CN116814563A - Duck enteritis virus strain and preparation method and application thereof - Google Patents

Duck enteritis virus strain and preparation method and application thereof Download PDF

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CN116814563A
CN116814563A CN202310624858.4A CN202310624858A CN116814563A CN 116814563 A CN116814563 A CN 116814563A CN 202310624858 A CN202310624858 A CN 202310624858A CN 116814563 A CN116814563 A CN 116814563A
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gene
duck enteritis
enteritis virus
strain
virus
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杨承槐
苗清新
宋亚芬
杨宵玥
张敏
张兵
陈玲
王静文
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China Institute of Veterinary Drug Control
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China Institute of Veterinary Drug Control
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Abstract

The invention relates to the technical field of biology, and particularly discloses a duck enteritis virus strain, a preparation method and application thereof. The US4 gene of the duck enteritis virus strain of the invention lacks the 468-1362 base. The anti-enteronitis virus vaccine is non-pathogenic to ducks, has good immunogenicity, and can realize immune protection against duck enteronitis viruses. The strain can be used as a vaccine candidate strain, can also be used as a live virus vector for expressing exogenous genes, and is used for developing and applying genetic engineering vaccines.

Description

Duck enteritis virus strain and preparation method and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a duck enteritis virus strain and a preparation method and application thereof.
Background
Duck Plague (DP) is also called Duck enteritis (Duck viral enteritis, DVE), and is an acute, febrile, contact infectious disease that only infects birds of the order anseriformes caused by Duck enteritis virus (Duck enteritis virus, DEV), which is widely spread and causes a great loss to the poultry farming industry. Duck viral enteritis can cause diseases to ducks of different sexes and ages, and has the most serious harm to adult ducks, and is characterized by vascular invasion, alimentary canal mucous membrane erosion, tissue hemorrhage, lymphoid organ lesions and degenerative changes of solid organs thereof. The recovered ducks can still carry and expel toxin, so that the disease is widely spread in the ducks, the development of the duck raising industry is seriously influenced, and the duck raising method is one of the most serious infectious diseases endangering the duck raising industry.
Duck enteritis virus belongs to the herpesviridae, the subfamily alpha-herpesviridae, the genus Marek's virus, and the duck herpesvirus type 1. Duck enteritis virus has a typical morphological structure of herpes virus, and can generate cross immunity between different strains. The virus particles are large spherical and provided with a capsule film, the diameter is 120-200 nm, and the structure mainly comprises a capsid, an outer film, a capsule film and a core 4 part. The DEV virus capsid is icosahedral symmetrical, has hexagonal appearance, is composed of 162 capsomers which are mutually connected and are arranged radially and are provided with hollow shaft holes, and the core is formed by winding double strand linear DNA and protein, and has the diameter of about 35-40 nm. Li Yufeng et al used the Shot-gun method to complete whole genome sequencing of duck plague commercial vaccine (DEV VAC) for the first time. The results show that the DEV VAC genome is of full length 158,089bp and has a G+C content of 44.91% and consists of a Long Unique region (UL) and a Short Unique region (U.S. pat), the U.S. region being flanked by a pair of inverted repeats, called Internal Repeat (IR) and Terminal Repeat (TR), respectively. The genomic structure is UL-IR-US-TR, and is typical of the D-type herpesvirus genome. At present, a certain progress is made on duck enteritis virus gene research, but the research on molecular virology is still further needed.
The recombinant live vector vaccine usually takes animal pathogenic attenuated or nontoxic strain as a vector, controls viral vector genome through homologous recombination, a Fosmid multi-fragment rescue system, a bacterial artificial chromosome (bacterial artificial chromosome, BAC), a CRISPR/Cas9 system and other gene editing technologies, inserts and expresses exogenous antigen genes, and can generate protective force on original viruses and obtain protective force on inserted gene related diseases after being inoculated with the recombinant vaccine. Meanwhile, one vector can express a plurality of immune genes to obtain multivalent vaccine or multiple vaccine. DEV has the following advantages as a live vaccine carrier: (1) The large and complex genome of DEV provides many insertionable sites for exogenous genes; (2) DEV has good immunogenicity, and can generate certain protection after 3 days of immunization; (3) long immune retention period, which can reach 1 year. However, the insertion site of the foreign gene directly affects several aspects such as DEV virulence, foreign gene expression and immunogenicity thereof, and there is no unified standard and rule when the optimum insertion site which can simultaneously achieve several effects is determined by research, so that further research on related vaccine vectors is necessary.
