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CN117925662B - Construction method of recombinant vesicular stomatitis virus and Ebola hemorrhagic fever infection animal model - Google Patents

Construction method of recombinant vesicular stomatitis virus and Ebola hemorrhagic fever infection animal model Download PDF

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Publication number
CN117925662B
CN117925662B CN202311747661.6A CN202311747661A CN117925662B CN 117925662 B CN117925662 B CN 117925662B CN 202311747661 A CN202311747661 A CN 202311747661A CN 117925662 B CN117925662 B CN 117925662B
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virus
recombinant
vesicular stomatitis
gene
stomatitis virus
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CN117925662A (en
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闫飞虎
杨婉莹
赵永坤
冯娜
王铁成
夏咸柱
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Military Veterinary Research Institute Academy Of Military Medical Sciences
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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Abstract

The invention relates to the technical field of biology, in particular to a method for constructing a recombinant vesicular stomatitis virus and an Ebola hemorrhagic fever infection animal model. The recombinant vesicular stomatitis virus expressing the ebola virus envelope Glycoprotein (GP) provided by the invention can produce lethal infection on female golden mice, and clinical characterization and biochemical and tissue lesions after infection are similar to those of ebola virus infected by human beings, and can be used as a substitute virus of ebola virus for constructing an ebola hemorrhagic fever infection animal model or for detecting neutralizing antibodies of the ebola virus. The invention establishes a golden yellow ground mouse lethal infection model based on the recombinant vesicular stomatitis virus expressing the ebola virus envelope glycoprotein, and the animal model is a safe, effective and economic tool, and can be used for rapidly and preclinically evaluating medical countermeasures such as vaccines, antibodies, medicines and the like aiming at the EBOV under the biosafety level 2 laboratory condition.

Description

Construction method of recombinant vesicular stomatitis virus and Ebola hemorrhagic fever infection animal model
Technical Field
The invention relates to the technical field of biology, in particular to a method for constructing a recombinant vesicular stomatitis virus and an Ebola hemorrhagic fever infection animal model.
Background
Ebola virus disease (Ebola virus disease, EVD) is a virulent infectious disease caused by Ebola virus (EBOV) with severe bleeding that is an acute fever. Ebola virus (EBOV) is one of the members of the genus ebola of the family filoviridae, and can cause severe hemorrhagic disease in humans and non-human primates (NHPs), with mortality rates as high as 90%. Currently, two monoclonal antibodies have been used to treat ebola virus disease: mAb114 (Ansuvimab) and REGN-EB3 (Inmazeb). However, the difficulty in researching the EBOV is that the EBOV is a quaternary pathogen, and needs to be operated under the laboratory condition with extremely high biological safety level, and the experimental condition is harsh and has high cost, thus preventing the development of drugs and vaccines aiming at the EBOV. Thus, there is an urgent need for economical and convenient animal models to facilitate the development of products for the prevention and treatment of EBOV.
The vesicular stomatitis virus (Vesicular Stomatitis Virus, VSV) reverse genetics technology is widely applied to recombinant virus construction, and has the advantage of high safety. At present, although the recombinant vesicular stomatitis virus (EBV) expressing the Ebola virus envelope Glycoprotein (GP) is reported, the recombinant vesicular stomatitis virus is a recombinant virus used for vaccine development and antibody preparation, has weak pathogenicity, and has not been reported for constructing an Ebola virus infection animal model and constructing an infection animal model thereof.
Disclosure of Invention
The invention provides a method for constructing a recombinant vesicular stomatitis virus expressing ebola virus envelope glycoprotein and an ebola hemorrhagic fever infection animal model.
Based on the problems of the prior art, the invention aims to construct a substitute ebola virus which can be operated under the biosafety secondary laboratory condition (BSL-2) and can be used for constructing an ebola hemorrhagic fever infection animal model based on a VSV reverse genetic platform, and further establishes an ebola hemorrhagic fever infection animal model which can be used for in vivo evaluation of anti-EBOV drugs, antibodies and vaccines based on the substitute ebola hemorrhagic fever virus.
Specifically, the invention provides the following technical scheme:
In a first aspect, the present invention provides a recombinant nucleic acid molecule comprising a recombinant vesicular stomatitis virus genome;
The recombinant vesicular stomatitis virus genome is obtained by deleting a G gene in the vesicular stomatitis virus genome and inserting an Ebola virus GP gene between an N gene and a P gene;
Or the recombinant vesicular stomatitis virus genome is obtained by deleting a G gene in the vesicular stomatitis virus genome, inserting an ebola virus GP gene between an N gene and a P gene, and inserting a marker protein coding gene between an M gene and an L gene.
