Porcine infectious diarrhea vaccine taking vesicular stomatitis virus as carrier

Abstract

The invention discloses a porcine infectious diarrhea vaccine taking vesicular stomatitis virus as a carrier, and relates to the technical field of research and development of biological products for prevention. The porcine infectious diarrhea vaccine taking the vesicular stomatitis virus as the vector comprises a recombinant virus or an inactivated virus thereof; the recombinant virus is obtained by virus rescue by a recombinant virus vector and helper plasmids through a reverse genetic technology; the recombinant virus vector is obtained by taking VSV as a vector and replacing a G protein gene of the VSV with an S protein gene of a porcine infectious diarrhea virus. The toxicity of the vesicular stomatitis virus losing the surface envelope protein is greatly reduced, and the vesicular stomatitis virus is very suitable for being used as a virus vector. Experiments prove that the recombinant vaccine provided by the invention has good safety and effectiveness, has no neurotoxicity to mice, and can generate high serum neutralizing antibody titer at the same time. And a good neutralizing antibody titer is also generated in a piglet immune test. The invention lays a solid foundation for further research and development of PEDV vaccines, and provides a new strategy for safely and effectively preventing and controlling PEDV for the pig industry.

Description

Technical Field

[0001] The invention relates to the technical field of research and development of preventive biological products, and in particular to a porcine infectious diarrhea vaccine using vesicular stomatitis virus as a carrier. Background Art

[0002] Porcine epidemic diarrhea (PED) is an acute, highly contagious intestinal infectious disease that is particularly targeted at piglets. It can occur in pigs of any age. The younger the age, the more severe the symptoms and the higher the mortality rate. Although adult pigs have milder symptoms after infection, they can still spread the virus. Once a pig in the pig herd becomes ill, the disease will spread rapidly, causing huge economic losses.

[0003] The pathogen PEDV that causes the disease is a positive-strand RNA virus of the genus Alphacoronavirus of the family Coronaviridae. Its genome encodes four structural proteins: spike protein (S), membrane protein (M), envelope protein (E) and nucleocapsid (N) protein. Among them, the S protein is a PEDV host protective antigen, which is responsible for virus attachment, receptor binding, cell membrane fusion and entry, and plays an important role in the virus's pathogenic mechanism. Its external domain contains antigenic epitopes that mediate the production of neutralizing antibodies by host cells and receptor binding sites that bind to host cells.

[0004] Currently, the main methods to combat PEDV are through biosafety measures and vaccination. Since 1994, the development and promotion of inactivated and live attenuated vaccines of CV777 strain have played an important role in controlling PEDV infection. However, with the mutation of the virus, the protection of existing PEDV inactivated vaccines is limited.

[0005] Vesicular Stomatitis Virus (VSV) belongs to the Rhabdoviridae family and can infect a variety of animals and insects. Among livestock, horses, cattle (sheep), and pigs are naturally infected with VSV. Because it can spread across synapses in the interganglionic direction, it has potential neurotoxicity. Under the condition of overcoming neurotoxicity and ensuring safety, VSV can efficiently express exogenous proteins and induce a high level of immune response in the host. It is a potential vaccine vector and has been well applied.

[0006] In view of the many advantages of VSV as a vaccine vector and the problem that the current PEDV inactivated vaccine is insufficient in protection, this application intends to use it to develop a PEDV vaccine for pigs. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a porcine infectious diarrhea vaccine with vesicular stomatitis virus as a carrier in view of the many problems existing in the current development and application of PEDV vaccines. The vaccine has the advantages of 1. high safety; 2. convenient production and the like.

[0008] In order to solve the above problems, the present invention proposes the following technical solutions:

[0009] In a first aspect, the present invention provides a recombinant viral vector, the genome of which comprises replacing a surface envelope protein encoding gene in the genome of a vesicular stomatitis virus with a spike protein encoding gene of a PEDV virus;

[0010] Alternatively, the genome of the recombinant viral vector includes replacing the surface envelope protein encoding gene in the genome of the vesicular stomatitis virus with the spike protein encoding gene of the PEDV virus, and inserting a foreign protein encoding gene into the genome of the vesicular stomatitis virus.