Disclosure of Invention
The invention aims at providing a novel duck enteritis virus strain capable of providing good immune protection and application thereof.
In order to achieve the object, the technical scheme of the invention is as follows:
the invention provides a duck enteritis virus strain, wherein the US4 gene of the duck enteritis virus strain lacks 468-1362 th bases.
The sequence of the wild US4 gene is shown in SEQ ID No.11.
The invention constructs a recombinant duck enteritis virus carrying fluorescent markers and xanthine guanine phosphotransferase (gpt) genes and having specific site deletion of US4 genes. The recombinant duck enteritis virus with the deletion of the specific site of the US4 gene can be obtained by reverse screening, fluorescent knockout and gpt marking. The virus has no pathogenicity to duck, has good immunogenicity, and can realize immunoprotection against duck enteritis virus. Can be used as vaccine seed virus for producing duck plague live vaccine; the virus also acts as a live viral vector, expressing foreign genes, such as genes other than duck plague virus antigens.
The duck enteritis virus strain also comprises a fluorescent protein gene and a gpt gene, wherein the fluorescent protein gene and the gpt gene are inserted into the position of missing bases of the US4 gene.
Insertion of fluorescent protein and gpt gene facilitates virus identification. Preferably, the protein expressed by the fluorescent protein gene produces red fluorescence, and the gpt gene expresses xanthine guanine phosphotransferase, and is positioned downstream of and in tandem with the fluorescent protein gene; more preferably, the sequence of the fluorescent protein gene and gpt gene is shown as SEQ ID No. 12.
The duck enteritis virus strain further comprises an exogenous virus gene, wherein the exogenous virus gene is inserted into the position of the deletion base of the US4 gene and is positioned at the downstream of the fluorescent protein gene and the gpt gene.
The exogenous viral gene refers to a viral gene except duck enteritis virus, preferably an avian paramyxovirus type 1F gene, an avian influenza virus HA gene, a viral hepatitis VP1 gene, a parvovirus VP3 gene or a duck tembusu virus E protein gene.
In the preparation of recombinant live vector vaccines, the phenomenon that a large amount of exogenous viral genes are incompatible after being introduced into live viral vectors can occur, so that the exogenous viral genes cannot be expressed in a large amount, the immunogenicity of the exogenous viruses is low, and the corresponding immune effect cannot be realized. The strain with specific site deletion of the US4 gene is used as a vector, and after the exogenous virus gene is inserted, good immunogenicity of the exogenous virus can be obtained. Therefore, the strain provided by the invention can be used as an ideal component for preparing the genetically engineered live vector vaccine.
The invention also provides a method for constructing duck enteritis virus strains, which comprises the following steps:
(1) Cultivation of parent viruses: the cloning and purifying attenuated strain obtained by the transfer of the duck enteritis virus virulent strain is used as a parent strain;
(2) Construction of transfer vector: constructing a transfer vector of the US4 gene, which is deleted from the 468 th to 1362 nd bases and is inserted with a fluorescent protein gene and a gpt gene at the deletion position;
(3) Construction of recombinant duck enteritis virus: co-transfecting the transfer vector obtained in the step (2) and the parent strain obtained in the step (1) into host cells, and obtaining duck enteritis virus strains with the US4 gene deleted from 468 th to 1362 nd bases and the fluorescent protein gene and gpt gene inserted into the deletion positions through clone screening;
alternatively, further comprising (4) knocking out the fluorescent protein gene and gpt gene: knocking out the fluorescent protein gene and the gpt gene in the duck enteritis virus strain obtained in the step (3) through reverse screening to obtain the duck enteritis virus strain with only the 468 th to 1362 nd bases deleted from the US4 gene.
When the invention prepares the strain for further expressing the exogenous virus gene, the step (3) is carried out to obtain the duck enteritis virus strain in which the 468 th to 1362 th bases of the US4 gene are deleted, and the fluorescent protein gene is inserted at the deleted position, and then the invention further comprises the step of inserting the exogenous virus gene at the downstream of the fluorescent protein gene.
