CN114196639B - Recombinant duck plague virus for expressing 3-type duck hepatitis A virus P1 and 3C genes, construction method and application thereof - Google Patents

Abstract

The invention discloses a recombinant duck plague virus for co-expressing 3 type duck hepatitis A virus P1 and 3C genes, a construction method and application thereof, and belongs to the technical field of medicine or veterinary medicine. Specifically, the invention utilizes a recombinant cloning technology to insert a gene fragment beta-actin-P13C containing a beta-actin promoter and 3 type duck hepatitis A virus P1 and 3C genes into a spacer region between the duck plague virus US7 and US8 genes, constructs a recombinant cosmid for obtaining a beta-actin-P13C expression frame inserted between the US7 and US8 genes, and saves the recombinant cosmid to obtain a recombinant duck plague virus vaccine strain rDEV-US78-P13C for co-expressing the 3 type duck hepatitis A virus P1 and 3C genes. The result shows that the recombinant virus vaccine strain rDEV-US78-P13C can induce complete protection of the virulent and lethal attack of 3 type duck hepatitis A virus after immunizing duckling for 7 days, and simultaneously shows good immune protection effect on duck plague virus attack, thus being a good bivalent vaccine for preventing duck viral hepatitis and duck plague.

Description

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

Technical Field

The invention relates to a recombinant duck plague virus vaccine strain for expressing 3-type duck hepatitis A virus P1 and 3C genes, a construction method and application thereof. The present invention is in the field of medical or veterinary technology.

Background

The water fowl raising amount in China is the first world. In 2019, the total yield of water fowl in China is about 2300 hundred million yuan, wherein 42.68 hundred million meat ducks are put in the field, which is 32% higher than that in 2018, and 1.87 hundred million egg ducks are put in the field and are equal to that in 2018. The duck meat yield in China is 35% of that of the poultry meat, and the duck meat is third-sized meat in China, so that the duck meat is a rich meat-egg food for people living.

Duck viral hepatitis is a major epidemic disease endangering the duck raising industry in China, which is an acute, highly infectious and rapidly fatal infectious disease caused by duck hepatitis virus (Duck Hepatitis Virus, DHV), and is usually a duckling within 1 month, and is mainly characterized by hepatomegaly and hemorrhage of the duckling. DVH in China is mainly caused by duck hepatitis A virus (DuckHepatitisAVirus, DHAV). DHAV belongs to the picornaviridae and avian hepatovirus genera, and was first reported in the united states in 1949. The DHAV virus particles are spherical, the nucleocapsid is of a symmetrical icosahedral structure, no envelope exists, the core is single-stranded RNA, the diameter size is 20-40nm, and the DHAV virus particles are resistant 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. Cleavage of the precursor protein P1 by viral protease 3C can produce structural proteins VP0, VP1, VP3, the above 3 structural proteins being assembled into viral nucleocapsids. The virus protein VP1 is mostly exposed on the surface of the virus, is a binding protein for adsorbing the virus to a cell-specific receptor, is also a main component for determining the antigenicity of the virus, and can induce the organism to generate neutralizing antibodies. DHAV strains are divided into three different serotypes, duck hepatitis a virus type 1 (DHAV 1), type 2 (DHAV 2) and type 3 (DHAV 3), respectively. Among them, the main epidemic type 1 and type 3 duck hepatitis A viruses in the continental areas of China, DHAV2 is reported only in Taiwan areas of China.

Currently, modified live DHAV1 vaccines attenuated by serial passage in chick embryos are available to control DHAV1 infection. However, no commercial DHAV3 live vaccine suitable for early immunization of duckling exists in the market at present, and developing a vaccine for effectively preventing DHAV3 is one of the most effective means for coping with DHAV3 epidemic situations in China.

Duck plague virus (DEV) belongs to the subfamily alphaherpesviridae of herpesviridae and can cause acute, febrile, and septic infections in ducks, geese and other anseriformes birds. Vaccination is one of the most effective means of controlling duck plague virus infection. For over half a century, duck plague live attenuated vaccines have been routinely used for prevention of duck plague, without safety problems for humans and any other animals. In addition, DEV is used as a herpesvirus, has larger genome and more replication unnecessary regions for inserting exogenous genes, and is an ideal vector for constructing recombinant live vector vaccines.

