CN114196639A

CN114196639A - Recombinant duck plague virus expressing genes P1 and 3C of type 3 duck hepatitis A virus and construction method and application thereof

Info

Publication number: CN114196639A
Application number: CN202111370551.3A
Authority: CN
Inventors: 李凯; 高玉龙; 王笑梅; 高立
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2021-11-18
Filing date: 2021-11-18
Publication date: 2022-03-18
Anticipated expiration: 2041-11-18
Also published as: CN114196639B

Abstract

The invention discloses a recombinant duck plague virus for co-expressing genes P1 and 3C of duck hepatitis A virus type 3 and a construction method and application thereof, belonging to the technical field of medicine or veterinary medicine. Specifically, the recombinant duck plague virus vaccine strain rDEV-US78-P13C which jointly expresses the genes P1 and 3C of the duck hepatitis A virus 3 is obtained by constructing and obtaining recombinant cosmids which insert a beta-actin-P13C expression frame between genes US7 and US8 by inserting a gene fragment beta-actin-P13C containing a beta-actin promoter and the genes P1 and 3C of the duck hepatitis A virus 3 into a spacer region between the genes US7 and US8 by using a recombinant cloning technology. The result shows that the recombinant virus vaccine strain rDEV-US78-P13C can induce complete protection against lethal attack of virulent virus of hepatitis A virus of the duck type 37 days after the duckling is immunized by the duckling, and simultaneously shows good immune protection effect on virus attack of duck plague virus, so that the recombinant virus vaccine strain is a good bivalent vaccine for preventing duck viral hepatitis and duck plague.

Description

Recombinant duck plague virus expressing genes P1 and 3C of type 3 duck hepatitis A virus and construction method and application thereof

Technical Field

The invention relates to a recombinant duck plague virus vaccine strain for expressing genes P1 and 3C of type 3 duck hepatitis A virus, a construction method and application thereof. The invention belongs to the technical field of medicine or veterinary medicine.

Background

The waterfowl feeding amount is the first world in China. In 2019, the total yield of waterfowl in China is about 2300 million yuan, wherein 42.68 million meat ducks are listed, the growth is 32% in 2018, 1.87 million laying ducks are listed, and the balance is equal to 2018. The duck meat yield in China accounts for 35% of poultry meat, is the third largest meat in China, and provides rich meat and egg food for the lives of people.

Duck viral Hepatitis is a major epidemic disease which harms the Duck breeding industry in China, is an acute, highly infectious and rapidly fatal infectious disease caused by Duck Hepatitis Virus (DHV), usually occurs in ducklings within 1 month, and is mainly characterized by liver swelling and bleeding of diseased ducks. DVH in China is mainly caused by Duck Hepatitis A Virus (DHAV). DHAV belongs to the family Picornaviridae and the genus avian hepatovirus, and was first reported in the United states in 1949. The DHAV virion is spherical, the nucleocapsid is a symmetrical icosahedral structure and has no cyst membrane, the core is single-stranded RNA, the diameter is 20-40nm, and the DHAV virion has tolerance to chloroform. The complete genome comprises a 5 'non-coding region, a large open reading frame, a 3' non-coding region, and a poly (a) tail, wherein the open reading frame encodes 3 precursor proteins: p1, P2 and P3. The precursor protein P1 can generate structural proteins VP0, VP1 and VP3 by viral protease 3C cleavage, and the above 3 structural proteins are assembled into viral nucleocapsid. The virus protein VP1 is mostly exposed on the surface of the virus, is a binding protein for the virus to be adsorbed to a cell-specific receptor, is also a main component for determining the antigenicity of the virus, and can induce the body to produce neutralizing antibodies. The DHAV strains are divided into three different serotypes, namely duck hepatitis A virus type 1 (DHAV1), type 2 (DHAV2) and type 3 (DHAV 3). Among them, the type 1 and type 3 duck hepatitis A virus prevails mainly in mainland areas of China, and DHAV2 has been reported only in Taiwan areas of China.

Currently, improved live DHAV1 vaccines attenuated by serial passage in chicken embryos are useful for controlling DHAV1 infection. However, no commercial DHAV3 live vaccine suitable for early duckling immunity exists in the market at present, and the development of a vaccine for effectively preventing DHAV3 is one of the most effective means for coping with the DHAV3 epidemic situation in China.

Duckentertis virus (DEV) belongs to the sub-family of alphaherpesviridae and is capable of causing acute, febrile, septic infectious diseases of ducks, geese and other anseriformes. Vaccination is one of the most effective means of controlling duck plague virus infection. For more than half a century, duck plague attenuated live vaccines have been routinely used for prevention of duck plague, with no safety issues for humans and any other animal. Furthermore, DEV, as a herpesvirus, has a large genome and many replication non-essential regions into which foreign genes can be inserted, and is an ideal vector for constructing recombinant live vector vaccines.