[0011] Studies have found that when the vesicular stomatitis virus loses its surface envelope protein G protein or the G protein is replaced by the envelope protein of other viruses, the cell budding process can still be completed. The vesicular stomatitis virus that loses the G protein has greatly reduced toxicity and is very suitable for use as a viral vector. The recombinant viral vector constructed by the present invention uses VSV as a vector, and replaces its G protein gene with the S protein gene of porcine infectious diarrhea virus. It has been experimentally verified that the recombinant VSV with the VSV envelope protein replaced can effectively induce a specific immune response.

[0012] Furthermore, the PEDV virus is derived from a virus of the G1 lineage or a virus of the G2 lineage.

[0013] Furthermore, the viruses of the G1 lineage include the PEDV SD-M strain, and the viruses of the G2 lineage include the NH-TA2020 strain.

[0014] The PEDV virus is PEDV SD-M strain, GeneBank Accession no. AFX98013.1, or NH-TA2020 strain, GeneBank Accession no. ON168803, etc.

[0015] Furthermore, the spike protein of the PEDV virus includes a truncation of the spike protein, and the truncation is selected from a protein corresponding to the C-terminal amino acid deletion of the spike protein, and the number of the C-terminal deleted amino acids is 5 to 19.

[0016] For example, for the PEDV SD-M strain, the amino acid sequence of its spike protein is shown as SEQ ID NO.1, the amino acid sequence of the corresponding truncation is shown as SEQ ID NO.3, or an isofunctional sequence that has at least 95% identity with SEQ ID NO.3.

[0017] For the NH-TA2020 strain, the amino acid sequence of its spike protein is shown in SEQ ID NO.2, the amino acid sequence of the corresponding truncation is shown in SEQ ID NO.4, or an isofunctional sequence that has at least 95% identity with SEQ ID NO.4.

[0018] According to the above-mentioned amino acid sequence and codon rules, those skilled in the art can obtain the nucleotide sequence of the spike protein (including truncations) of the PEDV virus. Due to the degeneracy of the codons, the nucleotide sequence encoding the above-mentioned spike protein is not unique. All nucleotide sequences that can encode the spike protein of the above-mentioned PEDV virus are within the scope of protection of the present invention.

[0019] For example, the nucleotide sequence of the gene encoding the truncated spike protein of the PEDV SD-M strain is shown in SEQ ID NO.5, or a functionally identical sequence having at least 95% homology to SEQ ID NO.5.

[0020] The nucleotide sequence of the gene encoding the truncated spike protein of the NH-TA2020 strain is shown in SEQ ID NO.6, or a functionally identical sequence having at least 95% homology with SEQ ID NO.6.

[0021] In a specific embodiment, the present invention clones the corresponding coding gene sequence of the S protein of the PEDV virus lacking the last 5-19 amino acids into the G protein gene position in the VSV virus by homologous recombination (Uniclone One Step Seamless Cloning Kit) and replaces the ORF region of the G protein gene to construct a viral vector for preventing PEDV.

[0022] Furthermore, the recombinant virus vector of the present invention further comprises introducing a foreign protein encoding gene into the vesicular stomatitis virus genome, wherein the foreign protein is a tracer protein or an antigen protein. The introduction of the tracer protein encoding gene facilitates the detection of virus amplification and neutralizing antibody detection by detecting signals such as fluorescence, and the introduction of the antigen protein is conducive to the development of multivalent multi-combination vaccines.

[0023] Furthermore, the tracer protein includes green fluorescent protein GFP.

[0024] The introduction of exogenous protein encoding genes will only further reduce the toxicity of the recombinant virus vector and increase the safety of the recombinant virus. The exogenous protein encoding gene is inserted between the nucleoprotein N encoding gene and the phosphoprotein P encoding gene in the vesicular stomatitis virus genome; or the exogenous protein encoding gene is inserted between the matrix protein M encoding gene in the vesicular stomatitis virus genome and the spike protein S encoding gene (or the truncated version of the spike protein S encoding gene) of the PEDV virus.