As a preferable scheme, the invention provides a recombinant virus which expresses the F protein and the Red Fluorescent Protein (RFP) of the avian paramyxovirus type 1 simultaneously, has good protection effect on the paramyxovirus type 1, carries a red fluorescent protein mark and is favorable for identification.
As a specific embodiment, the construction of the strain comprises the following steps:
(1) Cultivation of parent viruses: the clone purified attenuated strain obtained by continuously passaging duck plague virus CVCC AV1221 virulent strain through chick embryo fibroblasts and chick embryo is used as a parent strain;
(2) Construction of transfer vector: respectively amplifying an upstream sequence of a US4 gene and a downstream sequence of the US4 gene by PCR by taking a pUC18 cloning vector as a skeleton vector as a left and right homology arms of homologous recombination, and then inserting an RFP-gpt gene between the left and right homology arms to obtain a transfer vector pUC18-US4-RFP-gpt;
(3) Construction of red fluorescent protein marked recombinant duck enteritis virus: CEF cells are co-transfected by pUC18-US4-RFP-gpt transfer vector and recombinant duck enteritis virus parent strain, and the deletion recombinant virus duck enteritis virus rDEV-delta US4-RFP strain containing RFP marker protein is obtained through cloning and screening.
Or further comprises (4) obtaining duck enteritis virus rDEV delta US4 strain by reversely screening recombinant duck enteritis virus rDEV delta US4-RFP and knocking out red fluorescent marker (the vaccine prepared by the strain can be used for preventing duck plague by detecting US4 gene antibody, and distinguishing natural infection from immunized duck).
The invention also provides a recombinant live virus vector which is the duck enteritis virus strain.
The invention further provides application of the duck enteritis virus strain in preparation of duck enteritis virus vaccines, wherein the duck enteritis virus vaccines are duck enteritis virus single vaccine or duck enteritis virus combined vaccines.
The invention further provides a duck enteritis virus vaccine, which comprises the duck enteritis virus strain.
The invention also provides a preparation method of the duck enteritis virus live vaccine, which comprises the steps of inoculating host cells with a duck enteritis virus strain with the 468-1362 th base deleted from the US4 gene, and harvesting a virus culture for subsequent vaccine preparation.
As a specific embodiment, the vaccine is prepared by inoculating recombinant duck enteritis virus (rDEV delta US 4) into chicken embryo fibroblasts of a single-layer non-specific pathogen chicken, harvesting virus culture, adding a proper stabilizer, and freeze-drying in vacuum.
The invention has the advantages that:
the invention uses duck enteritis virus cell adaptive virus as parent virus, uses homologous recombination method to construct recombinant DEV which lacks specific US4 gene fragment and/or carries fluorescent protein mark, the recombinant virus has no pathogenicity to duck, and has good immunogenicity (100% immunity protection can be realized), can realize immunity protection against duck enteritis virus, can be used as vaccine candidate strain, can also be used as live virus vector, expresses exogenous gene, and is used for development and application of genetic engineering vaccine.
Drawings
FIG. 1 is a schematic diagram showing the structure of a vector pUC18-US4-RFP-gpt in example 1 of the present invention.
FIG. 2 is a schematic diagram showing the structure of the vector pUC18-US4-RFP-EF1a-NDVF in example 5 of the present invention.
FIG. 3 is a schematic diagram showing the structure of the vector pUC18-RFP-gpt used in example 1 of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and the like used in the examples below, unless otherwise indicated, are all those available commercially or may be prepared by methods conventional in the art.
The parent strain used in the invention is duck plague virus CVCC AV1221 strain (disclosed in Chinese patent CN 109022373B) which is attenuated by SPF Chick Embryo Fibroblasts (CEF) and chick embryo continuous passage C20E85 strain (see: arch Virol (2017) 162:3549-3550.DOI 10.1007/s 00705-017-3491-1).