The invention has the advantages that the insertion position of the GP gene of the Ebola virus in the genome of the vesicular stomatitis virus has a great influence on the lethality of the recombinant virus. Insertion of the ebola virus GP gene between the N and P genes of the vesicular stomatitis virus genome can significantly increase the lethality of the recombinant virus to golden mice compared to other insertion sites (e.g., between the M and L genes).
In the present invention, the G gene of vesicular stomatitis virus is the gene encoding vesicular stomatitis virus glycoprotein (G), and N, P, M, L genes are the genes encoding vesicular stomatitis virus N protein, P protein, M protein and L protein, respectively. The ebola virus GP gene is a gene encoding ebola virus envelope Glycoprotein (GP).
The recombinant nucleic acid molecules described above are preferably recombinant DNA.
In the present invention, the ebola virus is preferably zaire ebola virus.
Compared with other types of Ebola viruses, the GP gene of the zaire type Ebola virus is more favorable for generating lethal infection, and is more suitable for constructing an Ebola hemorrhagic fever infection animal model.
Preferably, the nucleotide sequence of the zaire ebola virus GP gene is shown as SEQ ID NO. 1.
In some embodiments of the invention, a marker protein encoding gene is also introduced into the recombinant nucleic acid molecule, so as to facilitate detection of the recombinant virus by detecting a marker signal such as fluorescence. The kind of the labeled protein is not particularly limited in the present invention.
Alternatively, the marker protein is a fluorescent marker protein, including but not limited to green fluorescent protein.
In some embodiments of the invention, the marker protein encoding gene is an eGFP gene.
In some embodiments of the invention, the vesicular stomatitis virus genome is the genome of the vesicular stomatitis virus Indiana vaccine strain (Indiana), having Genbank accession No. J02428.1.
Preferably, the recombinant nucleic acid molecule further comprises a promoter located upstream of the recombinant vesicular stomatitis virus genome.
Preferably, the recombinant nucleic acid molecule further comprises a hepatitis delta virus ribozyme sequence downstream of the recombinant vesicular stomatitis virus genome.
Preferably, the promoters are the CMV promoter and the T7 promoter.
The sequences of the CMV promoter and the T7 promoter are respectively shown as 1-204bp and 205-223bp of the sequence shown in SEQ ID NO. 2.
The hepatitis delta virus ribozyme sequence is shown as 11933-12008bp of the sequence shown as SEQ ID NO. 2.
Preferably, the nucleotide sequence of the recombinant nucleic acid molecule is shown as SEQ ID NO. 2.
In a second aspect, the invention provides a biological material that is a recombinant vector or host cell;
the recombinant vector comprises the recombinant nucleic acid molecule described above;
The host cell comprises the recombinant nucleic acid molecule described above, or comprises the recombinant vector.
Preferably, the recombinant vector is a mammalian expression plasmid.
The recombinant vector is obtained by inserting the recombinant nucleic acid molecule into a mammalian expression plasmid. The kind of the mammalian expression plasmid is not particularly limited in the present invention, and all plasmids capable of replication and protein expression in animal cells may be used.
In some embodiments of the invention, the mammalian expression plasmid is pcdna3.1.
The recombinant vector and the auxiliary plasmid are used for co-transfecting host cells, so that the recombinant vesicular stomatitis virus expressing the Ebola virus GP protein can be obtained through virus rescue, and the recombinant vesicular stomatitis virus can be subjected to replication, proliferation and stable passage.
The host cells described above include microbial cells or animal cells. The present invention is not particularly limited as to the kind of host cell, wherein the microbial cells include, but are not limited to, E.coli, yeast, etc.; animal cells include, but are not limited to, golden hamster kidney cells (BSR), and the like.
In a third aspect, the present invention provides a recombinant vesicular stomatitis virus, wherein the recombinant vesicular stomatitis virus genome is obtained by deleting a G gene in a vesicular stomatitis virus genome and inserting an ebola virus GP gene between an N gene and a P gene;
Or the recombinant vesicular stomatitis virus genome is obtained by deleting a G gene in the vesicular stomatitis virus genome, inserting an ebola virus GP gene between an N gene and a P gene, and inserting a marker protein coding gene between an M gene and an L gene.
