Recombinant duck enteritis virus live vector vaccine and application

Abstract

The invention provides a recombinant duck enteritis virus live vector vaccine and application, and belongs to the field of genetic engineering vaccines. The duck enteritis virus vaccine for expressing the duck parvovirus VP2 gene is constructed by inserting an expression framework containing the duck parvovirus VP2 gene between a US7 gene and a US8 gene of a duck enteritis virus genome by utilizing a recombinant cloning technology. After a specific pathogen-free SPF duck is immunized by the recombinant virus vaccine strain, the SPF duck can obtain immune protection on duck parvovirus and duck enteritis virus, and the recombinant virus vaccine strain can be used for preparing duck short beak dwarf syndrome and duck plague vaccines. The recombinant duck enteritis virus vaccine provided by the invention can provide multiple protection against duck parvovirus and duck enteritis virus infection at the same time, and has a good market application prospect.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering vaccines, and specifically relates to a recombinant duck enteritis virus live vector vaccine and application thereof. Background Art

[0002] Duck parvovirus (DPV) is a single-stranded DNA virus belonging to the Parvoviridae family and the Dependovirus genus. It mainly infects meat ducks such as Cherry Valley ducks and Muscovy ducks aged 10-25 days. The younger the infected ducks are, the higher the incidence rate is. The infected ducks will show obvious growth retardation, lameness or even paralysis. Although the mortality rate of duck parvovirus disease is not high, even after recovery, the infected ducks will show symptoms such as hair loss on the head and tail, reduced beaks, enlarged tongues, and severe growth retardation, which will cause them to lose their original economic value and seriously affect the economic benefits of my country's duck farming industry.

[0003] Duck virus enteritis (DVE) is an infectious disease caused by duck enteritis virus (DEV). The disease mainly infects ducks, geese and other Anseriformes. It is characterized by wide prevalence, rapid transmission, high mortality rate, and susceptibility to diseases of all age groups. Adult ducks and laying ducks are the most seriously affected, which seriously affects the development of waterfowl industry and brings huge losses to my country's duck farming industry. Duck enteritis virus belongs to the Herpesviridae family and the Marekvirus genus. Like other herpes viruses, the duck enteritis virus genome contains a large number of non-essential replication regions that can be used as insertion sites for exogenous genes, and is qualified to be used as a multi-link recombinant live vaccine vector. However, the existing technology will cause two or more recombinant virus vector vaccines to compete with each other in the duck after duck enteritis viruses that express different exogenous genes are simultaneously inoculated, thereby leading to immunization failure. Moreover, inserting different types of exogenous genes into the same duck enteritis virus vector will result in a problem of only having a good immune effect on one of the diseases, or an unsatisfactory immune effect on all diseases. Therefore, there is an urgent need for a recombinant duck enteritis virus vector vaccine that can simultaneously express multiple exogenous antigen genes, stably propagate, and provide good immune protection. Summary of the invention

[0004] The purpose of the present invention is to provide a recombinant duck enteritis virus.

[0005] The purpose of the present invention is to provide a method for constructing a recombinant duck enteritis virus.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] The recombinant duck enteritis virus comprises an expression frame of the VP2 gene coding sequence of duck parvovirus, and the nucleotide sequence of the expression frame is shown in SEQ ID NO.1.

[0008] Furthermore, the expression framework comprises a promoter, and the promoter is an mCMV promoter or a SV40ployA promoter.

[0009] A vaccine containing the recombinant duck enteritis virus.

[0010] Furthermore, the insertion position of the expression frame is the non-coding region between US7 and US8 of the duck viral enteritis virus genome.

[0011] The invention discloses an application of the recombinant duck enteritis virus in preparing a duck short-beak dwarfism syndrome vaccine.

[0012] The application of the recombinant duck enteritis virus in the preparation of duck plague vaccine.

[0013] The present invention utilizes recombinant cloning technology to insert an expression frame containing duck parvovirus VP2 gene into duck enteritis virus genome US7 and US8 genes, and constructs a duck enteritis virus vaccine expressing duck parvovirus VP2 gene. After immunizing specific pathogen-free (SPF) ducks with the recombinant virus vaccine strain, the SPF ducks can obtain immune protection against duck parvovirus and duck enteritis virus, and can be used for the preparation of duck short-beak dwarfism syndrome and duck plague vaccines.

[0014] The recombinant duck enteritis virus vaccine provided by the present invention can provide multiple protections against duck parvovirus and duck enteritis virus infection at the same time, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings, which constitute part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention. In the accompanying drawings:

[0016] Figure 1 This is the result of PCR detection of VP2 gene of rDEV-VP2, where M is DL2000 marker and 1 is VP2;

[0017] Figure 2 These are immunofluorescence images of rDEV-VP2 expressing VP2 protein, where the left image is a fluorescence image of VP2 protein expressing DPV, and the right image is a control cell. DETAILED DESCRIPTION

[0018] The following specific descriptions are exemplary and are intended to provide further explanation of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements fall within the protection scope of the present invention.

[0019] Unless otherwise specified, the test methods used in the following experimental examples are conventional methods.