The harm caused by mixed infection of DHAV and DEV is more serious, and the difficulty of prevention and control is increased. Vaccine immunization is an effective measure for controlling DEV, and DEV attenuated live vaccines in meat duck groups are vaccines which must be used. At present, no commercial bivalent vaccine capable of effectively preventing duck plague and duck viral hepatitis simultaneously exists in the world.

Therefore, the invention firstly establishes a DEV attenuated vaccine C-KCE strain recombinant cosmid rescue system, inserts a fragment beta-actin-P13C containing a beta-actin promoter and genes P1 and 3C of duck hepatitis A virus type 3 between the genes US7 and US8 of a DEV genome, constructs and obtains recombinant cosmids inserting a beta-actin-P13C expression frame between the genes US7 and US8, and obtains a recombinant duck plague virus vaccine strain rDEV-US78-P13C expressing the genes P1 and 3C of duck hepatitis A virus by rescue. The recombinant DEV vaccine strain is used for immunizing Specific Pathogen Free (SPF) ducks, so that the SPF ducks can generate good antibodies against DHAV3 without influencing the immune effect of DEV, and the recombinant DEV vaccine strain is expected to be used as a combined vaccine for preventing duck plague and duck viral hepatitis.

Disclosure of Invention

The invention aims to provide a recombinant duck plague virus capable of expressing genes P1 and 3C of Duck Hepatitis A Virus (DHAV) type 3 and a construction method and application thereof.

In order to achieve the purpose, the invention adopts the following technical means:

the invention relates to a recombinant duck plague virus (DEV) for expressing P1 and 3C genes of duck hepatitis A virus type 3 (DuckHeatitais Virus 3, DHAV3), which is obtained by inserting an expression frame beta-actin-P13C containing a beta-actin promoter and P1 and 3C genes of the duck hepatitis A virus type 3 into a spacer between US7 and US8 genes of a duck plague virus genome.

Preferably, the duck plague virus is a duck plague virus attenuated vaccine C-KCE strain.

Wherein, preferably, the nucleotide sequence of the expression frame beta-actin-P13C of the type 3 duck hepatitis A virus P1 and 3C genes is shown as SEQ ID NO. 1.

Furthermore, the invention also provides a method for constructing the recombinant duck plague virus, which comprises the following steps:

(1) construction of recombinant plasmid expressing DHAV 3P 1 and 3C genes

Amplifying by a PCR method to obtain a P13C gene segment of the duck hepatitis A virus type 3; carrying out enzyme digestion on the P13C gene fragment obtained by amplification through EcoR1 and ClaI, and then inserting the gene fragment into a pCAGGS vector subjected to the same enzyme digestion treatment to obtain a recombinant eukaryotic expression plasmid pCAGGS-P13C which jointly expresses DHAV 3P 1 and 3C genes;

(2) construction of entry plasmid expressing DHAV 3P 1 and 3C genes

Carrying out enzyme digestion on the constructed pCAGGS-P13C expression plasmid by using SalI and BamHI, and recovering a digestion product to obtain a P13C gene expression framework beta-actin-P13C, wherein the nucleotide sequence of the expression plasmid is shown as SEQ ID NO. 1; cloning the obtained expression frame beta-actin-P13C into pENTR1 entry vector through SalI and BamHI enzyme cutting sites to obtain entry plasmid pENTR1-P13C for expressing DHAV 3P 1 and 3C genes;

(3) recombinant cosmid constructs expressing DHAV 3P 1 and 3C genes

Inserting a Kan-ccdB expression frame between US7 and US8 genes of recombinant cosmid C343 containing DEV attenuated vaccine C-KCE strain genome DNA fragments to obtain recombinant mutant cosmid C343-US 78-KanccdB; mixing an entry plasmid pENTR1-P13C and a recombinant mutant cosmid C343-US78-KanccdB, carrying out LR reaction, and replacing a Kan-ccdB expression frame by a beta-actin-P13C expression frame to obtain the recombinant cosmid C343-US78-P13C with a beta-actin-P13C expression frame inserted between genes of US7 and US8 of the recombinant cosmid C343;

(4) rescue of recombinant DEVs expressing DHAV 3P 1 and 3C genes

Extracting recombinant cosmids C027, C018, C144 and C211 containing DEV genome DNA fragments and recombinant cosmids C343-US78-P13C containing P13C expression frames, co-transfecting five cosmids into CEF cells by a calcium phosphate transfection method, observing the appearance of cytopathic effect after transfecting for 4-5 days, and obtaining a rescued recombinant virus named as rDeV-US78-P13C, namely the recombinant duck plague virus expressing the genes P1 and 3C of the duck hepatitis A virus;

wherein, the recombinant cosmids C343, C027, C018, C144, C211 are five pCC1Fos cosmids which respectively comprise DEV attenuated vaccine C-KCE strain genome DNA fragments and can splice and cover the complete DEV genome, wherein, C027 comprises nucleotide fragments at 1-40133 site of C-KCE strain genome, C018 comprises nucleotide fragments at 28323-67264 site of C-KCE strain genome, C144 comprises nucleotide fragments at 59085-98008 site of C-KCE strain genome, C211 comprises nucleotide fragments at 81629-67264 site of C-KCE strain genome, and C343 comprises nucleotide fragments at 11385857-158014 site of C-KCE strain genome.

