CN110331135A - The recombinant herpesvirus of turkeys candidate vaccine strain and preparation method of expressing gene VII type newcastle disease virus fusion protein - Google Patents

Abstract

The invention belongs to recombinant vaccine technical fields, and in particular to a kind of the recombinant herpesvirus of turkeys candidate vaccine strain and preparation method of expressing gene VII type newcastle disease virus fusion protein.The recombinant herpesvirus of turkeys candidate vaccine strain of the expressing gene VII type newcastle disease virus fusion protein is named as rHVT-NDV-VII-F, its deposit number is CCTCC NO:V201904.The recombinant virus energy expressing gene VII type NDV F protein exactly matches with current domestic popular NDV street strain genotype, can be used as the recombinant vaccine Candidate Strain for preventing NDV infection.

Description

The recombinant herpesvirus of turkeys of expressing gene VII type newcastle disease virus fusion protein is waited Select vaccine strain and preparation method

Technical field

The invention belongs to recombinant vaccine technical fields, and in particular to a kind of expressing gene VII type newcastle disease virus The recombinant herpesvirus of turkeys construction method of fusion protein and the recombinant virus of building.

Background technique

Newcastle disease (Newcastle disease, ND) be by newcastle disease virus (Newcastle disease virus, NDV birds caused by) are acute, strong, highly contagious disease.NDV is sub-thread, non-segmented negative, the strand RNA disease for having cyst membrane Poison, genome 3'-NP-P-M-F-HN-L-5', successively encode nucleocapsid protein (NP), phosphoprotein (P), stromatin (M), Fusion protein (F), hemagglutinin-neuraminidase albumen (HN) and large protein (L).In addition, can go out during P genetic transcription Existing rna editing, generates non-structural protein V and W.F protein is located on the cyst membrane of virus, is the major structural protein of virus, can Body is induced to generate neutralizing antibody.The molecular basis of NDV Virulence Difference depends mainly on the protease cracking of F protein precursor (F0) The amino acid sequence in site (112-117 amino acids), the amino acid sequence of different type strain have very big difference, velogen strain It is¹¹²R/K-R-Q-K/R-R-F¹¹⁷, low virulent strain is¹¹²G/E-K/R-Q-G/E-R-L¹¹⁷。

Only one serotype of NDV, but genotype is more, it is homologous according to the specific restriction enzyme mapping of NDV and F gene nucleotide NDV, can be divided into two class of Class I and Class II by the feature of property sequence.Class I genoid group overall length 15198bp, can be into One step is divided into 9 genotype (1-9 type), is to separate from wild aquatic bird or from live-bird trade market, separation strains are generally For low virulent strain；Class II genoid group overall length is 15186bp or 15192bp, and domestic and foreign scholars are by Class II class at present 15 kinds of genotype (I-XV) are divided into, are the strains being separated to from poultry and wild bird, isolated strain has low virulent strain and velogen strain. Since mid-term the 1990s, the popular preponderant genotype of China ND is mainly genotype VII NDV strain.

FC126 plants of herpes turkey virus (HVT) is close with marek virus (MDV) in antigenicity, is usually used in chicken horse The prevention of vertical creutzfeldt jakob disease (MD).Since the genome of HVT contains the more Nonessencial region for foreign gene insertion, simultaneously Preservation can be lyophilized again, frequently as the carrier of fowl recombinant viral vaccine.In recent years, main in the NDV velogen strain of China's prevalence It is genotype VII, and widely used La Sota vaccine strain is gene II type.Although La Sota vaccine is to genotype VII NDV strain can provide good clinical protection, but the toxin expelling for the fowl that can not effectively prevent infections.Therefore, it is ground by carrier of HVT The recombinant vaccine to match with NDV prevalence strain genotype is made to be of great significance for the prevention and control of ND.

Currently, having there is some buildings about the recombinant virus for expressing NDV F protein using HVT as carrier.Chris's enlightening It is replicated between nonessential UL45-UL46 Deng recombinating NDV F protein to HVT, the recombinant virus of building expression NDV F protein； And F gene is inserted into HVT and replicates the nonessential area UL45-UL46 by Shi Hua company, France, constructs recombinant virus rHVT-ND-IBD； Verstegen co-expresses the recombinant virus that NDV clones -30 low virulent strain F proteins and IBDV VP2 albumen by vector construction of HVT； The amino acid mutation of VIId hypotype NDV F gene cracking site is low virulent strain by Bi Jianmin, and it is non-to be then inserted into HVT duplication The required area UL55, constructs recombinant virus rHVT-GB-NDV F；Bi Jianmin etc. by NDV F gene be inserted into HVT replicate it is nonessential The area US10 and SORF3, the recombinant virus of building expression NDV F protein；The bacterial artificial chromosome based on HVT such as Li Yongqing (BAC), NDV F gene is inserted into HVT and replicates the nonessential area US1 and US10, construct recombinant virus rHVT-VP2-F.Originally it grinds Study carefully the insertion point using the US2 Nonessencial region of HVT as foreign gene, it is complete to construct expressing gene VII type NDV respectively The recombinant virus of the genotype VII NDVF albumen of the recombinant virus and expression deletion transmembrane region of F protein.