[0020] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0021] Example 1. Establishment of rDEV-VP2 recombinant virus stably expressing VP2 protein

[0022] Using homologous recombination genetic manipulation technology, the homologous recombination arms of the US7-US8 region of the DEV / RPZT01 virus were constructed into the 5' and 3' regions of the mCMV-EGFP-SV40polyA gene fragment to produce "US7 homologous arm-mCMV-EGFP-SV40polyA-US8 homologous arm", which was simultaneously transfected into CEF cells with the DEV / RPZT01 virus genomic DNA. The mCMV-EGFP-SV40polyA gene was inserted into the US7-US8 region of the DEV / RPZT01 virus genome by homologous recombination, and the rDEV-EGFP virus with green fluorescence was purified by plaque purification. Similarly, the homologous recombination arms of the US7-US8 region of the DEV / RPZT01 virus were constructed into the 5' and 3' regions of the mCMV-VP2-SV40polyA gene fragment to produce the "US7 homologous arm-mCMV-VP2-SV40polyA-US8 homologous arm" gene fragment. This fragment and the rDEV-EGFP genomic DNA were transfected into CEF cells at the same time. The mCMV-VP2-SV40polyA fragment would replace the EGFP gene by homologous recombination, and the rDEV-VP2 virus without green fluorescence was purified by plaque purification. The nucleotide sequence of the mCMV-VP2-SV40polyA fragment is shown in SEQ ID NO.1.

[0023] Example 2. Verification of rDEV-VP2 recombinant virus

[0024] 1.PCR identification

[0025] The genomic DNA of the rDEV-VP2 recombinant virus was extracted, and the VP2 gene in the rDEV-VP2 recombinant virus was detected by PCR. Figure 1As shown, rDEV-VP2 can amplify the specific band of VP2.

[0026] 2. Indirect Immunofluorescence Identification

[0027] Infect cells with rDEV-VP2 recombinant virus. Discard the culture medium, wash the cells three times with washing solution, and remove the washing solution. Add cold methanol to each well and fix at 4°C for 30 minutes, discard the fixative, and wash three times with washing solution. Add PBS solution containing 2% bovine serum albumin to each well and block at 37°C for 30 minutes. Discard the blocking solution, wash three times with 1mL PBS solution, and absorb the excess washing solution with filter paper. Dilute the DPV polyclonal antibody with 2% BSA, add the polyclonal antibody, and incubate at 37°C for 2 hours. Discard the primary antibody, wash three times with PBS, add the goat anti-chicken secondary antibody diluted 200 times with 2% BSA, and incubate at 37°C for 1 hour. Wash three times with PBS, observe and record the results with an inverted fluorescence microscope. The results are as follows. Figure 2 The test results showed that the expression effect of DPV VP2 protein was good.

[0028] Example 3. Immune effect test of recombinant duck enteritis virus live vector vaccine

[0029] CEF cells were infected with rDEV-VP2. Three days after infection, the virus supernatant was collected by repeated freezing and thawing, and the virus titer was determined. Forty one-day-old SPF ducks were randomly divided into 6 groups, with 10 ducks in each group. rDEV-VP2 (containing 10 4 TCID 50 ), group 3 received subcutaneous injection of DEV CVCC AV1222 vaccine strain (containing 10 4 TCID 50 ) and group 4 received subcutaneous injection of DPV SBDS-HB01 vaccine strain (containing 10 4 TCID 50 ), Group 5 and Group 6 were not immunized and served as the control group. Each group was isolated and raised separately. 14 days after immunization, the leg muscles of Group 1, Group 3 and Group 5 were inoculated with the strong strain of DEV CVCC AV1221, and the amount of each inoculation was 100LD50. The leg muscles of Group 2, Group 4 and Group 6 were inoculated with the strong strain of DPV JS-01, and the amount of each inoculation was 100ID50. The rats were observed for 14 days, and the incidence and mortality were recorded every day to analyze the protective effect of rDEV-VP2 recombinant virus against DEV strong virus and duck parvovirus.

[0030] Table 1 Animal experiment grouping table

[0031]

[0032]

[0033] The results showed that: against the strong DEV virus, ducks immunized with recombinant rDEV-VP2 and DEV vaccine strains were 100% protected, and all the ducks in the control group died within 6 days. The results are shown in Table 2; against the strong duck parvovirus virus, ducks immunized with recombinant rDEV-VP2 were 100% protected, and the average neutralizing antibody titer of serum collected 14 days after immunization was 28, and no detoxification was detected in cloacal swabs collected 14 days after the virus attack; the protection rate of ducks immunized with DPV vaccine strains was 90%, and the average neutralizing antibody titer of serum collected 14 days after immunization was 27.6, and no detoxification was detected in cloacal swabs collected 14 days after the virus attack; the incidence rate of the control group was 7 / 10, and the serum collected 14 days after immunization showed no neutralizing antibodies, and 7 ducks were found to be shedding detoxified in cloacal swabs collected 14 days after the virus attack, the results are shown in Table 3.

[0034] Table 2 Protection of duck enteritis virus rDEV-VP2 strain against DEV virulent

[0035]

[0036] Table 3 Protection of duck enteritis virus rDEV-VP2 strain against DPV virulent

[0037]

[0038]

[0039] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A recombinant duck enteritis virus, characterized in that: The recombinant duck enteritis virus comprises the expression frame of the VP2 gene coding sequence of duck parvovirus, and the nucleotide sequence of the expression frame is shown in SEQ ID NO.

1.

2. The recombinant duck enteritis virus according to claim 1, characterized in that The expression framework comprises a promoter, and the promoter is an mCMV promoter and an SV40ployA promoter.

3. A vaccine containing the recombinant duck enteritis virus according to claim 1.

4. The recombinant duck enteritis virus according to claim 1, characterized in that The insertion position of the expression frame is the non-coding region between US7 and US8 of the duck viral enteritis virus genome.

5. Use of the recombinant duck enteritis virus as claimed in claim 1 in the preparation of a duck short-beak dwarfism syndrome vaccine.

6. Use of the recombinant duck enteritis virus as claimed in claim 1 in the preparation of duck plague vaccine.

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