[0025] In a second aspect, the present invention provides a recombinant virus obtained by rescuing the virus from the recombinant virus vector and helper plasmid described in the first aspect through reverse genetics technology;

[0026] The recombinant virus comprises vesicular stomatitis virus nucleoprotein, vesicular stomatitis virus phosphoprotein, vesicular stomatitis virus matrix protein, vesicular stomatitis virus RNA polymerase and PEDV virus spike protein, and does not comprise vesicular stomatitis virus surface envelope protein;

[0027] The recombinant virus has the ability to replicate and proliferate in cells without adding trypsin.

[0028] The recombinant virus provided by the present invention can effectively induce immune response after virus rescue, and has the ability to replicate and proliferate in cells without adding trypsin.

[0029] Furthermore, the cells include African green monkey kidney (Vero) cells and their derivative cell lines, such as Vero-E6, and hamster kidney fibroblast (BHK21) cells.

[0030] It should be noted that the above-mentioned replacement refers to replacing the ORF region of the surface envelope protein encoding gene in the vesicular stomatitis virus genome with the spike protein encoding gene of the PEDV virus strain.

[0031] Furthermore, the recombinant virus is highly safe and has no neurotoxicity to mice.

[0032] The present invention also provides a method for preparing the recombinant virus, comprising: infecting a sensitive cell strain with the recombinant virus, and harvesting the released virus.

[0033] The sensitive cell lines include African green monkey kidney (Vero) cells and their derivative cell lines, and BHK21 cells.

[0034] Furthermore, the method for preparing the recombinant virus does not require the addition of trypsin during the process of the recombinant virus infecting host cells.

[0035] In the production process of conventional PEDV inactivated vaccines, pancreatic enzymes are required to assist PEDV in infecting and amplifying cells normally, and the addition of pancreatic enzymes will increase the subsequent purification time and economic costs. In the preparation method of the recombinant virus provided by the present invention, high-titer viruses can be harvested without adding pancreatic enzymes, which greatly saves the production and purification costs of the virus.

[0036] The present invention also provides a vaccine strain prepared by using the recombinant virus vector.

[0037] In a third aspect, the present invention further provides a porcine infectious diarrhea vaccine using vesicular stomatitis virus as a carrier, the vaccine comprising the recombinant virus described in the second aspect; the vaccine strain described above;

[0038] Alternatively, the vaccine comprises an inactivated virus of the recombinant virus described in the second aspect.

[0039] The present invention also provides any of the following applications of the recombinant viral vector, the recombinant virus, the vaccine strain or the vaccine:

[0040] (1) Application in the preparation of recombinant vesicular stomatitis virus expressing PEDV virus spike protein;

[0041] (2) Use in the preparation of drugs for preventing and / or treating PEDV infection or diseases caused by PEDV infection;

[0042] (3) Application in the preparation of vaccines for preventing the spread and infection of porcine infectious diarrhea virus.

[0043] Compared with the prior art, the technical effects achieved by the present invention include:

[0044] The recombinant viral vector provided by the present invention replaces the surface envelope protein encoding gene in the genome of the vesicular stomatitis virus with the spike protein gene of the PEDV virus through molecular biological technology. The vesicular stomatitis virus that loses the surface envelope protein has its toxicity greatly reduced, and is very suitable for use as a viral vector.

[0045] The method for preparing the recombinant virus provided by the present invention infects host cells with the recombinant virus, and the recombinant virus can complete replication and proliferation without adding exogenous trypsin, and high-titer virus can be harvested, which will effectively reduce the cost of producing purified virus in the industrial production process.

[0046] The porcine infectious diarrhea vaccine with vesicular stomatitis virus as a carrier provided by the present invention uses VSV as a carrier, replaces its G protein gene with the S protein gene of porcine infectious diarrhea virus, and utilizes reverse genetic technology to construct a recombinant vaccine for preventing PEDV. Experimental verification shows that the recombinant vaccine of the present invention has good safety and effectiveness.