Summary of the invention

The object of the present invention is to provide a kind of recombinant turkey blister sores of expressing gene VII type newcastle disease virus fusion protein Malicious candidate vaccine strain.

The recombinant herpesvirus of turkeys candidate vaccine of expressing gene VII type newcastle disease virus fusion protein of the present invention Strain, is named as rHVT-NDV-VII-F, its deposit number is CCTCC NO:V201904.

The present invention also provides the preparation methods of the rHVT-NDV-VII-F, comprising the following steps:

(1) it using 2014 strain genome of genotype VII NDV DT as template, is expanded using RT-PCR such as Seq No.1 institute The F gene shown；F gene is inserted into carrier for expression of eukaryonIn-M3, then through PCR amplification containing CMV promoter, TK The F expression casette of ployA terminator and Flag label；

(2) F expression casette is cloned into middle interstitial granules pFR, constructs transfer vector pFR-F；

(3) by pFR-F and rHVT-EGFP genomic DNA cotransfection CEF, recombinant virus rHVT-NDV-VII-F is saved out.

During present invention test, using 2014 strain genome of genotype VII NDV DT as template, expanded using RT-PCR Increase F gene (Seq No.1).F gene is inserted into carrier for expression of eukaryonIn-M3, then through PCR amplification containing CMV starting The F expression casette of son, TK ployA terminator and Flag label.F expression casette is cloned into middle interstitial granules pFR (Wu Yan Great waves wait a kind of recombinant herpesvirus of turkeys strain number of patent application for expressing H9 HA Gene of H 9 Subtype AIV of: CN201710517830.5, application publication number: CN 107142280A), construct transfer vector pFR-F.To the transmembrane region of F gene It is analyzed, display F protein transmembrane region containing there are two is located at the position of 117-139 and 502-525 amino acids；With PFR-F is template, is lacked respectively to two transmembrane regions using the method for Overlap extension PCR, and building missing transmembrane region turns Transfer body pFR- Δ F.Respectively by pFR-F and pFR- Δ F and rHVT-EGFP genomic DNA cotransfection CEF, two plant weights are saved out Group virus rHVT-NDV-VII-F and rHVT-NDV-VII- Δ F.Further preferably gone out according to the test of SPF chicken immune Vaccine effectiveness Recombinant virus rHVT-NDV-VII-F is as candidate vaccine strain.

The present invention provides a kind of recombinant herpesvirus of turkeys structures of expressing gene VII type newcastle disease virus fusion protein Construction method, and utilize two plants of group virus rHVT-NDV-VII-F and rHVT-NDV-VII- Δ F of this method building, two plant weights Group virus carries the complete F gene of NDV (Seq No.1) and missing transmembrane region code sequence under the control of CMV promoter sequence respectively The expression cassette of the F gene △ F (Seq No.2) of column.

The equal expressing gene VII type NDV F protein of two plants of recombinant viruses that the present invention develops, with current domestic popular NDV The exact matching of street strain's genotype, is tested according to SPF chicken immune Vaccine effectiveness, and preferably out rHVT-NDV-VII-F plants of conduct is used for The recombinant vaccine Candidate Strain for preventing NDV infection.

The NDV F protein for the expression of recombinant virus that the present invention develops has Flag label in its C-terminal, facilitates recombinant virus Identification.

Detailed description of the invention

Fig. 1: transfer vector pFR-F building flow chart.

Fig. 2: NDV F gene transmembrane deletion schematic diagram.

Fig. 3: restriction enzyme digestion and electrophoresis figure (the M:DL10000DNA Marker of transfer vector pFR-F and pFR- △ F expression cassette；Swimming lane 1: purpose band after transfer vector pFR- △ F digestion；Swimming lane 2: the purpose band after transfer vector pFR-F digestion).

Fig. 4: transfer vector pFR-F and pFR- △ F transiently transfects Western-blot test for identification F protein expression after CEF Figure (M: albumen Marker；Swimming lane 1: transfer vector pFR-F, a are F protein bands, and b is F1 band after F protein cracking；Swimming lane 2: Transfer vector pFR- △ F, c are missing from transmembrane region F protein band).

(A: transfer carries IFA test for identification F protein expression figure after Fig. 5: transfer vector pFR-F and pFR- △ F instantaneously turns CEF Body pFR- △ F；B: transfer vector pFR-F；C:293T cell negative control).

Fig. 6: recombinant virus constructs flow chart.

Fig. 7: PCR identification recombinant virus rHVT-NDV-VII-F and rHVT-NDV-VII- Δ F target gene is inserted into electrophoretogram (M:DL10000DNA Marker；Swimming lane 1: missing transmembrane region F gene PCR product；Swimming lane 2:F gene PCR product).