Furthermore, the invention also provides application of the recombinant duck plague virus in preparing a medicine for preventing duck viral hepatitis and duck plague.

Wherein, preferably, the medicament is a vaccine.

Compared with the prior art, the invention has the beneficial effects that:

the P1 and 3C gene expression frames of DHAV3 are inserted into the genome of a DEV attenuated live vaccine strain, and recombinant viruses expressing DHAV 3P 1 and 3C proteins are constructed. Under the action of 3C protease, the expressed precursor protein P1 can be split into three DHAV structural proteins of VP0, VP1 and VP3, and the three DHAV structural proteins are assembled into virus-like particles, so that the expressed VP1 protein is facilitated to form a correct structure, and the immunogenicity of the protein is improved. The result of the invention shows that the recombinant virus rDEV-US78-P13C can induce complete protection against DHAV3 virulent lethal attack 7 days after the duckling is immunized by the recombinant virus rDEV-US78-P13C, and simultaneously shows good immune protection effect against DEV challenge, so that the vaccine is a good bivalent vaccine for preventing duck plague and duck viral hepatitis.

Drawings

FIG. 1 is a DEV vaccine strain virus genome Fosmid library cosmid and recombinant cosmid construction map inserted into DHAV 3P 1 and 3C gene expression frame;

FIG. 2 is a cytopathy produced by rescued parental virus rDEV on CEF cells;

FIG. 3 is a virion formed on CEF cells by rescued parental virus rDEV;

FIG. 4 shows DNAPCR identification of rescued rDEV genome of parental virus;

FIG. 5 is a structural map of a DHAV 3P 1 and 3C gene co-expression plasmid pCAGGS-P13C;

FIG. 6 is a diagram showing the construction of DHAV 3P 1 and 3C gene entry plasmid pENTR 1-P13C;

FIG. 7 is a map constructed by inserting a P13C expression frame into recombinant cosmid C343-US78-P13C between DEV genome US7 and US8 genes;

FIG. 8 is a cytopathy produced by recombinant virus rDEV-US78-P13C on CEF cells;

FIG. 9 is a schematic diagram showing virions formed on CEF cells by recombinant virus rDEV-US 78-P13C;

FIG. 10 shows the DNAPCR identification of the genome of recombinant virus rDEV-US 78-P13C;

FIG. 11 is an indirect immunofluorescence assay for detecting the expression of target genes P1 and 3C in recombinant virus rDEV-US78-P13C infected cells;

FIG. 12 is a graph showing the replication kinetics of recombinant virus rDEV-US78-P13C on CEF cells;

FIG. 13 is a PCR method for detecting the genetic stability of the foreign genes P1 and 3C in the rDEV-US78-P13C passage of the recombinant virus;

FIG. 14 is an indirect immunofluorescence assay for detecting the stable expression of exogenous genes P1 and 3C in recombinant virus rDEV-US78-P13C passages;

FIG. 15 shows the detection of neutralizing antibodies of blood DHAV 37 days after immunization of SPF duck with rDEV-US 78-P13C;

FIG. 16 is a detection of DEV neutralizing antibodies in blood 7 and 14 days after immunization of SPF duck with rDEV-US 78-P13C;

FIG. 17 shows the survival condition of the SPF duck after immunization with rDEV-US78-P13C and challenge with DHAV3 virulent virus;

FIG. 18 shows the survival of DEV after immunization of SPF duck with rDEV-US 78-P13C.

Detailed Description

The present invention is further described below, and the embodiments described in the present description are only exemplary and do not limit the scope of the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

Example 1: establishment of duck plague virus vaccine strain cosmid rescue system

1.1 construction of Duck plague vaccine strain viral genome Fosmid library

The DEV genome Library was constructed as described in the copy control plasmid Production Kit (Epicentre corporation). The method comprises the following steps:

genomic DNA of DEV vaccine strain virus (C-KCE strain) (GenBank accession No. KF263690) was aspirated repeatedly 50 to 100 times with a 200. mu.L pipette tip. The sheared DNA was blunt-ended and 5' phosphorylated using an End-Repair Enzyme Mix (Epicentre). The end-modified DNA was ligated with pCC1Fos vector (Epicentre). The ligation product was packaged with Packaging reagent MaxPlax Lambda Packaging Extracts (Epicentre Co.) and transfected into E.coli EPI 300-T1R. The bacterial liquid was spread on LB plate containing chloramphenicol and cultured overnight. 300 clones were picked from the culture plate, cosmid was extracted by alkaline lysis, and the plasmid was isolated using the primer pCC 1F: 5'-GGATGTGCTGCAAGGCGATTAAGTTGG-3' and pCC 1R: 5'-CTCGTATGTTGTGTGGAATTGTGAGC-3' genome sequencing of the recombinant cosmids. As a result, 217 recombinant cosmids in which DEV gene fragments were cloned were obtained in total, and the insert length was 30-45 kb.