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

To this end, the invention first establishes a recombinant cosmid rescue system for the DEV attenuated vaccine C-KCE strain, on the basis of which a fragment beta-actin-P13C containing a beta-actin promoter and the 3-type duck hepatitis A virus P1 and 3C genes is inserted between the US7 and US8 genes of the DEV genome, recombinant cosmid with a beta-actin-P13C expression frame inserted between the US7 and US8 genes is constructed, and the recombinant duck plague vaccine strain rDEV-US78-P13C expressing the 3-type duck hepatitis A virus P1 and 3C genes is obtained by rescue. The recombinant DEV vaccine strain is immunized with specific pathogen-free (SPF) ducks, so that the SPF ducks can generate good antibodies against DHAV3, the immunization effect of the DEV is not affected, and the recombinant DEV vaccine strain is expected to be used as a bivalent 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 3-type duck hepatitis A virus P1 and 3C genes, and a construction method and application thereof.

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

in the present invention, a recombinant duck plague virus (DEV) expressing Duckhepatitidisvirus 3, DHAV3) P1 and 3C genes is obtained by inserting a beta-actin promoter and an expression frame beta-actin-P13C containing P1 and 3C genes of Duckhepatitidisvirus genome US7 and US8 into a spacer region between genes of Duckhepatitidisvirus genome.

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

Preferably, the nucleotide sequences of the beta-actin-P13C gene expression frames of the 3-type duck hepatitis A virus P1 and 3C gene expression frames are 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 fragment of the 3-type duck hepatitis A virus; the amplified P13C gene fragment is digested by EcoR1 and ClaI and then inserted into a pCAGGS vector which is subjected to the same digestion treatment, so as to obtain a recombinant eukaryotic expression plasmid pCAGGS-P13C for co-expressing DHAV 3P 1 and 3C genes;

(2) Entry plasmid construction for expressing DHAV 3P 1 and 3C genes

The pCAGGS-P13C expression plasmid constructed in the above is subjected to enzyme digestion by SalI and BamHI, and the enzyme digestion product is recovered to obtain a P13C gene expression frame beta-actin-P13C, the nucleotide sequence of which is shown as SEQ ID NO. 1; cloning the obtained expression frame beta-actin-P13C into pENTR1 entry vector through SalI and BamHI cleavage sites to obtain entry plasmid pENTR1-P13C for expressing DHAV 3P 1 and 3C genes;

(3) Recombinant cosmid construction for expression of DHAV 3P 1 and 3C genes

Inserting a Kan-ccdB expression frame between the US7 and US8 genes of a recombinant cosmid C343 containing DEV attenuated vaccine C-KCE strain genome DNA fragments to obtain a recombinant mutant cosmid C343-US78-KanccdB; mixing an entry plasmid pENTR1-P13C with a recombinant mutant cosmid C343-US78-KanccdB and carrying out LR reaction to enable a beta-actin-P13C expression frame to replace a Kan-ccdB expression frame, so as to obtain a recombinant cosmid C343-US78-P13C with the beta-actin-P13C expression frame inserted between US7 and US8 genes 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 cosmid C343-US78-P13C containing P13C expression frames, co-transfecting CEF cells with five cosmids by a calcium phosphate transfection method, wherein cytopathic effect can be observed after 4-5 days of transfection, and the rescued recombinant virus is named rDEV-US78-P13C, namely the recombinant duck plague virus for expressing the P1 and 3C genes of the duck hepatitis A virus;

wherein, the recombinant cosmids C343, C027, C018, C144 and C211 are five pCC1Fos cosmids which respectively comprise DEV attenuated vaccine C-KCE strain genome DNA fragments and can splice and cover complete DEV genome, wherein, C027 comprises nucleotide fragments of 1-40133 positions of C-KCE strain genome, C018 comprises nucleotide fragments of 28323-67264 positions of C-KCE strain genome, C144 comprises nucleotide fragments of 59085-98008 positions of C-KCE strain genome, C211 comprises nucleotide fragments of 81629-67264 positions of C-KCE strain genome, and C343 comprises nucleotide fragments of 113857-158014 positions of C-KCE strain genome.