Fig. 8: PCR identification recombinant virus rHVT-NDV-VII-F and rHVT-NDV-VII- Δ F destination gene expression box insertion Electrophoretogram (M:DL10000DNA Marker；Swimming lane 1: missing transmembrane region F expression casette PCR product；Swimming lane 2:F gene expression Box PCR product).

Fig. 9: IFA test for identification recombinant virus rHVT-NDV-VII-F and rHVT-NDV-VII- Δ F destination protein expression figure (A: recombinant virus rHVT-NDV-VII- Δ F；B: recombinant virus rHVT-NDV-VII-F；C:HVT negative control).

Figure 10: Western-blot test for identification recombinant virus rHVT-NDV-VII-F and rHVT-NDV-VII- Δ F purpose Protein expression figure (M: albumen Marker；Swimming lane 1: recombinant virus rHVT-NDV-VII-F, a are F protein bands, and b is that F protein is split F1 band after solution；Swimming lane 2: recombinant virus rHVT-NDV-VII- Δ F c is missing from transmembrane region F protein band).

The growth of Figure 11: HVT parent's poison and recombinant virus rHVT-NDV-VII-F and rHVT-NDV-VII- Δ F on CEF Curve graph.

Figure 12: recombinant virus rHVT-NDV-VII-F and rHVT-NDV-VII- Δ F attacks respiratory tract toxin expelling detection in 3 days after poison As a result statistical chart.

Figure 13: recombinant virus rHVT-NDV-VII-F and rHVT-NDV-VII- Δ F attacks alimentary canal toxin expelling detection in 3 days after poison As a result statistical chart.

Figure 14: test chicken survivorship curve figure after poison is attacked.

Recombinant virus rHVT-NDV-VII-F is preserved in China typical culture collection center on January 18th, 2019, protects Hide unit address: Wuhan, China university.Deposit number: CCTCC NO:V201904；Classification naming are as follows: recombinant herpesvirus of turkeys rHVT-NDV-VII-F。

Recombinant virus rHVT-NDV-VII- Δ F is preserved in China typical culture collection center on December 10th, 2018, Depositary institution address: Wuhan, China university.Deposit number: CCTCC NO:V201872；Classification naming are as follows: recombinant turkey blister sore Malicious rHVT-NDV-VII- Δ F.

Specific embodiment

It will be specifically described, but be not to be construed as by the way that the drawings and specific embodiments are further to the present invention below Limiting the scope of the present invention.

Embodiment one: the building of transfer vector pFR-F and pFR- Δ F

1, the clone of newcastle disease virus strain (DT-2014) F gene and identification

1.1 design of primers

Sequence (the GenBank accession number: MN125616 of reference gene VII DT-2014 plants of F genes of type newcastle disease virus； Seq No.1), with the primer of a pair of of specificity of primer5.0 software design, wherein upstream primer includes KOZAK sequence, downstream Primer removes terminator codon.Primer sequence is as follows:

NDF-F(Seq No.3):5'-GCCACCATGGGCTCCAAACTTTCTACC-3'

NDF-R(Seq No.4):5'-TGCTCTTGTAGTGGCTCTCATCT-3'

The building of interstitial granules pM3-F in 1.2

DT-2014 plants of genotype VII NDV sterile 200 μ L of allantois are taken, RNA is extracted, using NDF-F/NDF-R as primer, into Row RT-PCR, carries out 1% agarose gel electrophoresis after reaction, and glue recycles the band of F gene, the product of recycling withThe connection of-M3 (Beijing Quanshijin Biotechnology Co., Ltd) carrier, with universal primer CMV-PA-F (Seq No.5) and NDF-R (Seq No.4) is that primer carries out bacterium solution PCR identification.Correctly positive bacterium solution is sent to the sequencing of Anhui General Corporation for identification. Correct bacterium solution is sequenced, expand bacterium and extracts plasmid, plasmid is named as pM3-F.

2, the building of transfer vector pFR-F

2.1 design of primers

Referring to carrier for expression of eukaryon- M3 sequence uses 5.0 software design pair for amplification expression cassette of Premier Specific primer.F gene cloning is to carrier for expression of eukaryonOn-M3, F expression casette is obtained after PCR amplification Sequence.Expression cassette is made of cytomegalovirus early promoter (CMV), F gene, TK poly A three parts.Design primer is sent Nanjing Genscript Biotechnology Co., Ltd.'s synthesis.

Primer sequence is as follows:

The PCR amplification of 2.2F expression casette

By plasmid pM3-F according to template is used as after 1:1000 times of dilution, with CMV-PA-F (Seq No.5) and CMV-PA-R It (SeqNo.6) is primer, PCR amplification F expression casette carries out 1% agarose gel electrophoresis, glue recycling after reaction The F expression casette band of 2700bp, recovery product are placed -20 DEG C and are saved backup.