1.2 rescue of DEV vaccine Strain Virus

According to the sequencing analysis of recombinant cosmid ends, 5 genomic DNA fragments cloned with DEV vaccine C-KCE strain (GenBank accession No. KF263690) are selected and can be spliced to recombinant cosmids C027, C018, C144, C211 and C343 covering the complete DEV genome. Wherein C027 comprises the nucleotide fragment at positions 1-40133, C018 comprises the nucleotide fragment at positions 28323-67264, C144 comprises the nucleotide fragment at positions 59085-98008, C211 comprises the nucleotide fragment at positions 81629-67264 and C343 comprises the nucleotide fragment at positions 113857-158014 of the genome of the C-KCE strain (FIG. 1).

The selected cosmid DNA was prepared using QIAGEN plasmid extraction kit. Five cosmids were co-transfected into CEF cells by calcium phosphate transfection method, specifically: taking SPF chick embryos of 9-10 days old, taking out the chick embryos aseptically, placing the chick embryos in a dish containing Hank's solution for washing, removing heads, limbs and internal organs, and shearing the chick embryos with scissors; washing twice with Hank's solution, adding 0.25% pancreatin (4 mL/embryo), and incubating at 37 deg.C for 10 min; pancreatin was discarded, DMEM medium containing 5% FBS and 1% double antibody was added, and the cells were dispersed by repeated pipetting. After filtration through 6 layers of gauze, a cell suspension of 8X 105 cells/mL was prepared and distributed in a cell culture flask for culture at 37 ℃. Sterile water, DNA, 2M CaCl were mixed in a 1.5ml EP tube₂(ii) a 2 × HBS buffer was added to another 1.5ml EP tube; adding CaCl₂The DNA mixture was slowly added dropwise to 2 XHBS buffer and incubated at room temperature for 30 min. The prepared calcium phosphate-DNA precipitate was added to the prepared CEF cells, mixed gently and cultured in an incubator at 37 ℃. Cytopathic effects were observed 4-5 days after transfection (FIG. 2), and the rescued parental vaccine strain virus was designated as rDEV.

By observation with an endoscope, it can be seen that the DEV vaccine strain virus obtained by rescue was packaged in infected CEF cells to obtain typical DEV virions (fig. 3). PCR identification of the viral genome of the rescued DEV parental vaccine strain by using DEV UL2 gene identification primers DUL2F: 5'-ATGACA GAA CCT GCCACG GAAACG C-3' and DUL2R: 5'-TTATAC TGT TCC ACAAGG AAG TTG C-3' can amplify a 473bp target band, and the size of the target band is consistent with that expected (FIG. 4). The results show that the establishment of a cosmid rescue system of the DEV vaccine strain virus C-KCE strain is successful.

Example 2: construction of recombinant cosmids expressing DHAV 3P 1 and 3C genes

2.1 construction of recombinant plasmids expressing DHAV 3P 1 and 3C genes

DHAV 3-P1F: 5'-GAATTC GCCACC ATG GATACT CTAACTAAAAAC ATT-3' and DHAV3-P1R: 5'-TGG TCC TGG ATT TTC TTC CAC GTC TCC TGC CTG CTT CAA CAA TGA GAA GTT AGT TGC TCC GCT TCC TTC AAT TTC TAGATG GAG CTC AA-3' as primers, cDNA obtained by reverse transcription of genome RNA of DHAV3-SD1001 strain as a template, and DHAV3-P1-2A fragment is obtained by PCR amplification; with DHAV3-3 CF: 5'-GGAAGC GGA GCAACT AAC TTC TCA TTG TTG AAG CAG GCA GGA GAC GTG GAA GAA AAT CCA GGA CCA AAT AGA TTG GTC AAT GTC TCT AG-3' and DHAV3-3 CR: 5'-ATC GAT CTATCC TGAATA CTT TTT CTC AAC-3' as a primer, and cDNA obtained by reverse transcription of genome RNA of DHAV3-SD1001 strain as a template, and carrying out PCR amplification to obtain a DHAV3-2A-3C fragment; DHAV3-P1F and DHAV3-3CR are used as primers, a DHAV3-P1-2A, DHAV3-2A-3C fragment is used as a template, fusion PCR amplification is carried out, and a fragment P1-2A-3C containing a type 3 duck hepatitis A virus P1 gene, 2A self-cleavage peptide and 3C gene is obtained, and the sequence of the fragment is shown in SEQ ID NO. 1. The DHAV3-P1-2A-3C was digested with EcoR1 and ClaI, and inserted into the pCAGGS vector digested with the same enzyme to obtain a recombinant plasmid pCAGGS-P13C expressing DHAV 3P 1 and 3C genes (FIG. 5).

