The structure of 4.2 pFOS5 us78 Kan ccdB clays:

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 ℃ of 5min-35* (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

4.3 the structure of pENTR 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 of Invitrogen company 2 T1 Competent Cells test kits: 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 4.4 pSI HA and pSI F plasmid:

With the primer pHaO31 shown in table 4, pHa ORF2, and pNDff, the pNDfr avian influenza virus that increases has respectively lacked HA gene (SEQ ID NO:5) and the F gene of NDV strain (SEQ ID NO:7) in alkaline bleach liquor cleavage site.In the H5N1 type bird flu strain from Guangdong separation in 2010, (its detailed name was called A/duck/Guangdong/S1322/2010 (H5N1) to HA gene source, national bird flu reference laboratory by inventor place is preserved, and this laboratory is the mechanism of domestic legal preservation avian influenza virus).F gene source is in Avian pneumo-encephalitis virus LaSota strain, and by Vet Biotechnology, National Key Laboratory preserves.With Not I and Nhe I (purchased from New England Biolabs) enzyme, cut the HA gene and the complete F that lack alkaline bleach liquor cleavage site and be connected in pSI plasmid (purchased from Promega company), successfully build respectively pSI HA and pSI F, 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 GGA AAA AAT AGT GCT CC-3’
pHa ORF2	5’-GAT GGC GGC CGC TTA GAT GCA AAT TCT GCA-3’
		pNDff	5’-AAT GCT AGC CGC CAC CAT GGG CTC CAG ACC TTC TAC-3’
pNDfr	5’-GAT GGC GGC CGC TCA CAT TTT TGT AGT GGC TC-3’

The structure of 4.5 pENTR sv40-ha and pENTR sv40-F plasmid:

With primer entryha1 and the entryha2 shown in table 5, HA gene and F gene increase respectively from the above-mentioned pSI HA successfully constructing and pSI F, and with BamHI restriction enzyme site, be cloned into (BamHI enzyme is purchased from New England Biolabs company) in the pENTR MCS having built, obtain pENTR sv40-ha and pENTR sv40-F, 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 AAA A-3’

[0084]

entryha2

5’-CGA GGA TCC CAG CCC GGA TCC TTA TCG A-3’

The structure of 4.6 pFOS5us78 SV40 HA and pFOS5us78 SV40 F clay:

Use respectively pENTR sv40-ha, pENTR sv40-F and pFOS5us78 Kan ccdB utilize the effect of the Gateway Vector Conversion System with One Shot ccdB Survival of Invitrogen company 2 T1 Competent Cells test kits, make respectively sv40-ha and sv40-F in pENTR sv40-ha and pENTR sv40-F express the kan ccdB gene in framework replacement pFOS5us78 Kan ccdB, thereby obtain the clay pFOS5us78 SV40 HA or the pFOS5us78 SV40 F that in the transcribed spacer between US7 and US8, insert sv40-ha or sv40-F expression cassette, as shown in Figure 8.Particularly, described sv40-ha or sv40-F express the tetranucleotide fragment of the 108th to 111 that framework is replaced the transcribed spacer (SEQ ID NO:6) 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 pFOS5us78 SV40 HA or five cosmid DNAs of pFOS5us78 SV40F (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 ^[22].PFOS1, pFOS2, pFOS3, pFOS4 and pFOS5us78 SV40 HA or pFOS5us78 SV40F 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) ^[22], after transfection 6-9 days, can be observed the appearance that the contracting of cell circle waits typical cytopathic.Save out recombinant virus, respectively called after rDEVus78Ha-Re6 and rDEV F.RDEVus78Ha-Re6 is preserved in Chinese Typical Representative culture collection center (CCTCC, Wuhan, China, Wuhan University) on July 15th, 2011, and deposit number is CCTCC V201125.RDEV F is preserved in Chinese Typical Representative culture collection center (CCTCC, Wuhan, China, Wuhan University, postcode: 430072), deposit number is CCTCC V201220 on May 24th, 2012.

Embodiment 6. expression of recombinant virus HA and F protein immunization Fluorescence Identification

The recombinant virus rDEVus78Ha-Re6 of rescue, rDEV F and parental virus DEV are inoculated respectively inferior to CEF.When appearring in 80% cell, pathology detects respectively the expression of HA and F gene with indirect immunofluorescence method.