The building of interstitial granules pCMV-F in 2.3

Glue recycling F gene expression cassette product with- Blunt (Beijing Quanshijin Biotechnology Co., Ltd) Cloning vector connection, carries out PCR identification to bacterium solution with universal primer M 13F (Seq No.7) and M 13R (Seq No.8).Identification Correct bacterium solution send Anhui General Corporation to be sequenced.The plasmid of building is named as pCMV-F, and saves backup in -20 DEG C.

M 13F (Seq No.7): GTAAAACGACGGCCAGT

M 13R (Seq No.8): CAGGAAACAGCTATGAC

The building of 2.4 transfer vector pFR-F

Plasmid pFR and pCMV-F carries out double digestion with Not I, Avr II.After 1% gel electrophoresis, glue recycles Not I- NDV-F-AvrII segment and Not I-pFR-Avr II segment, segment after the recovery are attached with T4 ligase, building transfer The process of carrier pFR-F is as shown in Figure 1.

The building of 2.5 transfer vector pFR- Δ F

2.5.1 the transmembrane region of forecast analysis F gene

Forecast analysis is carried out to the transmembrane region of F gene, F gene show there are two transmembrane region, be located at 117-139 with Then the position of 502-525 amino acids is designed two pairs of primer pair transmembrane regions and is lacked respectively.

2.5.2 the building of middle interstitial granules pFR- Δ F1

According to the position where transmembrane region 117-139 amino acids, transmembrane deletion primer sequence is as follows:

It is that template carries out Overlap extension PCR amplification with pFR-F (1:1000 times dilutes), takes 5 μ L1% agarose of PCR product Gel electrophoresis, after identification purpose band is correct, 2 μ LDpn I enzymes are to 37 DEG C of degradation 1.5h of remaining PCR product.After PCR degradation Product directly converts DMT competent cell, stands 30min, 42 DEG C of heat shock 45s, ice bath 2min on ice, is added 37 DEG C of nonreactive LB Shake bacterium culture 1h, the culture growth 12h on the solid plate containing ammonia benzyl resistance, picking monoclonal colonies, with CMV-PA-F and CMV-PA-R is that primer bacterium solution PCR identifies that correct purpose band send sequencing, and extracts plasmid, names pFR- Δ F1

The building of 2.6 transfer vector pFR- Δ F

According to the position where transmembrane region 502-525 amino acids, transmembrane deletion design of primers is as follows:

It is that template carries out Overlap extension PCR amplification, specific method with the above primer with pFR- Δ F1 (1:1000 times dilutes) Ibid, it identifies that purpose band correctly send sequencing using CMV-PA-F and CMV-PA-R as primer bacterium solution PCR, and extracts plasmid, construct The process of transfer vector pFR- Δ F is as shown in Figure 2.

The identification of 2.7 transfer vector pFR-F and pFR- Δ F

2.7.1 digestion is identified

Transfer vector pFR-F and pFR- Δ F is subjected to double digestion, 37 DEG C of digestions with restriction enzyme Not I, Avr II 1.5h.After 1% gel electrophoresis, the F expression casette band of the visible about 2700bp of pFR-F and the carrier ribbon of 5800bp, The carrier ribbon of missing transmembrane region the F expression casette band and 5800bp of the visible about 2600bp of pFR- Δ F, as shown in Figure 3.

2.7.2Western-blot test for identification

Transfer vector pFR-F and pFR- Δ F is transiently transfected into 293T cell, continues culture and collects cell afterwards for 24 hours, prepare egg White sample.The expression of Western-blot test for identification F protein, using Flag label monoclonal antibody as primary antibody, horseradish peroxidase mark The sheep anti-mouse antibody of note is secondary antibody, is developed the color with enhanced chemical luminescence method (ECL), observes protein band, as shown in Figure 4.2.4.3 Indirect immunofluorescence assay (IFA) identification

Transfer vector pFR-F and pFR- Δ F is transiently transfected in 48 orifice plates for being covered with 293T cell, laggard for 24 hours in the ranks to connect Immunofluorescent test, wherein using the positive serum of genotype VII NDV as primary antibody, marked by fluorescein isothiocyanate goat-anti chicken antibody For secondary antibody, fluorescence microscopy transfects the fluorescence of cell under the microscope, as shown in Figure 5.