Preferably, the recombinant vesicular stomatitis virus comprises vesicular stomatitis virus nucleoprotein, vesicular stomatitis virus phosphoprotein, vesicular stomatitis virus matrix protein, vesicular stomatitis virus RNA polymerase, and ebola virus envelope Glycoprotein (GP), and does not comprise vesicular stomatitis virus glycoprotein.
Preferably, the recombinant vesicular stomatitis virus has replication and proliferation capabilities.
Preferably, the ebola virus is zaire ebola virus.
Preferably, the nucleotide sequence of the GP gene of the zaire type ebola virus is shown as SEQ ID NO. 1.
In some embodiments of the invention, a marker protein encoding gene is also introduced into the recombinant nucleic acid molecule, so as to facilitate detection of the recombinant virus by detecting a marker signal such as fluorescence. The kind of the labeled protein is not particularly limited in the present invention.
Alternatively, the marker protein is a fluorescent marker protein, including but not limited to green fluorescent protein.
In some embodiments of the invention, the marker protein encoding gene is an eGFP gene.
In some embodiments of the invention, the vesicular stomatitis virus genome is the genome of the vesicular stomatitis virus Indiana vaccine strain (Indiana), having Genbank accession No. J02428.1.
Preferably, the nucleotide sequence of the recombinant vesicular stomatitis virus genome is shown as 224-11932bp of SEQ ID NO. 2.
In a fourth aspect, the present invention provides a method of preparing a recombinant vesicular stomatitis virus as described above, the method comprising: introducing the recombinant vector containing the recombinant nucleic acid molecule and the auxiliary plasmid into a host cell to obtain a recombinant host cell, culturing the recombinant host cell and harvesting the released virus.
Preferably, the helper plasmid is four mammalian expression plasmids that express RNA polymerase, nucleoprotein, phosphoprotein and glycoprotein, respectively, of vesicular stomatitis virus.
The helper plasmids described above can be obtained by ligating the N gene, P gene, L gene and G gene of vesicular stomatitis virus, respectively, to mammalian expression plasmids.
The recombinant vesicular stomatitis virus can be obtained by utilizing the recombinant vector and the auxiliary plasmid and adopting a conventional recombinant virus rescue method in the field.
In a fifth aspect, the invention provides the use of any one of the recombinant nucleic acid molecules or the biological material or the recombinant vesicular stomatitis virus described above:
(1) The application in preparing recombinant vesicular stomatitis virus expressing ebola virus GP protein;
(2) The application in preparing an Ebola virus infection animal model;
(3) Use of a neutralizing antibody for detecting ebola virus in the preparation of a reagent for the presence or level of neutralizing antibody in a sample;
(4) Use of neutralizing antibodies to detect ebola virus in samples for non-disease diagnosis and treatment purposes, or levels thereof;
(5) Use in the manufacture of a vaccine for the prevention and/or treatment of ebola virus infection or a disease caused by ebola virus infection;
(6) Use in the manufacture of a medicament for the prevention and/or treatment of an ebola virus infection or a disease caused by an ebola virus infection;
(7) Use in the preparation of a reagent for diagnosing an ebola virus infection or a disease caused by an ebola virus infection.
In the above (1), the use is to prepare a recombinant vesicular stomatitis virus expressing ebola virus GP protein using the recombinant nucleic acid molecule or the biological material described above.
In the above (2), the animal model is preferably a golden mouse model.
In the above (3) and (4), the application comprises detection of the ebola virus neutralizing antibody by using the recombinant vesicular stomatitis virus.
In (4) above, the detection of non-disease diagnostic and therapeutic purposes includes, but is not limited to, detection of chemically synthesized or in vitro expressed ebola virus neutralizing antibodies.
In the above (6), the drug includes a neutralizing antibody and the like.
In the above (7), it is possible to diagnose whether or not the specimen is infected with the Ebola virus or a disease caused by the Ebola virus infection by detecting the presence and/or the level of the neutralizing antibody in the specimen such as serum.
In such applications, the disease caused by ebola virus infection includes ebola virus disease.
In a sixth aspect, the present invention provides a method for constructing an animal model of ebola virus infection, the method comprising: and (3) using the recombinant vesicular stomatitis virus to attack the toxicity of female golden mice.