Wherein indirect immunofluorescence step is summarized as follows: waited to infect the inferior of rDEVus78Ha-Re6, rDEVF and DEV and occurred pathology for CEF 80% cell, with 4% paraformaldehyde fixed cell 30 minutes, PBS washes 3 times, add and use 1%BSA confining liquid by the anti-HA antibody of the chicken of the dilution proportion of 1: 100, the anti-F antibody of chicken (the national bird flu reference laboratory by inventor place is prepared and preserves), 37 ℃ act on 1 hour.With PBST, wash 3 times each 10 minutes.With the goat-anti chicken igg antibody (purchased from Sigma company) of green fluorescent protein (GFP) mark, at 37 ℃, act on 1 hour.With PBS, wash 3 times, observe and take pictures.

Embodiment 7. blood clottings suppress experiment and detect rDEVus78Ha-Re6 induction antibody (HI antibody) situation of tiring

To recombinate rDEVus78Ha-Re6 and parent DEV respectively by 10 ⁶tCID ₅₀15 of SPF chickens in 1 week age (being provided by Harbin veterinary institute Animal House) are provided, and set up phosphoric acid buffer (PBS) control group, blood sampling weekly, separation of serum, surveys its hemagglutination inhibition antibody (HI antibody).And whether affect SPF chicken healthy state after observing recombinant virus immunity simultaneously.

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/Guangdong/S1322/2010 (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 (You Zhe is prepared research department according to a conventional method).Observations.

Embodiment 8.rDEVus78Ha-Re6 experimentation on animals

To recombinate rDEVus78Ha-Re6 and parent DEV respectively by 10 ⁶ tCID ₅₀10 of immunity SPF chickens in 1 week age (each 5 of male and females, approximately 100 grams of body weight are provided by Harbin veterinary institute Animal House), and set up phosphoric acid buffer (PBS) control group.Two weeks Zhou Houyong 100DLD ₅₀a/duck/Guangdong/S1322/2010 (H5N1) influenza virus (the national bird flu reference laboratory by inventor place is preserved, and this laboratory is the mechanism of domestic legal preservation avian influenza virus) attack.Attack the rear Continuous Observation of poison 14 days, observe and respectively organize test chicken morbidity and death condition.And within 3,5,7 days, adopt larynx, cloaca swab after attacking poison, for titration toxin expelling situation.

Embodiment 9.rDEV F experimentation on animals

To recombinate rDEV F and parent DEV respectively by 10 ⁶ tCID ₅₀10 of immunity SPF chickens in 1 week age (each 5 of male and females, approximately 100 grams of body weight are provided by Harbin veterinary institute Animal House), and set up phosphoric acid buffer (PBS) control group.After two weeks with the strong malicious F48E9 of NDV (purchased from Vet Biotechnology National Key Laboratory) with 10 ⁵eLD ₅₀dosage is attacked through intramuscular injection approach, observes statistics morbidity and death condition 2 weeks.

Result

1.HA genetic expression detects

With the 5th generation recombinant virus rDEVus78Ha-Re6 and rDEV F, infect after CEF, when pathology appears in 80% cell, with indirect immunofluorescence method, detect respectively the expression of HA gene and F gene.Its result as shown in figure 10.Recombinant virus can be good in CEF expression HA and F albumen.

The 2.rDEVus78Ha-Re6 recombinant virus HI antibody extended period tests

Restructuring rDEVus78Ha-Re6 and parent DEV are respectively by 10 ⁶tCID ₅₀15 of the SPF chickens in immune 1 week age, its HI antibody is surveyed in blood sampling weekly.Its result as shown in figure 12.During 1 week age, all test chicken HI antibody is 0.After immunity, the HI antibody of one week most of chicken turns sun, and mean value is 1.7, and on average tiring of second week and the 3rd week is respectively 3.3 and 6.3.After this, all chicken HI antibody all maintains a higher level, until observe immunity latter 6 weeks, its HI antibody mean value is all greater than 6.And be 0 with parent DEV immune group and PBS group chicken HI antibody.At whole viewing duration, have no rDEVus78Ha-Re6 and parent DEV immune group chicken morbidity or dead.

3.rDEVus78Ha-Re6 recombinant virus results of animal

Challenge test result is as shown in table 6.Immune group chicken has no morbidity and dead after attacking poison, and all morbidities successively of DEV vaccine strain immune group and PBS control group chicken, and all in immunity death in latter about 5 days.The titration of virus situation of shouting swab and cloaca swab of 3,5,7 days from attacking poison, immune group is without toxin expelling phenomenon, and DEV vaccine strain immune group and the equal toxin expelling of PBS control group chicken.