[0047] The porcine infectious diarrhea vaccine with vesicular stomatitis virus as a carrier provided by the present invention uses VSV as a carrier, replaces its G protein gene with the S protein gene of porcine infectious diarrhea virus, or replaces its G protein gene with the S protein gene of porcine infectious diarrhea virus and inserts the exogenous protein encoding gene GFP, and is constructed by using reverse genetic technology. The recombinant virus has good growth characteristics at the cell level, and shows good genetic stability and growth characteristics after continuous passage. In terms of safety evaluation, the study was conducted by using Balb / C mice as an animal model. The results show that after the vaccine is administered by intramuscular and intracranial routes, the mice did not show adverse reactions and clinical symptoms, and the weight changes were stable, showing the good safety of the vaccine. In terms of effectiveness, experiments have shown that the preliminary research results of the porcine infectious diarrhea vaccine provided by the present invention are excellent, and the serum neutralizing antibody titers are significantly higher than the serum neutralizing antibody levels of mice in the control group: after immunization on piglets, the porcine infectious diarrhea vaccine provided by the present invention can induce piglets to produce higher antibody levels. 33 days after the first immunization, the level of neutralizing antibodies in the serum of piglets was significantly higher than the antibody level measured from clinical positive serum. This result not only proves the potential effectiveness of the porcine infectious diarrhea vaccine provided by the present invention, but also lays a solid foundation for further research and development of PEDV vaccines. We expect that this vaccine can provide a safe and effective new strategy for the prevention and control of PEDV in the pig industry in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 A schematic diagram of the structure of a PEDV vaccine constructed using a VSV vector provided in an embodiment of the present invention;

[0049] Figure 2 A schematic diagram of the structure of a PEDV vaccine constructed using a VSV vector provided in another embodiment of the present invention;

[0050] Figure 3 The fluorescence microscope and white light photography results of the passage and amplification of the recombinant virus provided in the embodiment of the present invention in the sensitive cell line, the red circles are marked diseased cells;

[0051] Figure 4 A growth dynamics curve diagram of the recombinant virus provided in an embodiment of the present invention;

[0052] Figure 5 The results of the weight changes of mice immunized with the recombinant vaccine provided by the embodiment of the present invention by intramuscular injection;

[0053] Figure 6 The results of the weight changes of mice after intracranial inoculation of the recombinant vaccine provided in the embodiment of the present invention;

[0054] Figure 7The recombinant vaccine provided in the embodiments of the present invention is used to measure the survival rate of mice after intracranial inoculation. DETAILED DESCRIPTION

[0055] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments. Obviously, the embodiments described below are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0057] It should also be understood that the terms used in this description of the embodiments of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present invention. As used in the description of the embodiments of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms of "a", "an" and "the" are intended to include plural forms.

[0058] Understandably, according to the phylogenetic tree constructed from the whole genome of PEDV, porcine infectious diarrhea virus can be divided into two groups: the "classic strain" represented by CV777 and the "variant strain" discovered after 2010, which are distantly related. According to the gene phylogenetic tree constructed based on its antigenic protein S, PEDV can be divided into two gene groups, G1 and G2. Among them, G1 mainly includes early discovered strains (such as CV777) and some classic strains (such as LZC and SM98), as well as vaccine-adapted strains from China and South Korea, and G2 covers most of the variant strains after 2010.

[0059] The following examples take the G1 lineage PEDV SD-M strain and the G2 lineage NH-TA2020 strain as examples, replace the G protein gene in VSV with the S protein gene (S protein truncation gene) of the above virus strains, construct PEDV vaccines, and verify safety and effectiveness. It can be expected that the PEDV vaccines constructed by the method of the present invention using other PEDV virus strains are also safe and effective. Those skilled in the art replace the above PEDV SD-M strain and NH-TA2020 strain with other PEDV virus strains, and the recombinant viruses or PEDV vaccines constructed by the method of the present invention belong to the scope of protection of the present invention.

[0060] Example 1

[0061] Method for preparing VSV vector PEDV vaccine candidate strains (virus construction and rescue)

[0062] See also Figure 1 , which is a schematic diagram of the structure of the PEDV vaccine constructed using the VSV vector of the present invention. In this embodiment, VSV is used as a vector, its G protein gene is replaced with the S protein gene of porcine infectious diarrhea virus, and a recombinant vaccine for preventing PEDV is constructed using reverse genetic technology.

[0063] See also Figure 2 In another embodiment of the present invention, based on the above embodiment, a gene encoding green fluorescent protein GFP is further inserted into the vesicular stomatitis virus genome to facilitate virus amplification by detecting fluorescent signals and to facilitate the detection of neutralizing antibodies.