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

Wherein, preferably, the medicine is a vaccine.

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

the invention inserts the P1 and 3C gene expression frames of DHAV3 into the DEV attenuated live vaccine strain genome to construct the recombinant virus for expressing the DHAV 3P 1 and 3C proteins. Under the action of 3C protease, the expressed precursor protein P1 can be cracked into three DHAV structural proteins VP0, VP1 and VP3 and assembled into virus-like particles, so that the expressed VP1 protein can form a correct structure, and the immunogenicity of the VP1 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 immunization of duckling, and simultaneously shows good immune protection effect on DEV, thus being a good bivalent vaccine for preventing duck plague and duck viral hepatitis.

Drawings

FIG. 1 is a diagram of construction of cosmids from a DEV vaccine strain viral genome Fosmid library and recombinant cosmids inserted into the DHAV 3P 1 and 3C gene expression cassettes;

FIG. 2 is a cytopathic effect of the rescued parent virus rDEV on CEF cells;

FIG. 3 is a viral particle of the rescued parent virus rDEV formed on CEF cells;

FIG. 4 is a graph showing the identification of the rescued parent virus rDEV genome DNAPCR;

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

FIG. 6 is a construction map of the DHAV 3P 1 and 3C gene entry plasmids pENTR1-P13C;

FIG. 7 is a recombinant cosmid C343-US78-P13C construct with a P13C expression cassette inserted between the DEV genomes US7 and US8 gene;

FIG. 8 is a cytopathic effect of recombinant virus rDEV-US78-P13C on CEF cells;

FIG. 9 is a viral particle formed on CEF cells by recombinant virus rDEV-US 78-P13C;

FIG. 10 shows the identification of recombinant virus rDEV-US78-P13C genome DNAPCR;

FIG. 11 shows the detection of the expression of the genes of interest P1 and 3C in recombinant virus rDEV-US78-P13C infected cells by an indirect immunofluorescence assay;

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

FIG. 13 shows the detection of the genetic stability of the foreign genes P1 and 3C in the passage of recombinant virus rDEV-US78-P13C by PCR;

FIG. 14 shows the detection of stable expression of foreign genes P1 and 3C in rDEV-US78-P13C passage of recombinant virus by indirect immunofluorescence assay;

FIG. 15 is a blood DHAV3 neutralizing antibody assay 7 days after SPF duck immunization of rDEV-US 78-P13C;

FIG. 16 shows detection of blood DEV neutralizing antibodies 7, 14 days after SPF duck immunization of rDEV-US 78-P13C;

FIG. 17 shows survival of SPF ducks immunized with rDEV-US78-P13C against virulent DHAV 3;

FIG. 18 shows survival of SPF ducks after immunization with rDEV-US78-P13C against virulent virus of virulent DEV.

Detailed Description

The invention is further described below, the embodiments presented in this description are only exemplary and do not limit the scope of the invention. It will be understood by those skilled in the art that the details and forms of the invention may be modified or substituted without departing from the spirit and scope of the invention.

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

1.1 construction of Fosmid library of Duck plague vaccine strain viral genome

The construction of the DEV genome Fosmid library was performed according to Epicentre company CopyControlTM Fosmid Library Production Kit kit instructions. The method comprises the following steps:

DEV vaccine strain virus (C-KCE strain) (GenBank accession No. KF 263690) viral genomic DNA was repeatedly aspirated 50-100 times with 200. Mu.L pipette tips. The sheared DNA was blunt ended and 5' phosphorylated with End-Repair Enzyme Mix (Epicentre). The end-modified DNA was ligated with pCC1Fos vector (Epicentre Co.). The ligation product was packaged with packaging reagent MaxPlax Lambda Packaging Extracts (Epicentre Co.) and E.coli EPI300-T1R transfected. The bacterial solution was spread on LB plate containing chloramphenicol and cultured overnight. 300 clones were picked from the culture plate, cosmids were extracted by alkaline lysis and primers pCC1F:5'-GGATGTGCTGCAAGGCGATTAAGTTGG-3' and pCC1R:5'-CTCGTATGTTGTGTGGAATTGTGAGC-3' genomic sequencing of recombinant cosmids. As a result, 217 recombinant cosmids cloned with DEV gene fragments were obtained, and the insert length was between 30 and 45 kb.