Embodiment two: the rescue and purifying of recombinant virus rHVT-NDV-VII-F and rHVT-NDV-VII- Δ F

1, the expansion culture of recombinant virus rHVT-EGFP

Recombinant virus rHVT-EGFP is saved and is provided by livestock and poultry pestology emphasis open laboratory, the Ministry of Agriculture, Yangzhou University (Wu Yantao waits a kind of recombinant herpesvirus of turkeys strain number of patent application for expressing H9 HA Gene of H 9 Subtype AIV of: CN201710517830.5, application publication number: 107142280 A of CN).Recover the recombinant virus rHVT- being stored in liquid nitrogen EGFP, the cytopathy venom recovered with 10 times of doubling dilutions of M199 culture medium containing 5% class fetal calf serum.Prepare second generation CEF paving Into 6 porocyte culture plates, it is inoculated with diluted virus liquid again after cultivating 2h, every 900 μ L of hole is placed in cell incubator and cultivates, It discards culture solution afterwards for 24 hours, 1% agar culture solution of 45 DEG C of preheatings is added, stand 10min, after waiting agar culture solution to solidify, be inverted It is put into cell incubator and cultivates 3-5d.In fluorescence microscopy microscopic observation and pure green fluorescence plaque is marked, with 20 μ The pancreatin of L0.25% is added drop-wise on the virus plaques of label, is inhaled and is beaten until plaque is digested completely repeatedly with pipette tips.Virus is sucked out Liquid uses 10 times of doubling dilutions of limiting dilution assay again.Preparation second generation CEF is taped against in 96 porocyte culture plates, and each plaque is arranged three Dilution, 100 μ L of inoculation cultivate 3-5d in 96 porocyte culture plates.Microscopically observation select only one in a hole and All with the plaque of fluorescence, and expand culture.

2, the extraction of the genome of rHVT-EGFP recombinant virus

(1) maintaining liquid is discarded, PBS is cleaned three times, and 5mL PK lysate (200mMTris- is added in every bottle of T-75 cell bottle Hcl, pH 8.0；10%SDS；1.5M NaCl；0.5mM EDTA, pH 8.0；The PK of 1mg), 10min is acted on, cell is sucked out It is added in 50mL centrifuge tube, 37 DEG C of incubators crack 1.5h；

(2) balance phenol of 1/2 volume is added into the cell after cracking, is gently mixed by inversion, makes albuminous degeneration；It adds Chloroform/isoamyl alcohol (24:1) solution of 1/2 volume, is gently mixed by inversion, and 8000r/min is centrifuged 15min；

(3) water phase on upper layer is drawn onto new centrifuge tube, add isometric chloroform/isoamyl alcohol (24:1) solution into Row extracting, 8000r/min are centrifuged 15min；

(4) step (3) are repeated 3-4 times, up to no foreign protein is extracted out；The water phase on upper layer is drawn into new centrifuge tube, is added The 3M sodium acetate (pH 5.2) of 1/10 volume mixes gently, and adds the dehydrated alcohol of 2 times of volumes pre-cooling, gently overturns until going out Until the existing cotton-shaped pockets of genome of milky, -20 DEG C of standings 1h, 5000r/min are centrifuged 5min；

(5) the 70% ethyl alcohol 5mL cleaning genome being pre-chilled in advance, 5000r/min centrifugation is added in supernatant is outwelled gently 5min is discarded supernatant, wink from and blot supernatant.

(6) genome is dissolved in TE buffer (pH 8.0), 4 DEG C save backup.

3, the rescue and purifying of recombinant virus rHVT-NDV-VII-F and rHVT-NDV-VII- Δ F

The transfer vector pFR-F and pFR- Δ F of linearisation respectively with rHVT-EGFP genomic DNA coprecipitation of calcium phosphate Method cotransfection CEF cell, the technical principle of homologous recombination is as shown in Figure 6.Rotaring redyeing system is prepared, is stood in super-clean bench 30min.By above-mentioned system, with pipettor, gently pressure-vaccum is mixed after standing, is added in the culture dish of 60mm.Then it places 37 DEG C, 5%CO₂It suffers a shock after cultivating 4h in cell incubator.

Glycerol shock liquid is prepared, culture medium in culture dish is discarded, cleans cell 3 with the M199 culture medium for not adding serum It is secondary, formulated glycerol shock liquid 1mL is added and is stored at room temperature 1min, it is clear with the M199 culture medium for the serum not added again It washes 3 times, M199 growth-promoting media, 37 DEG C, 5%CO is added₂5-7d is cultivated in cell incubator.Fluorescence microscopy microscopic observation picking without The plaque of fluorescence or part without fluorescence plaque, by limiting dilution assay in 96 porocyte culture plates purified virus, directly To an only plaque in a certain hole and plaque is without fluorescence, recombinant virus after purification be named as rHVT-NDV-VII-F and rHVT-NDV-VII-ΔF。

4, the identification of recombinant virus rHVT-NDV-VII-F and rHVT-NDV-VII- Δ F

4.1PCR identifies recombinant virus

The recombinant virus rHVT-NDV-VII-F and rHVT-NDV-VII- Δ F of preliminary purification is seeded in respectively long On full single layer CEF cell, 37 DEG C, 5%CO₂Culture extracts disease after cytopathy up to after 50%, digesting and collecting virus liquid Malicious DNA.Using this DNA as template, with CMV-PA-F (Seq No.5)+CMV-PA-R (Seq No.6) and NDF-F (SeqNo.3)+ Two pairs of primers of NDF-R (Seq No.4) carry out PCR identification respectively, as shown in Figure 7 and Figure 8.And send Nanjing golden its amplified production The sequencing of Si Rui Biotechnology Co., Ltd.