[0064] The S protein sequence of this example is derived from PEDV SD-M strain (GeneBank Accessionno.AFX98013.1) and NH-TA2020 strain (GeneBank Accessionno.ON168803), and the amino acid sequences are SEQ ID NO.1 and SEQ ID NO.2, respectively. The C-terminus is truncated (the resulting amino acid sequences are SEQ ID NO.3 and SEQ ID NO.4, respectively) and the genes are synthesized after codon optimization. The nucleotide sequences after codon optimization are SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0065] The vesicular stomatitis virus strain in this example is the Indiana strain.

[0066] After gene synthesis, the above synthetic fragments and VSV vector were PCR amplified according to the instructions of Primer Star enzyme. The primer sequence information of amplification is shown in Table 1.

[0067] The coding gene sequences of the S protein SEQ ID NO.3 and SEQ ID NO.4 with the last 19 amino acids missing were cloned into the G protein gene position of the viral vector VSV by homologous recombination (Uniclone One Step Seamless Cloning Kit) and replaced the ORF region of the G protein gene to form a full-length recombinant virus plasmid carrying PEDV-S protein of VSV, which was named pVSV-G1S Δ19 and pVSV-G2S Δ19On this basis, another embodiment of the present invention clones the green fluorescent protein GFP gene between the VSV-M coding gene and the PEDV-S coding gene of the above-mentioned viral vector by homologous recombination to form a recombinant virus full-length plasmid carrying VSV GFP and PEDV-S protein coding genes, which are named pVSV-GFP-G1S Δ19 and pVSV-GFP-G2S Δ19 .

[0068] Table 1 Amplification primer sequences

[0069]

[0070]

[0071] The specific plasmid construction process is as follows: 1. Use DNA polymerase (Primer Star) to perform PCR amplification to obtain the corresponding fragments; 2. Each fragment is recombined using homologous recombinase (Uniclone One Step Seamless Cloning Kit) and transformed into competent cells; 3. Pick a single colony and use universal vector primers and Taq enzyme to perform bacterial liquid PCR, and send the PCR product with the correct band size for testing; 4. Extract the plasmid from the colony clone with correct sequencing.

[0072] The virus rescue method is as follows: BHK-21 cells are infected with poxvirus expressing T7 polymerase, followed by transfection of full-length plasmids and auxiliary plasmids expressing VSV-N, VSV-P, VSV-L, and VSV-G. After 48 hours, the cells and supernatant are collected and filtered through a 0.22 μm filter to obtain the supernatant for later use. The virus stock solution is inoculated into new Vero cells (no need to add trypsin), and the cultured cells are observed for lesions or fluorescence. If lesions or fluorescence occur, the cells are collected again and repeatedly frozen and thawed three times, filtered through a 0.45 μm filter, and then frozen at -80°C to obtain the virus stock solution. The collected recombinant viruses are named VSV-G1S Δ19 and VSV-G2S Δ19 , VSV-GFP-G1S Δ19 and VSV-GFP-G2S Δ19 And measure the recombinant virus titer.

[0073] The recombinant virus titer was measured using the Reed-Muench method. The virus was diluted 10 times and inoculated into a 96-well plate with Vero cells. After 48 hours of culture, the cytopathic effect or fluorescence was observed, and the number of positive and negative wells was recorded, and the virus TCID was calculated. 50 After measurement, the virus titer of the recombinant virus of the present invention can reach 10 6.5 TCID50 / ml and above. It can be seen that the recombinant virus without VSV-G envelope protein of the present invention can be rescued by virus reverse genetics technology.

[0074] Example 2 Stable passage of recombinant virus and determination of growth kinetic curve

[0075] 1. Stability verification

[0076] Vero cells were plated in 6-well plates, and 100 μl of virus was inoculated in each well. The virus could be continuously subcultured and replicated. It can be seen that the recombinant virus harvested in Example 1 can be stably subcultured in Vero cells. To verify the replication ability of the recombinant virus in sensitive cell lines, Vero cells, Vero-E6 cells, and BHK-21 cells were plated in 12-well cell plates and cultured at 37°C and 5% CO2 for 12-24 hours. When the growth density reached 90-100% under a microscope, the recombinant virus was inoculated at a dose of MOI = 0.1. After 72 hours, the cell fluorescence expression and pathological conditions were observed.