1.2 rescue of DEV vaccine strains of virus

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

The cosmid DNA selected was prepared using QIAGEN plasmid extraction kit. Five cosmids were co-transfected with CEF cells by the calcium phosphate transfection method, specifically: taking SPF chick embryo of 9-10 days old, aseptically taking out chick embryo, placing into a plate containing Hank's liquid, washing, 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℃for 10min; the pancreatin was discarded, DMEM medium containing 5% fbs and 1% diabody was added, and the cells were dispersed by repeated pipetting. Filtering with 6 layers of gauze to obtain 8×105 cells/mL cell suspension, and packaging in cell culture flask for culturing at 37deg.C. Sterilized water, DNA, 2M CaCl were mixed in 1.5ml EP tube ₂ The method comprises the steps of carrying out a first treatment on the surface of the 2 XHBS buffer was added to another 1.5ml EP tube; caCl is added with ₂ The DNA mixture was slowly added dropwise to 2 XHBS buffer and incubated at room temperature for 30min. The prepared calcium phosphate-DNA precipitate was added to the prepared CEF cells, and the mixture was slightly mixed and cultured in an incubator at 37 ℃. Cytopathic appearance was observed 4-5 days after transfection (FIG. 2), and the rescued parental vaccine strain virus was designated rDEV.

When observed by electron microscopy, it was seen that the DEV vaccine strain virus obtained by rescue was packaged in infected CEF cells to obtain typical DEV virions (FIG. 3). The DEV parent vaccine strain viral genome obtained by rescue was subjected to PCR identification 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', and a target band of 473bp was obtained by amplification, the size of which was in accordance with the expectations (FIG. 4). The result shows that the cosmid rescue system of the DEV vaccine strain virus C-KCE strain is successfully established.

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

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

In DHAV3-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' are used as primers, cDNA obtained by reverse transcription of genome RNA of DHAV3-SD1001 strain is used as a template, and a DHAV3-P1-2A fragment is obtained by PCR amplification; in DHAV3-3CF: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 DHAV3-3CR:5'-ATC GAT CTATCC TGAATA CTT TTT CTC AAC-3' as primer, cDNA obtained by reverse transcription of DHAV3-SD1001 strain genome RNA as template, and PCR amplification to obtain DHAV3-2A-3C fragment; taking DHAV3-P1F, DHAV3-3CR as a primer and taking a DHAV3-P1-2A, DHAV3-2A-3C fragment as a template, carrying out fusion PCR amplification to obtain a fragment P1-2A-3C containing a 3-duck hepatitis A virus P1 gene, a 2A self-cleaving peptide and a 3C gene, wherein the sequence of the fragment P1-2A-3C is shown as SEQ ID NO. 1. After cleavage of DHAV3-P1-2A-3C with EcoR1 and ClaI, the same cleavage-treated pCAGGS vector was inserted to obtain recombinant plasmid pCAGGS-P13C expressing DHAV 3P 1 and 3C genes (FIG. 5).

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

The pCAGGS-P13C expression plasmid constructed in the above is digested with SalI and BamHI, and the digested product is recovered to obtain the expression frame beta-actin-P13C (abbreviated as P13C). The obtained expression frame beta-actin-P13C was cloned into pENTR1 entry vector through SalI and BamHI cleavage sites (this plasmid and its preparation method are described in patent application with publication No. CN105695423A, entitled "recombinant chicken Marek's disease virus vaccine strain expressing infectious bursal disease virus VP2 gene and its construction method and application"), obtaining entry plasmid pENTR1-P13C expressing DHAV 3P 1 and 3C genes (FIG. 6).