2.2 construction of an entry plasmid expressing the DHAV 3P 1 and 3C genes

The pCAGGS-P13C expression plasmid constructed above was digested with SalI and BamHI, and the digested product was recovered to obtain expression cassette beta-actin-P13C (P13C). The obtained expression frame beta-actin-P13C was cloned into pENTR1 entry vector through SalI and BamHI restriction sites (this plasmid and its preparation method are described in the patent application with publication number CN105695423A, entitled "recombinant chicken Marek's disease virus vaccine strain expressing infectious bursal disease virus VP2 gene and its construction method and application"), and an entry plasmid pENTR1-P13C (FIG. 6) expressing DHAV 3P 1 and 3C genes was obtained.

2.3 recombinant cosmid construction for expression of DHAV 3P 1 and 3C genes

A recombinant plasmid pKS-KanccdB (the plasmid and a preparation method thereof are recorded in a patent application with the publication number of CN105695423A and the invention name of ' recombinant chicken Marek's disease virus vaccine strain for expressing infectious bursal disease virus VP2 gene and a construction method and application thereof ') containing a Kan-ccdB expression frame is taken as a template, and DUS78 hmF: 5'-ATT AAC ATC CAAATATAT TTG TAC ATG AGG TAA TAG GCT ATG GGT GGA GCA TCA CAA GTT TGT ACA AAA AAG CTG-3' and DUS78 hmR: 5'-TCC GCG CAT AAT ACA GTT AAC CAG GCT GCA CAC TTA AATTAG TACAGATCATCACCACTT TGTACAAGAAAG-3' is used as a primer, and the Kan-ccdB expression frame with the homologous arms of the genes of US7 and US8 is obtained by PCR amplification. The amplified fragment was cloned between the genes US7 and US8 of recombinant cosmid C343 containing genomic DNA fragment of DEV attenuated vaccine C-KCE strain using Counter-Selection BAC Modification Kit to obtain recombinant mutant cosmid C343-US 78-KanccdB. The entry expression plasmid pENTR1-P13C and the recombinant mutant cosmid C343-US78-KanccdB are utilized

LR Clonase^TMII Enzyme Mix was subjected to LR reaction to replace the Kan-ccdB expression frame with P13C expression frame, to obtain recombinant cosmid C343-US78-P13C in which P13C expression frame was inserted between DEV genome US7 and US8 genes (FIG. 7).

Example 3: rescue and identification of recombinant viruses expressing DHAV 3P 1 and 3C genes

3.1 rescue of recombinant DEVs expressing DHAV 3P 1 and 3C genes

Recombinant cosmids C027, C018, C144, C211 containing DEV genomic DNA fragments and recombinant cosmid C343-US78-P13C containing P13C expression framework were extracted with a plasmid kit. Five cosmids were co-transfected into CEF cells by calcium phosphate transfection method, specifically: taking 9-10 days old SPF chick embryo, taking out the chick embryo aseptically, and placing the chick embryo in a containerWashing in a dish of Hank's solution, removing head, limbs and viscera, and cutting with scissors; washing twice with Hank's solution, adding 0.25% pancreatin (4 mL/embryo), and incubating at 37 deg.C for 10 min; pancreatin was discarded, DMEM medium containing 5% FBS and 1% double antibody was added, and the cells were dispersed by repeated pipetting. After filtration through 6 layers of gauze, a cell suspension of 8X 105 cells/mL was prepared and distributed in a cell culture flask for culture at 37 ℃. Sterile water, DNA, 2M CaCl were mixed in a 1.5ml EP tube₂(ii) a 2 × HBS buffer was added to another 1.5ml EP tube; adding CaCl₂The DNA mixture was slowly added dropwise to 2 XHBS buffer and incubated at room temperature for 30 min. The prepared calcium phosphate-DNA precipitate was added to the prepared CEF cells, mixed gently and cultured in an incubator at 37 ℃. Cytopathic appearance was observed 4-5 days after transfection, and the rescued recombinant virus was named rDeV-US 78-P13C.

3.2 identification of recombinant DEVs expressing DHAV 3P 1 and 3C genes

When CEF cells were inoculated with the recombinant virus rDEV-US78-P13C, plaque lesions were observed on the infected cells after 2-3 days of culture, as shown in FIG. 8. The rescued recombinant virus rDeV-US78-P13C was packaged in infected CEF cells to obtain typical DEV virions as observed by transmission electron microscopy (FIG. 9).