The recombinant vesicular stomatitis virus can not produce lethal infection on mice, rats and guinea pigs, but the invention surprisingly discovers that the recombinant vesicular stomatitis virus can produce lethal infection on female golden mice, the body weight of the female golden mice is obviously reduced after infection, coagulation disorder and serious liver function damage occur, inflammation or damage occur on organs such as liver, spleen, lung, kidney and the like, secretion occurs on eyes, and the recombinant vesicular stomatitis virus is developed into serious systemic diseases similar to human Ebola virus patients; female golden mice die completely after 2-3 days of infection, histopathology shows that recombinant viruses target hepatocytes, and their tissue targeting is consistent with wild-type EBOV. Therefore, the recombinant vesicular stomatitis virus can be used for carrying out virus attack on female golden mice to construct an Ebola virus infection animal model, and is used for developing medicines such as Ebola virus vaccines, antibodies and the like and prevention and treatment means.
In the above construction method, the female golden mice are preferably 2-4 weeks old. More preferably 3 weeks of age. The present invention has found that female golden mice of the above ages are more susceptible to recombinant viruses than female golden mice of other ages.
In the above construction method, the toxicity counteracting mode is preferably intraperitoneal injection. The invention discovers that in different toxin attacking modes, golden mice can be all killed only by intraperitoneal injection; the pathogenicity of the recombinant vesicular stomatitis virus described above has been demonstrated to be species specific, age-related, sex-related and injection route-dependent.
In a seventh aspect, the present invention provides an animal model of ebola virus infection constructed by the method described above.
In an eighth aspect, the present invention provides any one of the following applications of the ebola virus infection animal model constructed by the construction method described above:
(1) Application in screening or effectiveness evaluation of Ebola virus antibodies;
(2) Application in screening or effectiveness evaluation of Ebola virus vaccines;
(3) Use in screening or efficacy evaluation of a drug for the prevention and/or treatment of ebola virus.
The beneficial effects of the invention at least comprise: the invention provides a recombinant vesicular stomatitis virus expressing ebola virus envelope Glycoprotein (GP), which can produce lethal infection to female golden mice, has clinical symptoms and biochemical and tissue lesions similar to those of human infected ebola virus after infection, can be used as a substitute virus of ebola virus, is used for constructing an ebola hemorrhagic fever infection animal model or is used for detecting an ebola virus neutralizing antibody, and solves the problems that vaccine and medicine development are difficult and no recombinant virus capable of producing lethal infection to animals is available at present because the ebola virus needs high biosafety level laboratory conditions.
The invention establishes a golden-yellow mice lethal infection model based on the recombinant vesicular stomatitis virus expressing Ebola virus envelope Glycoprotein (GP), and the Ebola hemorrhagic fever infection animal model shows disease signs of fever, weight loss, multi-organ failure, severe uveitis, high virus load and the like and develops into severe systemic diseases similar to human Ebola virus patients. The invention verifies the availability of the equine anti-EBOV immunoglobulin and the EBOV subunit vaccine by using the animal model, proves that the animal model is a safe, effective and economic tool, and can be used for rapidly and preclinically evaluating the medical countermeasures such as vaccines, antibodies, medicines and the like aiming at the EBOV under the condition of BSL-2.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of double digestion of recombinant plasmid p 3.1-VSV.DELTA.G-eGFP-EBOV/GP in example 1 of the present invention, wherein M: DNA MARKER,1: p 3.1-VSV.DELTA.G-eGFP-EBOV/GP.
FIG. 2 is a fluorescence microscope observation result of rescue and subculture of EBOV GP recombinant VSV virus in example 1 of the present invention.
FIG. 3 shows the results of RT-PCR identification of recombinant viruses VSV-EBOV/GP in example 1 of the present invention, wherein M: DNA MARKER,1: VSV-EBOV/GP,2: WT-VSV.
FIG. 4 shows the Western blot identification result of the recombinant virus VSV-EBOV/GP in example 1 of the present invention, wherein M: protein Marker,1: VSV-EBOV/GP,2: WT-VSV.
FIG. 5 shows the immunofluorescence assay of the recombinant virus VSV-EBOV/GP of example 1 of the present invention, wherein A: VSV-EBOV/GP, B: WT-VSV.
FIG. 6 is a morphological observation under a transmission electron microscope of the recombinant virus VSV-EBOV/GP in example 1 of the present invention, wherein (a): VSV-EBOV/GP, (b): WT-VSV.
FIG. 7 shows the growth kinetics of the recombinant virus VSV-EBOV/GP of example 1 of the present invention, wherein the left graph shows the growth kinetics of WT-VSV and VSV-EBOV/GP compared, and the right graph shows the growth kinetics of VSV-EBOV/GP at different MOI.