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

Recombinant plasmid pKS-KanccdB containing Kan-ccdB expression frame (the plasmid and the preparation method thereof are described in patent application with publication number of CN105695423A and the invention name of recombinant chicken Marek's disease virus vaccine strain expressing infectious bursal disease virus VP2 gene and construction method and application thereof) is taken as a template, and DUS78hmF is taken as a template: 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 DUS78hmR:5'-TCC GCG CAT AAT ACA GTT AAC CAG GCT GCA CAC TTA AATTAG TACAGATCATCACCACTT TGTACAAGAAAG-3' is a primer, and the Kan-ccdB expression frame with US7 and US8 gene homology arms is obtained through PCR amplification. The amplified fragment was cloned between the US7 and US8 genes of recombinant cosmid C343 containing the genomic DNA fragment of DEV attenuated vaccine C-KCE strain using Counter-Selection BAC Modification Kit kit to obtain recombinant mutant cosmid C343-US78-KanccdB. Utilization of the entry expression plasmid pENTR1-P13C and the recombinant mutant cosmid C343-US78-KanccdBLR Clonase ^TM II Enzyme Mix was subjected to LR reaction with the P13C expression frame replaced with the Kan-ccdB expression frame to obtain recombinant cosmid C343-US78-P13C with P13C expression frame inserted between the genes of DEV genome US7 and US8 (FIG. 7).

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

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

Recombinant cosmids C027, C018, C144, C211 containing DEV genomic DNA fragments and recombinant cosmids C343-US78-P13C containing P13C expression cassettes were extracted with plasmid kits. Five cosmids were co-transfected with CEF cells by the calcium phosphate transfection method, specifically: taking SPF chick embryo of 9-10 days old, aseptically taking out chick embryo, placing into a plate containing Hank's liquid, washing, 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℃for 10min; the pancreatin was discarded, DMEM medium containing 5% fbs and 1% diabody was added, and the cells were dispersed by repeated pipetting. Filtering with 6 layers of gauzeThen, cell suspension of 8X 105 cells/mL was prepared, and the suspension was packed in a cell culture flask and cultured in a 37℃incubator. Sterilized water, DNA, 2M CaCl were mixed in 1.5ml EP tube ₂ The method comprises the steps of carrying out a first treatment on the surface of the 2 XHBS buffer was added to another 1.5ml EP tube; caCl is added with ₂ The DNA mixture was slowly added dropwise to 2 XHBS buffer and incubated at room temperature for 30min. The prepared calcium phosphate-DNA precipitate was added to the prepared CEF cells, and the mixture was slightly mixed and cultured in an incubator at 37 ℃. Cytopathic appearance was observed 4-5 days after transfection and the rescued recombinant virus was designated rDEV-US78-P13C.

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

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

The 4.1kb target fragment comprising the P13C-polyA expression frame and the downstream homology arm sequence can be obtained by PCR identification of recombinant virus rDEV-US78-P13C genomic DNA using the 5' -end sequence of the P13C gene fragment as the upstream primer (DHAV 3-P1F:5'-GAATTC GCCACC ATG GATACT CTAACTAAAAACATT-3') and the insertion site US8 gene sequence as the downstream primer (DUS 78R:5'-TCC GCG CAT AAT ACA GTT AAC CAG-3'), as shown in FIG. 10. The above results indicate that recombinant virus rDEV-US78-P13C rescue was successful with the insertion of a P13C gene expression framework between the DEV genomes US7, US8 genes.

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

4.1 expression of the genes P1 and 3C of recombinant viral interest

Recombinant virus rDEV-US78-P13C obtained by the above rescue was inoculated to CEF cells cultured in six well plates, and after 48 hours of culture, the cells were collected and subjected to an indirect immunofluorescence assay (IFA) using DHAV3-VP1 and 3C polyclonal antibodies and TRITC-labeled anti-rabbit fluorescent secondary antibody. The process is as follows: the virus-inoculated cells were fixed with absolute ethanol at room temperature for 20min. The fixed plates were washed once with PBST. A1:100 dilution of DHAV3-VP1 or 3C polyclonal antibody was added and incubated in a 37℃wet box for 1 hour. Wash 5 times with PBST. A1:100 dilution of TRITC-labeled anti-rabbit fluorescence secondary antibody was added and incubated in a 37℃wet box for 1h. The results were observed under a fluorescence microscope by washing with PBST 5 times. As a result, as shown in FIG. 11, a clear red fluorescent signal was detected in the recombinant virus rDEV-US 78-P13C-infected cells, indicating that the recombinant virus rDEV-US78-P13C successfully expressed the P1 and 3C proteins in the infected cells.