4.2 indirect immunofluorescence assay (IFA) identify recombinant virus

Recombinant virus after purification is inoculated in 96 porocyte plates for being covered with second generation CEF cell, while inoculation is set The CEF cell of HVT parent's poison is as negative control.Using genotype VII newcastle disease virus positive serum as primary antibody and different sulphur The fluorescein-labeled goat-anti chicken fluorescence antibody of cyanic acid carries out indirect immunofluorescence assay as secondary antibody.Fluorescence microscopy is felt under the microscope Clearly green fluorescence can be seen in the cell hole of dye recombinant virus, green fluorescence is not seen by the HVT cell infected, such as Fig. 9 institute Show, it was demonstrated that the F protein of recombinant virus being capable of successful expression.

4.3Western-blot test for identification recombinant virus

Recombinant virus is inoculated in the 6 orifice plates for the second generation CEF cell completed, after lesion occurs in 90% cell Harvest cell simultaneously prepares protein sample.NDV F protein can be expressed with Western-blot detection recombinant virus.In test with Flag label monoclonal antibody is as primary antibody, and the sheep anti-mouse antibody of horseradish peroxidase-labeled is secondary antibody, with enhanced chemical luminescence method (ECL) it develops the color, the results show that recombinant virus rHVT-NDV-VII-F, which corresponds to swimming lane, can be observed two specific band sizes point Not about 55kDa and 60kDa；Recombinant virus rHVT-NDV-VII- Δ F, which corresponds to swimming lane, can be observed a specific band size about 60kDa, as shown in Figure 10.

5, growth curve of the recombinant virus on CEF

2 plants of recombinant viruses and parent HVT are trained with the dose inoculation of 100PFU to the 6 hole cells for being covered with single layer CEF respectively It supports in plate, each strain is inoculated with 6 holes, 3 plants of poison 3 tissue culture plates of total inoculation.37 DEG C, 5%CO₂Under the conditions of trained It supports.Respectively the 0h after virus infection, for 24 hours, 48h, 72h, 84h and 96h digestion collect cell.The cell of collection is placed in 2mL EP pipe in M199 culture medium be settled to 2mL, 100 μ L virus liquids are drawn after mixing well and carry out 10 times of doubling dilution (altogether Count 8 dilution gradients), it is inoculated into 24 orifice plates with single layer CEF cell respectively, sets 37 DEG C, 5%CO₂Incubator culture 4- 5d carries out plaque counting, measures the virus titer of each time point, goes out recombinant virus and parent using GraphPad Software on Drawing Growth curve of the HVT in CEF analyzes growth characteristics of the recombinant virus in CEF, such as Figure 11.

Embodiment three: the immune efficacy evaluation of recombinant virus rHVT-NDV-VII-F and rHVT-NDV-VII- Δ F

1, the Immunoprotection test of recombinant virus

Experimental design is divided into 5 groups, and the grouping of all test chickens is put in isolator and raises by 20 SPF chickens of every group of selection It supports.Recombinant virus rHVT-NDV-VII-F immune group and rHVT-NDV-VII- Δ F immune group, in 1 age in days, neck is subcutaneously injected Corresponding recombinant virus, injection dosage are 5000 plaque forming units.La Sota vaccine immunity group then in 7 age in days of chicken, connects The La Sota vaccine of 1 plumage part of kind；It is nonimmune to attack malicious group and be inoculated with PBS respectively in 1 age in days and 7 ages in days；Setting is not inoculated with any simultaneously The blank control group of vaccine.In addition to blank control group, other each groups are in 21 age in days of chicken, by NDV velogen strain with 10⁵EID₅₀Agent Amount is carried out attacking poison with the mode of collunarium eye droppings.See Table 1 for details.

Immunoprotection test of table 1rHVT-NDV-VII-F and rHVT-NDV-VII- the Δ F to NDV

2, toxin expelling monitors

The 3rd day after attacking poison, 10 chickens are selected at random from each test group, acquire oropharynx and cloaca cotton swab sample respectively Product, extract the RNA in sample and reverse transcription is cDNA, and the toxin expelling situation of each group is detected by absolute fluorescence quantifying PCR method.Knot Fruit is as shown in Figures 12 and 13, and the nonimmune copy number for attacking virus cDNA in poison group oropharynx and cloaca cotton swab sample is significantly higher than two Plant weight group virus immunity group (P < 0.001)；Recombinant virus rHVT-NDV-VII-F immune group oropharynx and cloaca cotton swab sample disease The copy number of malicious cDNA is substantially less than La Sota vaccine immunity group (P < 0.001)；Recombinant virus rHVT-NDV-VII- Δ F is immune The copy number of virus cDNA is lower than La Sota vaccine immunity group (P < 0.01) in group oropharynx and cloaca cotton swab sample.To sum up institute It states, attacks shedding virus of the two plant weight group virus immunity groups in respiratory tract and alimentary canal after poison and be substantially less than and nonimmune attack malicious group.La The shedding virus of Sota vaccine immunity group detection attacks malicious group and is higher than two plant weight group virus immunity groups lower than nonimmune, and recombinant virus The shedding virus of rHVT-NDV-VII-F immune group is lower than recombinant virus rHVT-NDV-VII- Δ F immune group