EXAMPLE 1 construction of Duck enteritis Virus rDEV.DELTA.US 4-RFP Strain
1. Primer design
The following primers were designed and synthesized based on the DEV sequences published on GenBank:
U.S. Pat. No. 4 left homology arm amplification primer:
U-US4-1(SEQ ID NO.1)：
5'CGGGGTACCCCTGTGGTCTACAATCGT 3'；
U-US4-2(SEQ ID NO.2)：
5'GCGTCGACAGATCT ATAACTTCGTATAATGTATGC TATA CGAAGTTAT GTGTCCATTTGTCCATCA3'；
US4 gene right homology arm amplification primer:
D-US4-3(SEQ ID NO.3)：
5'GCGTCGACACGCGTATAACTTCGTATAGCATACATTATAC GAAGTTAT CGCGTTCCATTAGTTTAA3'；
D-US4-4(SEQ ID NO.4)：
5'CCCAAGCTTGACATACGTTGCAGACCC 3'。
DEV genomic DNA extraction
Taking 437.5 mu L of duck enteritis virus C20E85 strain virus liquid, adding 12.5 mu L of proteinase K (20 mg/mL) and 50 mu L of 10% SDS; water bath at 56 ℃ for 1h; extracting with phenol, phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform for 1 time respectively; taking the supernatant, adding 1/10 volume of 3M sodium acetate and 2 times of absolute ethyl alcohol, and standing at-20 ℃ for 30min; centrifuge 12000 Xg for 10min, wash the precipitate with 70% ethanol for 1 time, dissolve the precipitate in 30 μl deionized water, and store at-20deg.C for use.
3. Construction of the transfer vector pUC18-US4-RFP-gpt
The duck enteritis virus CVCC AV1221 genome DNA is used as a PCR amplification template, the primers U-US4-1 and U-US4-2 are used for amplifying the US4 gene to obtain a left homology arm, and the primers D-US4-3 and D-US4-4 are used for amplifying the US4 gene to obtain a right homology arm. The left and right homology arms are connected to pUC18 vector through cleavage site KpnI, salI, hindIII to construct corresponding recombinant plasmid pUC18-US4 (deletion of US4 gene 468-1362 nt). The recombinant plasmid pUC18-US4 is digested with Mlu I, and is dephosphorylated after electrophoresis recovery; pUC18-RFP-gpt (preparation method: RFP sequence was synthesized from cloning vector pSAT6-DsRed2-C1 (GenBank: AY 818375). Refer to GenBank M12907 (E.coli gpt gene encoding xanthine guanine phosphoribosyl transferase, complex cds) the gpt sequence was synthesized, promoter and Poly A sequence were derived from pEGFP-C1 vector (GenBank U55763). The above-mentioned seamless cloning was performed into pUC-18 vector by total gene synthesis. The structure schematic diagram is shown in FIG. 3, the sequence is shown as SEQ ID No.13, which can be obtained from the national institute of veterinary medicine) was digested with Mlu I, 2.1kb fragment was recovered by electrophoresis, and RFP-gpt expression cassette (shown as SEQ ID No. 12) was inserted into pUC18-US4 to obtain transfer vector pUC18-US4-RFP-gpt. The structure of the structure is shown in figure 1.
4. Homologous recombination
According to Lipofectamine TM 3000 transfection kit instructions, transfer vector pUC18US4-RFP-gpt was transfected with DEV into Chick Embryo Fibroblasts (CEF). The method comprises the following specific steps:
culturing CEF in a 12-hole plate until the CEF grows into 80% cell monolayer, inoculating a proper amount of DEV C20E85, and adsorbing for 2-3 h; mixing 3 μl Lipofectamine3000 with 50 μl opti-MEM free of serum and antibiotics; another 5. Mu.g of transfer vector was added to 50. Mu.L of opti-MEM without serum and antibiotics, followed by 2. Mu. L P3000 solution; adding the mixed liquid containing the transfer carrier and the P3000 into the mixed liquid of Lipofectamine3000 and opti-MEM, and lightly and uniformly mixing; incubate at room temperature for 15min. During this period, cells in the twelve well plates were gently washed twice with serum-free opti-MEM, then Lipofectamine 3000/DNA complex was added dropwise to the 12 well plates, 0.5mL of serum-free opti-MEM was added, and the mixture was gently shaken to mix them uniformly, incubated at 37℃for 6 to 7 hours, the supernatant was discarded, 10% fetal bovine serum M199 medium was added, incubated at 37℃for 24 hours, and then incubated at 37℃for 3 to 5 days with 1% fetal bovine serum M199 medium, and observed daily until recombinant viral spots appeared.