The 4.1kb target fragment containing the P13C-polyA expression frame and the downstream homology arm sequence can be obtained by carrying out PCR identification on recombinant virus rDEV-US78-P13C genome DNA by taking the 5 ' end sequence of the P13C gene fragment as an upstream primer (DHAV 3-P1F: 5'-GAATTC GCCACC ATG GATACT CTAACTAAAAACATT-3') and the insertion site US8 gene sequence as a downstream primer (DUS 78R: 5'-TCC GCG CAT AAT ACA GTT AAC CAG-3'), as shown in FIG. 10. The results show that the recombinant virus rDEV-US78-P13C inserting the P13C gene expression frame between DEV genome US7 and US8 genes has been successfully rescued.

Example 4: in vitro biological characterization of recombinant virus rDEV-US78-P13C expressing DHAV 3P 1 and 3C genes

4.1 expression of genes P1 and 3C of interest in recombinant viruses

The recombinant virus rDEV-US78-P13C obtained by rescue was inoculated to CEF cells cultured in a six-well plate, and after 48 hours of culture, the cells were collected and subjected to an indirect immunofluorescence assay (IFA) using DHAV3-VP1 and 3C polyclonal antibody and TRITC-labeled anti-rabbit fluorescent secondary antibody. The process is as follows: the inoculated cells were fixed with absolute ethanol at room temperature for 20 min. The well fixed plates were washed once with PBST. DHAV3-VP1 or 3C polyclonal antibody was added at a 1:100 dilution and incubated in a wet box at 37 ℃ for 1 hour. Wash 5 times with PBST. Add 1:100 dilution of TRITC labeled anti-rabbit fluorescent secondary antibody and incubate 1h in 37 ℃ wet box. Washed 5 times with PBST and observed under a fluorescent microscope. As shown in FIG. 11, a clear red fluorescence signal was detected in the cells infected with the recombinant virus rDeV-US78-P13C, indicating that the recombinant virus rDeV-US78-P13C successfully expressed P1 and 3C proteins in the infected cells.

4.2 analysis of the replication Properties of recombinant viruses in vitro

Recombinant virus rDEV-US78-P13C and parental virus rDEV-WT were combined at 100TCID₅₀CEF cells cultured in 6-well plates were dose-inoculated and virus was collected every 24 hours post-infection (3 replicate wells per virus per time point) until 96 hours post-infection. The titer of the collected virus was determined at each time point, a growth curve was plotted, and the replication capacity of the recombinant virus rDEV-US78-P13C on CEF was examined. The results are shown in fig. 12, and the replication titer of rDEV-US78-P13C at each time point was not significantly different from that of the parental virus (P > 0.05), indicating that the recombinant virus was able to replicate well on CEF cells and that the insertion of the P13C expression frame had no effect on the in vitro replication characteristics of DEV.

4.3 genetic stability of recombinant viruses

The recombinant virus rDEV-US78-P13C was passed in CEF for 20 consecutive passages. Extracting the 5 th, 10 th, 15 th and 20 th generation recombinant virus genome DNA, and performing PCR identification by using a target gene upstream primer DHAV3-P1F and a downstream homology arm primer DUS 78R. As a result, the above generation virus amplified the target fragment of 4.1kb bp, which is consistent with the expectation (FIG. 13). CEF cells were inoculated with the 20 th virus and expression of the recombinant protein was detected by indirect immunofluorescence assay. The results showed that the 20 th virus stably expressed both P1 and 3C proteins of interest (fig. 14). The results show that the recombinant virus rDEV-US78-P13C has good genetic stability.

Example 5: immune protection effect of recombinant virus vaccine strain rDEV-US78-P13C on DHAV3 and DEV virulent virus

5.1 detection of neutralizing antibody in serum after immunizing recombinant vaccine strain rDEV-US78-P13C of SPF duck

Recombinant virus rDEV-US78-P13C was expressed as 1000ELD₅₀Dose inoculation of SPF duck of 1 day age, blood collection 7 and 14 days after immunization, serum separation, and detection of DHAV3 and DEV neutralizing antibody. The results are shown in FIG. 15 and FIG. 16, 7 days after immunization, the average neutralizing antibody titer of the recombinant virus rDEV-US78-P13C immune duck serum DHAV3 can reach 2^6.7The neutralizing antibody against the duck DHAV3 was negative (FIG. 15). 14 days after immunization, the average titer of neutralizing antibodies of recombinant virus rDEV-US78-P13C immune duck serum DEV can reach 2^4.1The non-immune control duck DEV neutralizing antibody was negative (fig. 16). The results show that the recombinant virus vaccine strain rDEV-US78-P13C induces and generates good immune response after immunizing the ducklings.