4.2 in vitro replication characterization of recombinant viruses

Recombinant virus rDEV-US78-P13C and parent virus rDEV-WT were treated with 100TCID ₅₀ CEF cells cultured in 6-well plates were dosed and virus was collected every 24 hours after infection (3 replicate wells per virus at each time point) until 96 hours after infection. The titer of the collected virus at each time point was determined, a growth curve was drawn, and the replication capacity of the recombinant virus rDEV-US78-P13C on CEF was examined. The results are shown in FIG. 12, where the replication titer of rDEV-US78-P13C at each time point was not significantly different from that of the parent 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 framework had no effect on the in vitro replication characteristics of DEV.

4.3 genetic stability of recombinant viruses

Recombinant virus rDEV-US78-P13C was serially transferred in CEF for 20 passages. And extracting the genome DNA of the 5 th, 10 th, 15 th and 20 th generation recombinant viruses, and carrying out PCR identification by using a target gene upstream primer DHAV3-P1F and a downstream homology arm primer DUS 78R. The results showed that the previous generation of the secondary virus could be amplified to obtain the desired fragment of 4.1kb bp in size, in agreement with the expectations (FIG. 13). The 20 th generation virus was inoculated with CEF cells and the expression of recombinant proteins was detected using an indirect immunofluorescence assay. The results showed that the 20 th generation virus can stably express the P1 and 3C target proteins (FIG. 14). The results show that the recombinant virus rDEV-US78-P13C has good genetic stability.

Example 5: immunoprotection effects of recombinant viral vaccine strain rDEV-US78-P13C against DHAV3 and DEV virulence

5.1 detection of serum neutralizing antibody after SPF Duck immune recombinant vaccine strain rDEV-US78-P13C

Will recombineVirus rDEV-US78-P13C at 1000ELD ₅₀ 1 day old SPF duck was inoculated at each dose, blood was collected 7 and 14 days after immunization, serum was separated, and DHAV3 and DEV neutralizing antibodies were detected, respectively. As shown in FIGS. 15 and 16, the average titer of neutralizing antibody against recombinant virus rDEV-US 78-P13C-immunized duck serum DHAV3 could reach 2 at 7 days after immunization ^6.7 The non-immunized control duck DHAV3 neutralizing antibody was negative (fig. 15). The average titer of neutralizing antibodies of the recombinant virus rDEV-US78-P13C immunized duck serum DEV can reach 2 after 14 days of immunization ^4.1 The non-immunized control duck DEV neutralizing antibodies were negative (fig. 16). The result shows that the recombinant virus vaccine strain rDEV-US78-P13C induces good immune response after the duckling is immunized.

5.2 immunoprotection against virulent DHAV3 challenge following immunization with recombinant vaccine strain rDEV-US78-P13C

7 days after immunization, the experimental ducks of the immune recombinant vaccine strain rDEV-US78-P13C and the non-immune control ducks are respectively challenged with the DHAV3 virulent A3 strain, and the survival condition of the ducks after the challenge of each experimental group is shown in figure 17. During observation period after the immune recombinant vaccine strain rDEV-US78-P13C is subjected to virus-killing DHAV3, no clinical symptoms are seen, the immune duck is subjected to virus-killing DHAV3 and fully survives after strong virus, and no obvious eye lesions are observed in the section inspection; the non-immunized control ducks die all 3 days after the virus of the DHAV3 is removed. The result shows that the protection rate of the recombinant vaccine strain rDEV-US78-P13C on the DHAV3 virulent attack after immunization is 100%.