3, survivorship curve and protective rate of the different tests group after attacking poison

21 ages in days of observation attack the state of the test chicken group chicken after poison daily, are drawn according to test chicken group's death condition of record Survivorship curve processed, as shown in figure 14.2 statistical data of table shows, La Sota vaccine immunity group up to 90% protective rate；Recombination Viral rHVT-NDV-VII-F immune group has 40% protective rate.

The measurement of DT-2014 strain Vaccine effectiveness is attacked after 2 each group chicken immune of table

According to immuning effect test as a result, recombinant virus rHVT-NDV-VII-F can provide DT-2014 plants of NDV velogen strain Better immunoprotection.

Sequence table

<110>Yangzhou University

<120>the recombinant herpesvirus of turkeys candidate vaccine strain of expressing gene VII type newcastle disease virus fusion protein and preparation Method

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1659

<212> DNA

<213>newcastle disease virus (Newcastle disease virus)

<400> 1

atgggctcca aactttctac caggatccca gtacctctaa tgctaatcac tcggattatg 60

ctgacattga gctgcatccg tctgacaagc tctcttgacg gcaggcccct tgcagctgca 120

ggaattgtag taacgggaga taaggcagtc aatgtataca cctcgtctca gacagggtca 180

atcatagtca agttgctccc gaatatgccc agagataagg aggcatgtgc aagagcccca 240

ctggaggcat ataacagaac actgactact ctgctcactc ctcttggtga ctccatccgc 300

aagatccaag ggtctgtatc cacgtccgga ggaaggagac aaagacgttt tataggtgct 360

gttattggca gtgtagctct tggggttgca acagcggcac agataacagc agctgcggcc 420

ctgatacaag ccaaacagaa tgccgccaac atcctccggc ttaaggagag cattgctgca 480

accaatgaag ctgtgcatga agtcaccgac ggattatcac aactatcagt ggcagttggg 540

aagatgcagc agtttgtcaa tgaccagttt aataatacgg cgcgagaatt ggactgcata 600

aaaatcacac aacaggtcgg tgtagaactc aacctatacc taactgaatt aactacagta 660

ttcgggccac agatcacctc ccctgcatta actcagctga ccatccaggc actttataat 720

ttagctggtg gcaatatgga ctacttatta actaagttag gtataggaaa caatcaactc 780

agctcgttaa ttggtagcgg cctgatcact ggttacccta tactgtatga ctcacatact 840

caactcttgg gcatacaagt aaatctgccc tcagtcggga acttaaataa tatgcgtgcc 900

acctatttgg agaccttatc tgtaagtaca accaaaggat atgcctcagc actagtcccg 960

aaagtagtga cacaagttgg ttctgtgata gaagagcttg acacctcata ctgtatagag 1020

tccgatctgg atttatattg tactagaata gtgacatttc ccatgtcccc aggtatttat 1080

tcctgtttga gcggcaacac atcagcctgc atgtattcaa agactgaagg cgcactcact 1140

acgccatata tggcccttag aggctcagtt attgccaatt gtaagataac aacgtgcaga 1200

tgtacagacc ctcctggtat catatcgcaa aattacggag aagctgtatc cctgatagat 1260

agacattcgt gtaatgtctt atcgttagac ggaataactc tgaggctcag tggggaattt 1320

gatgcaactt atcaaaagaa catctcaata ctagattctc aggtcatcgt gacaggcaat 1380

cttgatatat caactgaact tggaaacgtc aacaattcaa tcagcaatgc cttggatagg 1440

ttggcagaaa gcaacagcaa actagaaaaa gtcaatgtca gactaactag cacatccgct 1500

ctcattacct atattgttct aactgtcatt tccctaattt tcggtgcact tagtctggct 1560

ttagcgtgtt acctgatgta caaacagaag gcacaacaaa agaccttgct atggcttggg 1620

aataataccc tcgatcagat gagagccact acaagagca 1659

<210> 2

<211> 1521

<212> DNA

<213>newcastle disease virus (Newcastle disease virus)