5.2 immune protection effect of recombinant vaccine strain rDEV-US78-P13C on DHAV3 virulent attack after immunization

7 days after immunization, the experimental duck of the immune recombinant vaccine strain rDEV-US78-P13C and the non-immune control duck are respectively attacked by DHAV3 virulent A3 strain, and the survival situation of the duck after attacking is shown in figure 17. During observation period after the duck is tested to challenge the DHAV3 by the immune recombinant vaccine strain rDEV-US78-P13C, no clinical symptoms are seen, the immune duck survives after the duck is challenged to strong toxicity by the DHAV3, and no obvious eye-watching lesion exists in the autopsy; the non-immunized control duck is killed 3 days after being attacked by DHAV 3. The results show that the protection rate of the recombinant vaccine strain rDEV-US78-P13C after immunization on DHAV3 virulent attack is 100%.

5.3 immune protection effect of recombinant vaccine strain rDEV-US78-P13C against DEV virulent challenge after immunization

14 days after immunization, the experimental duck of the immune recombinant vaccine strain rDEV-US78-P13C and the non-immune control duck are respectively attacked by the DEV virulent CSC strain, and the survival situation of the duck after attacking is shown in figure 18. After 10 experimental ducks of the immune recombinant vaccine strain rDEV-US78-P13C challenge DEV, 1 duck dies 6 days after immunization; the rest 9 ducks survived healthily, no obvious clinical symptoms are seen in the observation period after toxin attack, and no obvious ocular lesions are observed in the autopsy. The non-immunized duck killed all the DEVs after being attacked by virulent virus for 4 days. The results show that the protection rate of the recombinant vaccine strain rDEV-US78-P13C after immunization on DEV virulent challenge is 90%.

Sequence listing

<110> Harbin veterinary institute of Chinese academy of agricultural sciences (Harbin center of Chinese center of animal health and epidemiology)

<120> recombinant duck plague virus expressing genes P1 and 3C of duck hepatitis A virus type 3, and construction method and application thereof

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tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960

cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020

ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080

tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140

cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200

gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260

ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320

gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380

gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440

tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500

cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560

acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620

gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680

acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attcatggat actctaacta 1740