5. Plaque purification of recombinant viruses
The preliminarily obtained recombinant virus is inoculated with CEF cells of a 6-well plate full of cell monolayers, the adsorption liquid is removed after adsorption for 1 hour, and 1% agarose is paved for continuous culture. Red fluorescence was observed under a fluorescence microscope 48 hours after inoculation, and single fluorescent plaques were selected in 0.5ml m199 nutrient solution, frozen and thawed 1 time, and 6-well plate CEF cells were inoculated, and plaque purification was performed 4 to 6 times, and the obtained recombinant virus was named rdevΔus4-RFP.
EXAMPLE 2 evaluation of safety of recombinant Duck enteritis Virus rDEV DeltaUS 4-RFP
8 susceptible sheldrake of 4 weeks of age were randomly divided into 2 groups of 4. Group 1 rDEV-. DELTA.US 4-RFP injections were performed with 1mL of virus solution (10 in each muscle 6 TCID 50 ). Group 2 was injected with saline and 1mL of each muscle was inoculated. After 14 days of isolated feeding observation, both groups are 100% healthy, which shows that rDEV-delta US4-RFP has good safety.
EXAMPLE 3 evaluation of immunogenicity of recombinant Duck enteritis Virus rDEV.DELTA.US 4-RFP Strain
12 susceptible sheldrake of 4 weeks of age were randomly divided into 3 groups of 4. Group 1 injections of rDEV-. DELTA.US 4-RFP, each inoculated with 1mL (containing 100TCID 50 ) Group 2 injections of rDEV-. DELTA.US 4-RFP, each inoculated with 1mL (containing 1000 TCID) 50 ) Group 3 was injected with saline and 1mL of each muscle was inoculated. After 14 days of isolated feeding, each leg was vaccinated with DEV CVCC AV1221 by intramuscular injection, 1mL each (1000 MLD in) and 14 days of observation, the onset of death was recorded daily. 100% of the virus-attack control group dies, and 100% of the immune group is protected, which proves that the rDEV delta US4-RFP strain has good immunogenicity and is immunized with 100TCID 50 Recombinant viruses can induce 100% protection by 100% against lethal virulent challenge. The specific results are shown in Table 1.
TABLE 1 immunogenicity of Duck enteritis Virus rDEV.DELTA.US 4-RFP strains
EXAMPLE 4 construction of recombinant Duck enteritis Virus rDEV.DELTA.US 4 Strain
According to Lipofectamine TM 3000 transfection kit Specification, the transfer vector pUC18-US4 constructed in example 1 was transfected with recombinant duck enteritis virus rDEV.DELTA.US 4-RFP into Chicken Embryo Fibroblasts (CEF). The method comprises the following specific steps:
culturing CEF in a 12-hole plate until the CEF grows into 80% cell monolayer, inoculating a proper amount of recombinant duck enteritis virus rDEV delta US4-RFP, and adsorbing for 2-3 h; mixing 3 μl Lipofectamine3000 with 50 μl opti-MEM free of serum and antibiotics; another 5. Mu.g of the transfer vector pUC18-US4 was added to 50. Mu.L of opti-MEM without serum and antibiotics, followed by 2. Mu.L of the solution LP 3000; adding the mixed liquid containing the transfer carrier and the P3000 into the mixed liquid of Lipofectamine3000 and opti-MEM, and lightly and uniformly mixing; incubate at room temperature for 15min. During this period, cells in the twelve well plates were gently washed twice with serum-free opti-MEM, then Lipofectamine 3000/DNA complex was added dropwise to the 12 well plates, 0.5mL of serum-free opti-MEM was added, and the mixture was gently shaken to mix them uniformly, incubated at 37℃for 6 to 7 hours, the supernatant was discarded, 10% fetal bovine serum M199 medium was added, incubated at 37℃for 24 hours, and then incubated at 37℃for 3 to 5 days with 1% fetal bovine serum M199 medium, and daily observation was performed until CPE reached about 80%.
The preliminarily obtained recombinant virus is inoculated with CEF cells of a 6-well plate full of cell monolayers, the adsorption liquid is removed after adsorption for 1 hour, and 1% agarose is paved for continuous culture. After 48 hours of inoculation, cytopathy is observed under a fluorescence microscope, single non-fluorescent plaques are selected in 0.5mL M199 nutrient solution, 6-hole plate CEF cells are inoculated after 1 time of freeze thawing, plaque purification is carried out for 4-6 times, and the obtained recombinant virus is named rDEV delta US4 strain. After the red fluorescent marker is knocked out, the strain can be used for preparing duck enteritis virus live vaccines.