5.3 immunoprotection effects of recombinant vaccine strains rDEV-US78-P13C against DEV virulent challenge after immunization

The experimental ducks of the immune recombinant vaccine strain rDEV-US78-P13C and the non-immune control ducks were challenged with DEV virulent CSC strains respectively 14 days after immunization, and the survival conditions of the ducks after the challenge of each experimental group are shown in FIG. 18. After 10 experimental ducks of the immune recombinant vaccine strain rDEV-US78-P13C attack the virus DEV, 1 duck dies 6 days after immunization; the rest 9 ducks survive healthily, no obvious clinical symptoms are seen in the observation period after the toxin is removed, and no obvious eye lesions are seen in the section inspection. The non-immunized control ducks die all 4 days after the virus attacks the virulent DEV. The result shows that the protection rate of the recombinant vaccine strain rDEV-US78-P13C on DEV virulent attack after immunization is 90%.

Sequence listing

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

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

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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 (4)

1. Recombinant duck plague virus (DEV) expressing duck hepatitis a virus type 3 (Duck Hepatitis A Virus, dhav 3) P1 and 3C genes, characterized in that said recombinant duck plague virus is obtained by inserting an expression frame β -actin-P13C comprising β -actin promoter, duck hepatitis a virus type 3P 1 and 3C genes into the spacer region between the genes of duck plague virus genomes US7, US 8; wherein the duck plague virus is a duck plague virus attenuated vaccine C-KCE strain, and the nucleotide sequences of the 3-type duck hepatitis A virus P1 and 3C gene expression frames beta-actin-P13C are shown as SEQ ID NO. 1.

2. A method of constructing the recombinant duck plague virus of claim 1, comprising the steps of:

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

(2) Entry plasmid construction for expressing DHAV 3P 1 and 3C genes

The pCAGGS-P13C expression plasmid constructed in the above is subjected to enzyme digestion by SalI and BamHI, and the enzyme digestion product is recovered to obtain a P13C gene expression frame beta-actin-P1-3C, the nucleotide sequence of which is shown as SEQ ID NO. 1; cloning the obtained expression frame beta-actin-P13C into pENTR1 entry vector through SalI and BamHI cleavage sites to obtain entry plasmid pENTR1-P13C for expressing DHAV 3P 1 and 3C genes;

(3) Recombinant cosmid construction for expression of DHAV 3P 1 and 3C genes

Inserting a Kan-ccdB expression frame between the US7 and US8 genes of a recombinant cosmid C343 containing DEV attenuated vaccine C-KCE strain genome DNA fragments to obtain a recombinant mutant cosmid C343-US78-KanccdB; mixing an entry plasmid pENTR1-P13C with a recombinant mutant cosmid C343-US78-KanccdB and carrying out LR reaction to enable a beta-actin-P13C expression frame to replace a Kan-ccdB expression frame, so as to obtain a recombinant cosmid C343-US78-P13C with the beta-actin-P13C expression frame inserted between US7 and US8 genes 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 cosmid C343-US78-P13C containing P13C expression frames, co-transfecting CEF cells with five cosmids by a calcium phosphate transfection method, wherein cytopathic effect can be observed after 4-5 days of transfection, and the rescued recombinant virus is named rDEV-US78-P13C, namely the recombinant duck plague virus for expressing the P1 and 3C genes of the duck hepatitis A virus;

wherein, the recombinant cosmids C343, C027, C018, C144 and C211 are five pCC1Fos cosmids which respectively comprise DEV attenuated vaccine C-KCE strain genome DNA fragments and can splice and cover complete DEV genome, wherein, C027 comprises nucleotide fragments of 1-40133 positions of C-KCE strain genome, C018 comprises nucleotide fragments of 28323-67264 positions of C-KCE strain genome, C144 comprises nucleotide fragments of 59085-98008 positions of C-KCE strain genome, C211 comprises nucleotide fragments of 81629-67264 positions of C-KCE strain genome, and C343 comprises nucleotide fragments of 113857-158014 positions of C-KCE strain genome.

3. Use of the recombinant duck plague virus of claim 1 for the preparation of a medicament for preventing duck viral hepatitis and duck plague.

4. The use according to claim 3, wherein the medicament is a vaccine.