[0077] The results are as follows Figure 3 As shown, compared with the control group, the recombinant virus VSV-G1S Δ19 and VSV-G2S Δ19 Different forms of cell lesions can be observed in the cells. In Vero cells and Vero-E6 cells, the cells aggregate and become round. In BHK-21 cells, the cell morphology changes from long strips to round. Recombinant virus VSV-GFP-G1S Δ19 and VSV-GFP-G2S Δ19 Infected cells can be observed for fluorescence expression and cytopathic effects. The results show that the above recombinant viruses can be stably propagated and replicated in Vero cells, Vero-E6 cells, and BHK-21 cells.

[0078] 2. Growth dynamics curve

[0079] In order to verify the recombinant virus VSV-G1S Δ19 and VSV-G2S Δ19 , VSV-GFP-G1S Δ19 and VSV-GFP-G2S Δ19In terms of intracellular replication dynamics, we established a growth curve in Vero cells. The specific steps are as follows: The recombinant virus and the control VSV wild-type virus (VSV-WT) were inoculated into a 6-well cell plate of Vero monolayer cells at a dose of MOI = 0.1, and the infected cells were cultured at 37°C. After infection, the corresponding cell plates were repeatedly frozen and thawed three times at 12, 24, 48, 72, and 96 hours, and the virus solution was collected. Frozen at -80°C. The virus solution at each time point was diluted 10 times in a gradient manner and inoculated into a 96-well plate with Vero cells, and the virus TCID was calculated according to the Reed-Muench method. 50 , a one-step virus growth curve was drawn with time as the horizontal axis and virus titer as the vertical axis.

[0080] The growth kinetics curve of the recombinant virus is as follows Figure 4 ,VSV-G1S Δ19 、VSV-G2S Δ19 , VSV-GFP-G1S Δ19 , VSV-GFP-G2S Δ19 The overall growth trend of VSV-WT was similar. After 24 hours of infection with the parent virus VSV-WT, the virus growth titer reached the highest level, which was 10 8.8 TCID 50 / ml;VSV-G1S Δ19 and VSV-GFP-G1S Δ19 The virus titer reached its highest level at 48 h after infection, which was 10 6.8 TCID 50 / ml;VSV-G2S Δ19 The growth kinetic curve is close to that of VSV-GFP-G2S Δ19 The virus titer reached its highest level at 72 hours after infection, which was 10 6.5 TCID 50 / ml. VSV-GFP-G1S Δ19 and VSV-GFP-G2S Δ19 It carries the reporter gene GFP and can be used in subsequent serum neutralization pseudovirus tests.

[0081] Example 3 Safety and effectiveness evaluation of mouse model

[0082] The inoculation reagent is the virus stock solution harvested in Example 1.

[0083] 1. Body weight changes of Balb / C mice after intramuscular injection:

[0084] The mice were inoculated by intramuscular injection, and the dose was 10^6TCID 50 / 50μl, stock solution diluted 10 times 10^5TCID 50 / 50μl, the body weight and status of Balb / C mice were monitored for 14 consecutive days, and the weight changes were plotted as follows: Figure 5 The results show that the weight of mice in each test group changed steadily 14 days after immunization, and no significant weight loss or abnormal behavior occurred, indicating that the recombinant virus VSV-G1S Δ19 and VSV-G2S Δ19 As a potential vaccine strain, it has good safety in small animal trials.

[0085] 2. Observation of body weight and neurological symptoms in Balb / C mice after intracranial inoculation

[0086] Since VSV virus has potential neurotoxicity, in order to verify the recombinant virus strain VSV-G1S prepared by the method of the present invention, Δ19 and VSV-G2S Δ19 With good safety, this experiment used intracranial inoculation to inoculate wild-type VSV-WT and VSV-G1S Δ19 and VSV-G2S Δ19 Directly inoculated into the mouse brain, the inoculation dose is 10^4TCID 50 / mouse, this dose of wild-type VSV inoculation can cause experimental central nervous system infection symptoms in mice, and a negative control (injection water group) was set up at the same time. The neurological symptoms of mice were recorded after the virus challenge to evaluate the safety of the virus.