EXAMPLE 5 construction of recombinant Duck enteritis Virus live vector
(1) Transfer vector construction
The F gene expression cassette EF1a-NDVF-BGHpolyA of the avian paramyxovirus type 1 is synthesized (shown as SEQ ID NO. 5), and a transfer vector pUC18-US4-RFP-EF1a-NDVF is obtained by cloning EagI and PmeI restriction enzyme sites between pUC18-US4-RFP-gpt vectors. The structure of the structure is shown in figure 2.
(2) Homologous recombination
The transfer vector was transfected with CEF with the recombinant duck plague virus according to Lipofectamine TM2000 transfection kit instructions. The method comprises the following specific steps:
culturing CEF in a six-hole plate until the CEF grows into a cell monolayer of 70-90%, inoculating a proper amount of duck enteritis virus C20E85, and adsorbing for 1-4 hours; diluting 1 mu g of recombinant transfer vector in opti-DMEM without serum and antibiotics to make the final volume of the mixed solution 50 mu L, and incubating for 5min at room temperature; mixing 2 μl Lipofectamine 2000 with 48 μl opti-DMEM free of serum and antibiotics, and incubating at room temperature for 5min; dripping 50 mu L of Lipofectamine 2000 diluent into 50 mu L of plasmid diluent respectively, and mixing uniformly while adding; incubate at room temperature for 20min. During this period, cells in the six-well plate were gently washed twice with serum-free OPTI-MEM, 0.5mL of serum-free OPTI-MEM was added to each well, 100. Mu.L of Lipofectamine 2000/DNA complex was added dropwise to the 6-well plate, and the mixture was gently shaken to mix them uniformly, incubated at 37℃for 4 hours, the supernatant was discarded, 10% fetal bovine serum M199 medium was added, incubated at 37℃for 24 hours, and then incubated at 37℃for 3 to 5 days with 1% fetal bovine serum M199 medium.
(3) Plaque purification of recombinant viruses
The preliminarily obtained recombinant virus is inoculated to CEF cells of a 6-well plate which grow into a good cell monolayer, the adsorption liquid is removed after adsorption for 1 hour, and 1% of low-melting agarose is paved for continuous culture. Observing under a fluorescence microscope after 48-72 hours of inoculation, selecting single red fluorescent plaque in 0.5ml M199 nutrient solution, inoculating 6-hole plate CEF cells after freezing and thawing for 1 time, and performing plaque purification for 3-4 times to obtain recombinant duck enteritis virus rDEV-RE-NDVF expressing avian paramyxovirus type 1F protein.
(4) Protection effect on paramyxovirus type 1
9 susceptible sheldrake ducks of 25 days of age were randomly divided into 2 groups. Group 1, 5, immunized group, immune recombinant virus rDEV-RE-NDVF, each intramuscular injection was inoculated with 0.5mL (containing 3000TCID 50 ). Group 2, 4, control, 0.5mL of saline was inoculated per intramuscular injection. After 47 days of isolated feeding, virus challenge was performed in a biosafety tertiary laboratory with paramyxovirus type 1 (available from China veterinary drug administration), each injected with 0.5mL (containing 10) 5.0 ELD 50 ) After the virus is removed, the patient is observed for 10 days, and the morbidity and mortality are recorded every day, wherein on the 3 rd day and the 5 th day after the virus is removed, cloaca swabs are collected for virus separation. 1/4 of the result of the toxicity attack control group dies, and 5/5 of the immune group is protected; on day 3 after virus challenge, virus separation of virus challenge control group is 4/4 positive, virus separation of immune group is 1/5 positive; on day 5 after virus challenge, virus isolation of the virus challenge control group was 1/3 positive, virus isolation of the immune group was 0/5 positive, indicating that rDEV-RE-NDVF has good immunogenicityImmunization 3000TCID 50 The recombinant virus can resist the attack of lethal virulence, and 80% of immunized ducks do not expel the virulence. The specific results are shown in Table 2.