<400> 2

atgggctcca aactttctac caggatccca gtacctctaa tgctaatcac tcggattatg 60

ctgacattga gctgcatccg tctgacaagc tctcttgacg gcaggcccct tgcagctgca 120

ggaattgtag taacgggaga taaggcagtc aatgtataca cctcgtctca gacagggtca 180

atcatagtca agttgctccc gaatatgccc agagataagg aggcatgtgc aagagcccca 240

ctggaggcat ataacagaac actgactact ctgctcactc ctcttggtga ctccatccgc 300

aagatccaag ggtctgtatc cacgtccgga ggaaggagac aaagacgtgc cctgatacaa 360

gccaaacaga atgccgccaa catcctccgg cttaaggaga gcattgctgc aaccaatgaa 420

gctgtgcatg aagtcaccga cggattatca caactatcag tggcagttgg gaagatgcag 480

cagtttgtca atgaccagtt taataatacg gcgcgagaat tggactgcat aaaaatcaca 540

caacaggtcg gtgtagaact caacctatac ctaactgaat taactacagt attcgggcca 600

cagatcacct cccctgcatt aactcagctg accatccagg cactttataa tttagctggt 660

ggcaatatgg actacttatt aactaagtta ggtataggaa acaatcaact cagctcgtta 720

attggtagcg gcctgatcac tggttaccct atactgtatg actcacatac tcaactcttg 780

ggcatacaag taaatctgcc ctcagtcggg aacttaaata atatgcgtgc cacctatttg 840

gagaccttat ctgtaagtac aaccaaagga tatgcctcag cactagtccc gaaagtagtg 900

acacaagttg gttctgtgat agaagagctt gacacctcat actgtataga gtccgatctg 960

gatttatatt gtactagaat agtgacattt cccatgtccc caggtattta ttcctgtttg 1020

agcggcaaca catcagcctg catgtattca aagactgaag gcgcactcac tacgccatat 1080

atggccctta gaggctcagt tattgccaat tgtaagataa caacgtgcag atgtacagac 1140

cctcctggta tcatatcgca aaattacgga gaagctgtat ccctgataga tagacattcg 1200

tgtaatgtct tatcgttaga cggaataact ctgaggctca gtggggaatt tgatgcaact 1260

tatcaaaaga acatctcaat actagattct caggtcatcg tgacaggcaa tcttgatata 1320

tcaactgaac ttggaaacgt caacaattca atcagcaatg ccttggatag gttggcagaa 1380

agcaacagca aactagaaaa agtcaatgtc agactaacta gcacatccgc tctcattatg 1440

tacaaacaga aggcacaaca aaagaccttg ctatggcttg ggaataatac cctcgatcag 1500

atgagagcca ctacaagagc a 1521

<210> 3

<211> 27

<212> DNA

<213>artificial sequence (manual sequence)

<400> 3

gccaccatgg gctccaaact ttctacc 27

<210> 4

<211> 23

<212> DNA

<213>artificial sequence (manual sequence)

<400> 4

tgctcttgta gtggctctca tct 23

<210> 5

<211> 35

<212> DNA

<213>artificial sequence (manual sequence)

<400> 5

gtatagcggc cgccgatgta cgggccagat atacg 35

<210> 6

<211> 30

<212> DNA

<213>artificial sequence (manual sequence)

<400> 6

gatcctaggt ggggataccc cctagagccc 30

<210> 7

<211> 17

<212> DNA

<213>artificial sequence (manual sequence)

<400> 7

gtaaaacgac ggccagt 17

<210> 8

<211> 17

<212> DNA

<213>artificial sequence (manual sequence)

<400> 8

caggaaacag ctatgac 17

<210> 9

<211> 35

<212> DNA

<213>artificial sequence (manual sequence)

<400> 9

gacaaagacg tgccctgata caagccaaac agaat 35

<210> 10

<211> 35

<212> DNA

<213>artificial sequence (manual sequence)

<400> 10

gtatcagggc acgtctttgt ctccttcctc cggac 35

<210> 11

<211> 35

<212> DNA

<213>artificial sequence (manual sequence)

<400> 11

cgctctcatt atgtacaaac agaaggcaca acaaa 35

<210> 12

<211> 35

<212> DNA

<213>artificial sequence (manual sequence)

<400> 12

gtttgtacat aatgagagcg gatgtgctag ttagt 35

Claims (2)

1. the recombinant herpesvirus of turkeys candidate vaccine strain rHVT-NDV- of expressing gene VII type newcastle disease virus fusion protein VII-F, its deposit number are CCTCC NO:V201904.

2. a kind of recombinant herpesvirus of turkeys of expressing gene VII type newcastle disease virus fusion protein described in claim 1 is waited Select the preparation method of vaccine strain, which comprises the following steps:

(1) using 2014 strain genome of genotype VII NDV DT as template, the F as shown in Seq No.1 is expanded using RT-PCR Gene；F gene is inserted into carrier for expression of eukaryonIn-M3, then it is whole containing CMV promoter, TK ployA through PCR amplification The only F expression casette of son and Flag label；

(2) F expression casette is cloned into middle interstitial granules pFR, constructs transfer vector pFR-F；

(3) by pFR-F and rHVT-EGFP genomic DNA cotransfection CEF, recombinant virus rHVT-NDV-VII-F is saved out.