[0087] The results of the weight changes of mice after the virus attack are as follows Figure 6 The results showed that VSV-G1S Δ19 Group and VSV-G2S Δ19 After intracranial injection, the mice in the VSV-WT group showed no neurological symptoms and their weight changed steadily, indicating that the vaccine was not pathogenic to mice. However, the mice in the positive control VSV-WT group showed significant weight loss and obvious symptoms of dullness, ruffled hair, curling up and trembling, and died on the 4th day after vaccination ( Figure 7 ), and the negative control PBS group mice showed stable physical signs.

[0088] 3. Balb / C mouse immune test

[0089] The mice were immunized twice by intramuscular injection, and the immunization procedure was "0+14". There were 4 experimental groups, namely, VSV-G1S Δ19 Stock solution immune group, VSV-G2S Δ19 The stock solution immune group, composed of VSV-G2S Δ19 The inactivated vaccine immunization group and PBS control group were prepared. Each mouse in each test group was injected with 50 μl, and the titer of the stock solution was 10^6 TCID 50After immunization, blood was collected from the eyeballs to obtain mouse serum on days 14, 21, and 28 to detect the neutralizing antibody titer against the recombinant virus.

[0090] The specific method of the neutralization experiment is as follows:

[0091] 1. Cell preparation: Vero cells were plated into 96-well cell plates and ready for use when the confluence reached 80%.

[0092] 2. Serum treatment: Inactivate the serum to be tested at 56°C for 30 minutes.

[0093] 3. Use DMEM medium containing 2% FBS to culture VSV-GFP-G1S Δ19 Pseudovirus and VSV-GFP-G2S Δ19 Pseudovirus was diluted to 200 TCID 50 / 100μL, which is the virus dilution solution for detection.

[0094] 4. Dilute the serum to be tested in a 3-fold ratio, add an equal volume of virus dilution solution to each dilution gradient, shake to mix, and incubate in a 37°C 5% CO2 incubator for 60 minutes.

[0095] 5. Take a 96-well cell plate, wash it twice with PBS solution, remove floating cells, add the incubated serum-virus mixture to the cell plate, and make 3 control wells for each dilution gradient, 100μl / well. At the same time, use the virus dilution as the virus control, and use 2% FBS DMEM culture medium as the negative control.

[0096] 6. Place the cell plate in a 37°C 5% CO2 incubator and culture for 72 hours.

[0097] 7. After the culture is completed, observe the percentage of cell fluorescence expression under a microscope and calculate the neutralizing antibody titer according to the Reed-Muench method.

[0098] The results of neutralizing antibody titers of each test group are shown in Table 2. It can be seen from the results that compared with the control group, the mouse sera of the test groups all produced high levels of neutralizing titers, among which VSV-G2S Δ19 The neutralization titer can reach 537 after 28 days of immunization, and the inactivated vaccine formed by inactivating the recombinant virus has a serum neutralization titer of up to 2148 after 21 days of the first immunization. From the above test results, it can be seen that the recombinant vaccine strains VPSQ and VPSQ1 rescued by the method of the present invention have good immunogenicity and safety.

[0099] Table 2 Neutralizing antibody titer results of each test group

[0100]

[0101]

[0102] Note: VSV-G1S Δ19 Group and VSV-G2S Δ19 The inactivated vaccine is VSV-G2S Δ19 It is prepared by inactivating it with β-propiolactone and adding adjuvant.

[0103] Example 4 Piglet Immunization Experiment

[0104] Five 4-day-old piglets with PEDV neutralizing antibody levels below the detection limit were selected for animal experiments: the virus (VSV-G1S Δ19 ) After the stock solution was inactivated, an inactivated vaccine was prepared with an adjuvant at 10^6.5 TCID 50 / dose vaccination, immunization was performed by intramuscular injection. Boosting immunization was performed 14 days after the first immunization. Serum was collected on the 14th, 21st and 33rd days after the first immunization, and the serum antibody level at each time point was detected by neutralization test.

[0105] The test results are shown in Table 3. According to the test results, the DIFF-PEDV recombinant inactivated vaccine provided by the present invention can stimulate the production of high-level neutralizing antibodies in 4-day-old piglets. Under the scheme of the first immunization and the booster immunization two weeks later, the serum neutralizing antibody titers at 21d and 33d after the first immunization were both higher than 1:214, which is an excellent candidate strain for the recombinant inactivated vaccine.