TABLE 2 Duck enteritis Virus rDEV-RE-NDVF protective Effect on paramyxovirus type 1 attack
(5) Toxicity-counteracting protection effect on duck plague virus
10 susceptible sheldrake of 4 weeks of age were randomly divided into 2 groups of 5. Group 1 injections of recombinant virus rDEV-RE-NDVF were each inoculated with 1mL (containing 1000TCID 50 ) Group 2 was injected with saline and 1mL of each muscle was inoculated. After 14 days of isolated feeding, each leg was vaccinated with DEV CVCC AV1221 by intramuscular injection, 1mL each (1000 MLD in) and 14 days of observation, the onset of death was recorded daily. 100% of the virus-attack control group dies, and 100% of the immune group is protected, which shows that the insertion of the exogenous gene does not affect the immunogenicity of DEV. The specific results are shown in Table 3.
TABLE 3 attack protection of Duck enteritis Virus rDEV-RE-NDVF against DEV
The invention also uses the seamless cloning technology to replace the NDV F gene in the transfer vector pUC18-US4-RFP-EF1a-NDVF respectively by using the avian influenza virus HA gene (H9 subtype is shown as SEQ ID NO.6, H5 subtype is shown as SEQ ID NO. 7), viral hepatitis VP1 gene (SEQ ID NO. 8), parvovirus VP3 gene (SEQ ID NO. 9) and duck tembusu virus E protein gene (SEQ ID NO. 10) to obtain the recombinant transfer vector containing the corresponding exogenous antigen genes. According to the same method, the recombinant duck plague virus which respectively expresses protective antigens of influenza virus HA gene, viral hepatitis VP1 gene, parvovirus VP3 gene and duck tembusu virus E protein gene is obtained.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (10)

1. The duck enteritis virus strain is characterized in that the US4 gene of the duck enteritis virus strain lacks 468-1362 bases.
2. The duck enteritis virus strain of claim 1, further comprising a fluorescent protein gene and gpt gene inserted at the location of the US4 gene deletion base.
3. The duck enteritis virus strain of claim 2, wherein the protein expressed by the fluorescent protein gene produces red fluorescence, and the gpt gene is positioned downstream of and in tandem with the fluorescent protein gene; preferably, the sequence of the fluorescent protein gene and the gpt gene is shown as SEQ ID No. 12.
4. A duck enteritis virus strain according to claim 2 or 3, further comprising an exogenous viral gene inserted at the position of the US4 gene deletion base and downstream of the fluorescent protein gene and gpt gene.
5. The duck enteritis virus strain of claim 4, wherein the exogenous viral gene is avian paramyxovirus type 1F gene, avian influenza virus HA gene, viral hepatitis VP1 gene, parvovirus VP3 gene, or duck tembusu virus E protein gene.
6. A method of constructing a strain of duck enteritis virus comprising:
(1) Cultivation of parent viruses: the cloning and purifying attenuated strain obtained by the transfer of the duck enteritis virus virulent strain is used as a parent strain;
(2) Construction of transfer vector: constructing a transfer vector of the US4 gene, which is deleted from the 468 th to 1362 nd bases and is inserted with a fluorescent protein gene and a gpt gene at the deletion position;
(3) Construction of recombinant duck enteritis virus: co-transfecting the transfer vector obtained in the step (2) and the parent strain obtained in the step (1) into host cells, and obtaining duck enteritis virus strains with the US4 gene deleted from 468 th to 1362 nd bases and the fluorescent protein gene and gpt gene inserted into the deletion positions through clone screening;
alternatively, further comprising (4) knocking out the fluorescent protein gene and gpt gene: knocking out the fluorescent protein gene and the gpt gene in the duck enteritis virus strain obtained in the step (3) through reverse screening to obtain the duck enteritis virus strain with only the 468 th to 1362 nd bases deleted from the US4 gene.
7. A recombinant live viral vector which is the duck enteritis viral strain of any one of claims 1-5.
8. The use of the duck enteritis virus strain of any one of claims 1-5 in the preparation of a duck enteritis virus vaccine, wherein the duck enteritis virus vaccine is a duck enteritis virus single vaccine or a duck enteritis virus combined vaccine.
9. A duck enteritis virus vaccine comprising the duck enteritis virus strain of any one of claims 1-5.
10. The preparation method of the duck enteritis virus live vaccine is characterized by comprising the steps of inoculating host cells with a duck enteritis virus strain with the 468-1362 th base deleted from the US4 gene, and harvesting a virus culture for subsequent vaccine preparation.
CN202310624858.4A 2023-05-30 2023-05-30 Duck enteritis virus strain and preparation method and application thereof Pending CN116814563A (en)

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