Table 6: results of animal table

Note: 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.

4.rDEVF recombinant virus results of animal

Within after rDEV F and DEV immunity 2 weeks, with the strong malicious F48E9 strain of NDV, attack.There is not any morbidity or death after attacking poison in rDEV F immune group, survival rate is respectively whole Mortalities (survival rate 0/10) of 10/10, DEV and PBS control group.

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 vaccine strain CCTCC V201125 of recombinant fowl influenza HA gene, this recombinant vaccine strain is applied for a patent on September 26th, 2011, application number is 201110286743.6, which describes in detail the construction process of the restructuring duck viral enteritis virus vaccine strain of expressing avian flu virus hemagglutinin (HA) gene, and proved that this recombinant vaccine strain can prevent by duck viral enteritis virus and avian influenza virus effectively duck, the transmissible disease causing in goose and other Anseriformes bird.

Meanwhile, it is model virus that the inventor be take rDEVus78Ha-Re6 (CCTCC V201125), proves that it can be for immune chicken, and can in chicken body, induce good HI antibody, and can make immune chicken protect completely.This discovery at home and abroad still belongs to the first time.In order further to verify that the reproducibility of above-mentioned discovery, the inventor have built again the restructuring duck viral enteritis virus vaccine strain CCTCC V201220 that expresses F gene of NDV strain, experimental result shows that it also can be for immune chicken.Above result of study explanation DEV attenuated vaccine strain virus (CVCC AV1222) can carry out necessarily copying of degree in chicken body, and can it be carrier, express the genes involved of Galliformes bird cause of disease, in order to development of new vaccine, and for preventing the disease of Galliformes bird.

By to model virus recombinant virus rDEVus78Ha-Re6 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 the HA gene expression construct that SV40 is promotor.This recombinant virus is good representation HA gene in vitro.With it, chicken is carried out after primary immune response, 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 V201125 (its called after rDEVus78Ha-Re6) of the expression Avian Influenza Virus HA Gene that the present invention obtains can cause for the preparation of the avian influenza virus in prevention Galliformes bird, the transmissible disease causing Galliformes bird for birds flu-preventing virus.And, the vaccine of the transmissible disease that the restructuring duck viral enteritis virus vaccine strain CCTCC V201220 of expression F gene of NDV strain also can cause for the preparation of the avian influenza virus in prevention Galliformes bird, for preventing the newcastle disease disease of Galliformes bird.

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 (8)

1. duck viral enteritis virus attenuated vaccine strain is for the preparation of the application of the recombinant viral vaccine strain of prevention Galliformes poultry disease, and wherein said duck viral enteritis virus attenuated vaccine strain is as carrying the expression vector that exogenous Galliformes bird pathogenic genes is expressed in Galliformes bird.

2. the application of claim 1, wherein said duck viral enteritis virus attenuated vaccine strain is CVCC AV1222.

3. the application of claim 1, wherein said exogenous Galliformes bird pathogenic genes is selected from the gene of the Galliformes bird common transmittable disease pathogens such as avian influenza virus, Avian pneumo-encephalitis virus, bursa of Fabricius in poultry virus or avian infectious bronchitis virus.

4. the application of claim 3, wherein said exogenous Galliformes bird pathogenic genes is selected from the F gene of avian flu virus hemagglutinin HA gene, Avian pneumo-encephalitis virus or the VP2 gene of HN gene or bursal disease virus etc.

5. the application of claim 1, the disease in wherein said Galliformes bird is selected infectious bronchitis that bird flu, the fabricius bursa being caused by bursal disease virus, the Newcastle disease being caused by Avian pneumo-encephalitis virus, avian infectious bronchitis virus that free avian influenza virus causes cause etc.

6. a restructuring duck viral enteritis virus vaccine strain of expressing F gene of NDV strain, its deposit number is CCTCC V201220.

7. the application of the restructuring duck viral enteritis virus vaccine strain CCTCC V201125 of expression avian flu virus hemagglutinin HA gene in the recombinant viral vaccine of the bird flu disease for the preparation of in prevention Galliformes bird.

8. the application of the restructuring duck viral enteritis virus vaccine strain CCTCC V201220 of expression F gene of NDV strain in the recombinant viral vaccine of the newcastle disease disease for the preparation of in prevention Galliformes bird.

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