[0106] Table 2 Results of piglet immunization experiment

[0107] Piglet number Before immunization 14d antibody titer 21d antibody titer 33d antibody titer 1 0 1:163 1:677 1:515 2 0 1:214 1:2142 1:3910 3 0 0 1:1627 1:1627 4 0 1:708 1:677 1:391 5 0 1:22 1:214 1:1627

[0108] In summary, the porcine infectious diarrhea vaccine with vesicular stomatitis virus as a carrier provided by the present invention has good safety and effectiveness. After immunization in piglets, only two immunizations are needed to induce piglets to produce high antibody levels. 33 days after the first immunization, the neutralizing antibody level in the piglet serum was significantly higher than the antibody level measured from clinical positive serum. This result not only proves that the porcine infectious diarrhea vaccine provided by the present invention (such as VSV-G1S Δ19 and VSV-G2S Δ19 The potential effectiveness of this vaccine has also laid a solid foundation for further research and development of PEDV vaccines. We hope that this vaccine can provide a safe and effective new strategy for the prevention and control of PEDV in the pig industry in the future.

[0109] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0110] The above is a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A recombinant viral vector, characterized in that: The genome of the recombinant viral vector includes replacing the surface envelope protein encoding gene in the genome of the vesicular stomatitis virus with the spike protein encoding gene of the PEDV virus; Alternatively, the genome of the recombinant viral vector includes replacing the surface envelope protein encoding gene in the genome of the vesicular stomatitis virus with the spike protein encoding gene of the PEDV virus, and inserting a foreign protein encoding gene into the genome of the vesicular stomatitis virus.

2. The recombinant viral vector according to claim 1, characterized in that The PEDV virus includes a virus derived from the G1 lineage or a virus from the G2 lineage.

3. The recombinant viral vector according to claim 2, characterized in that The G1 lineage viruses include the PEDV SD-M strain, and the G2 lineage viruses include the NH-TA2020 strain.

4. The recombinant viral vector according to any one of claims 1 to 3, characterized in that The spike protein of the PEDV virus includes a truncation of the spike protein, wherein the truncation is selected from a protein corresponding to a C-terminal amino acid deletion, and the number of C-terminal deleted amino acids is 5 to 19.

5. The recombinant viral vector according to claim 1, characterized in that The exogenous protein includes green fluorescent protein GFP.

6. A recombinant virus, characterized in that The recombinant virus is obtained by rescuing the genome of the recombinant virus vector described in claims 1 to 5 and the auxiliary plasmid through reverse genetics technology; The recombinant virus comprises vesicular stomatitis virus nucleoprotein, vesicular stomatitis virus phosphoprotein, vesicular stomatitis virus matrix protein, vesicular stomatitis virus RNA polymerase and PEDV virus spike protein, and does not comprise vesicular stomatitis virus surface envelope protein; The recombinant virus has the ability to replicate and proliferate in cells without adding trypsin.

7. The recombinant virus according to claim 6, characterized in that The cells include hamster kidney fibroblast cells, African green monkey kidney cells or cells derived from African green monkey kidney cells.

8. The method for preparing a recombinant virus according to any one of claims 6 to 7, characterized in that: include: The recombinant virus according to any one of claims 6 to 7 is used to infect a sensitive cell line, and the released virus is harvested.

9. A porcine infectious diarrhea vaccine using vesicular stomatitis virus as a carrier, characterized in that: The vaccine comprises the recombinant virus according to any one of claims 6-7 or the recombinant virus prepared by the preparation method according to claim 8; or, the vaccine comprises an inactivated virus of the recombinant virus according to any one of claims 6-7.

10. Any of the following uses of the recombinant viral vector according to any one of claims 1 to 5, the recombinant virus according to any one of claims 6 to 7, the recombinant virus prepared according to claim 8, or the vaccine according to claim 9: (1) Application in the preparation of recombinant vesicular stomatitis virus expressing PEDV virus spike protein; (2) Use in the preparation of drugs for preventing and / or treating PEDV infection or diseases caused by PEDV infection; (3) Application in the preparation of vaccines to prevent the transmission and infection of porcine infectious diarrhea virus.

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