aaaacattga agatgaaact gtcaagatta ttggctcatg tgctgagaag gcacaagaag 1800

caatctctgg tcttggagca gttgagagtg ttgcttcaac caactctgtg gttgctactg 1860

caaatgctac aacaacacaa acgattcctg atccaacaaa tggttccaca gatgactttt 1920

attcatgttc ctatgaggtt ggggcccggg gtgataacat ttcacgctta gtccatctac 1980

acactggaca gtggtccaca cagcatggtg tcactacatg ccttagatgg ttggccactc 2040

ctggatgttt ttatacagtt aatacccaac cagcatatgg acaaaccagg tattttaggt 2100

tcatcagatg tggctaccac ttccgccttc ttgtgaatgc accatctggt gctgctggtg 2160

gactaatgat ggtgtggatg ccttatccat attgccgggt tctcactgga tcttacaatg 2220

tggatgcatc agtagatcgc aggtcattgt tgaatcttcc ctatgccatc ttggatctgc 2280

gcaccgacac tgaaattgac ttggttattc catatgtaaa ttttagaaat tatgttgaaa 2340

tcactgccac agatagtgtt ggtggggcca tatgtgtctt tgtgttggga gcttttacac 2400

atgggtcagg aacctccaat actgttgatt acactctctt tggtgagatg ctagaaactg 2460

acttacaatg tcctcggcct tttaatgacc agggtaagaa gaaaccacgg cggaggccaa 2520

ttcataaacc aaagagccct cctcaagaat ctcgcatcat tattcagcct ggaccaggag 2580

ctgcaaatct atccaactct agtgtggtta ccatggctga gagtgtggct ctagctaatg 2640

agggtactgc agttgactac tcaacagccg ggtgtgcatc gtctgtggat gatgtagtca 2700

tggtgcttag acgctggcag attgtgggtg attttcagtg ggctaacaca gtgacccctg 2760

gcaatagaat taataggttt caggtggttt tcaatcgaat gccaaccttt gctctctttt 2820

ttgataagtt ccagtattgg agggggtccc tggaggttaa attattgacc tttggaagcc 2880

agtttaatac tggccgctat caaatgtcat ggtaccctgt ctctgatgga gagcaaactc 2940

tcgcccagtg ccagaactct gtgtttgtca cctgcgatgt ttgtgctaca ccagccactc 3000

tcatcttgcc cttcaccaat accacatggc gtaaaagcac acgtgaaaat tatggctata 3060

taacctggca tgttgtgaat cgcctaacag ttaactcaac atctccatct acaatcagct 3120

gtgttattct gatgcgagtt ggtaaggact ttcagtttac agctcccctg tatggggccc 3180

tgcagatggc tgccaataac cagggtgatt ccaatcagct tggcgatgat gaaccagtgt 3240

gttttctcaa ttttgagact gcaaatgtgc caatacaagg agagtcacac actttggtga 3300

aacatctttt tggtcgtcaa tggctggttc gtactgttca acatactaat gaggtacaag 3360

agttggattt gccagtacct gaccagggtc acgcatctct gttgcgcttc tttgcctact 3420

tctctggaga agtgattctg accattgtca ataatggaac aacaccatgc atggttgcac 3480

actcttatac aatggacaat ctcacttctg aatatgctgt cactgccatg gggggtattc 3540

ttatcccagc aaactctgcc aagaatatta atattccatt ttattctgtt acacctttac 3600

gccccacacg acccatgcca gcatttcagg ggggtggttt gacttttggc aggttgtata 3660

tttggacaca atcaggaagc gtttctgttt ttatgggcct ccacaagcca gctttgtttt 3720

ttccactgcc tgcaccaact tatacaacac acacacagtt gaataatatt gaaaccatga 3780

atctgcataa tcaatcagat cagccagact gtcacctgtg taagatttgc aagaaaatga 3840

agaaatggtc tcgtaatcat cgcccatttc gcttctgttt gagacttaaa acacttgcct 3900

ttgagctcca tctagaaatt gaaggaagcg gagcaactaa cttctcattg ttgaagcagg 3960

caggagacgt ggaagaaaat ccaggaccaa atagattggt caatgtctct agtgaaaatg 4020

aagttgccac aggattggca gttggtggca agtatgtctt aacttttggg cattctaagt 4080

ttactcaatt ggattccatc agggatatgg tctttaattc acctgctaaa ggaacaccga 4140

tcacttatga tgggttacca accgacctcc aactcttgga ttgtgatata ccccaccaat 4200

ttaaagatgt gtctaagctc atagctacag atgattatcg aggcaatggc tggttagtgt 4260

ggaaagatga tgaccaatat atgattcaag aggttactaa aatcagaccc tttggacaaa 4320

ctacaactgc ttctggcaca acatcatgcc aaacatatat ttataattgt aaaacaggcc 4380

ctggttcatg tggtggagtg cttgtagccc ttattggagg aaacctaaaa attcttggca 4440

tacacaccag tggtaatggt acaatgggtg caagtaatcg catcttccct gtgtttaatc 4500

aaggagccat tgttgagaaa aagtattcag gatagttttc cctctgccaa aaattatggg 4560

gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt 4620

gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat 4680

catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc catatgctgg 4740

ctgccatgaa caaaggtggc tataaagagg tcatcagtat atgaaacagc cccctgctgt 4800

ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt 4860

tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac tagccagatt 4920

tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat gaagatccct 4980

cgacctgcag cccaagctt 4999

Claims

1. A recombinant Duck plague virus (DEV) for expressing 3-type Duck hepatitis A virus (Duck hepatitis A Virus 3, DHAV3) P1 and 3C genes is characterized in that the recombinant Duck plague virus is obtained by inserting an expression frame beta-actin-P13C containing a beta-actin promoter and 3-type Duck hepatitis A virus P1 and 3C genes into a spacer between Duck plague virus genomes US7 and US8 genes.

2. The recombinant duck plague virus of claim 1, wherein said duck plague virus is a duck plague virus attenuated vaccine C-KCE strain.

3. The recombinant duck plague virus of claim 1, wherein the nucleotide sequences of the duck hepatitis A virus type 3P 1 and 3C gene expression framework beta-actin-P13C are shown in SEQ ID No. 1.

4. A method for constructing the recombinant duck plague virus of any one of claims 1 to 3, comprising the following steps:

(1) construction of recombinant plasmid expressing DHAV 3P 1 and 3C genes

Amplifying by a PCR method to obtain a P13C gene fragment of the duck hepatitis A virus type 3, carrying out enzyme digestion on the amplified P13C gene fragment by EcoR1 and ClaI, and inserting the gene fragment into a pCAGGS vector subjected to the same enzyme digestion treatment to obtain a recombinant eukaryotic expression plasmid pCAGGS-P13C for commonly expressing DHAV 3P 1 and 3C genes;

(2) construction of entry plasmid expressing DHAV 3P 1 and 3C genes

Carrying out enzyme digestion on the constructed pCAGGS-P13C expression plasmid by using SalI and BamHI, and recovering a digestion product to obtain a P13C gene expression framework beta-actin-P1-3C, wherein the nucleotide sequence of the beta-actin-P1-3C is shown in SEQ ID NO. 1; cloning the obtained expression frame beta-actin-P13C into pENTR1 entry vector through SalI and BamHI enzyme cutting sites to obtain entry plasmid pENTR1-P13C for expressing DHAV 3P 1 and 3C genes;

(3) recombinant cosmid constructs expressing DHAV 3P 1 and 3C genes

(4) rescue of recombinant DEVs expressing DHAV 3P 1 and 3C genes

5. Use of the recombinant duck plague virus of any one of claims 1-3 in the preparation of a medicament for preventing duck viral hepatitis and duck plague.

6. The use of claim 5, wherein the medicament is a vaccine.