FIGS. 8 and 9 show susceptibility of recombinant virus VSV-EBOV/GP in rodents according to example 2 of the present invention.
FIG. 10 shows the effect of glycoprotein correlation and GP gene insertion position on the lethality of recombinant viruses according to example 2, wherein VSV-EBOV/GP (NP) is the recombinant virus VSV-EBOV/GP constructed in example 1 and VSV-EBOV/GP (ML) is the control recombinant virus.
FIG. 11 shows the clinical characterization changes of the golden yellow mice of different sexes after challenge in example 2 of the present invention.
FIGS. 12 and 13 show the results of biochemical and routine blood treatment of the golden mice of different sexes in example 2 of the present invention.
FIG. 14 shows the results of virus load detection of golden yellow mice of different sexes after challenge in example 2 of the present invention.
Fig. 15 and 16 show the virus change and immunohistochemical results after challenge of golden yellow rats of different sexes in example 2 of the present invention, wherein the scale of fig. 15 is 100 times (100×) magnification, and the scale of fig. 16 is 100 times (100×) magnification.
FIG. 17 is a diagram showing the appearance of secretions on the eyes of female golden yellow mice in example 2 of the present invention, wherein the left graph shows uninfected normal female golden yellow mice, and the right graph shows infected female golden yellow mice.
FIGS. 18 and 19 show the sensitivity of different ages of golden mice to VSV-EBOV/GP in example 2 of the present invention, wherein FIG. 18 shows the change in clinical characterization after challenge, and FIG. 19 shows the result of viral load detection.
Fig. 20, 21, 22 and 23 show the sensitivity of golden mice to VSV-EBOV/GP in different challenge modes in example 2 of the present invention, wherein fig. 20 shows survival rate and weight change after challenge, fig. 21 shows tissue viral load, fig. 22 shows pathological changes, and fig. 23 shows immunohistochemical results. FIG. 24 shows the use of the golden mouse infection model in the evaluation of antibodies in example 3 of the present invention. FIG. 25 shows the use of the golden mouse infection model in vaccine evaluation in example 3 of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The experimental scheme of the invention mainly comprises the following three aspects:
1. Construction and identification of EBOV GP recombinant VSV and application thereof in neutralizing antibody detection
The recombinant plasmid carrying the recombinant nucleic acid molecule shown in SEQ ID NO.2 is respectively co-transfected with 4 auxiliary plasmids into BSR cells by a calcium phosphate transfection method, an EBOV GP recombinant VSV (VSV-EBOV/GP) carrying an eGFP tag is obtained by rescue, the genome of the recombinant plasmid is obtained by deleting a G gene in a vesicular stomatitis virus genome, an Ebola virus GP gene is inserted between an N gene and a P gene, a marker protein coding gene is inserted between an M gene and an L gene, and the genome sequence is shown as 224-11932bp of SEQ ID NO. 2. Identifying whether the recombinant virus is constructed correctly by Western immunoblotting (Western-blot), indirect immunofluorescence (Indirect immunofluorescence, IF), reverse transcription polymerase chain reaction (Reverse Transcription Polymerase Chain Reaction, RT-PCR); measuring recombinant virus titer, drawing a growth dynamics curve, and detecting neutralizing antibody titer by using the recombinant virus. The result shows that the replication type EBOV GP recombinant VSV carrying the eGFP label is successfully rescued and successfully applied to antibody neutralization titer detection, and the neutralization titer of the equine anti-EBOV refined immunoglobulin is measured to be 1:64.
2. Establishment of VSV-EBOV/GP golden mice infection model
Infecting the recombinant virus with 10 7TCID50 intraperitoneal injection to 3-week-old BALB/c mice, SD rats, guinea pigs and golden mice, observing clinical signs, and determining pathogenicity of EBOV GP glycoprotein; detecting viral load and pathological changes, blood routine and blood biochemical changes and clinical signs of the virus in each organ after VSV-EBOV/GP infection of golden mice; detecting the lethality of VSV-EBOV/GP to golden yellow rats of different ages and sexes; exploring the influence of different toxicity attack ways on the pathogenicity of golden mice; the results show that: VSV-EBOV/GP produced lethal infection only to golden mice and was related to sex, age and infection route. Post-infection golden-field mice have extensive tissue tropism and pronounced hepatotropism, and cause severe liver function impairment, presenting clinical symptoms similar to those after human infection with EBOV.
3. Evaluation of anti-EBOV antibodies and vaccines in golden mouse model
The therapeutic and prophylactic effects of equine anti-EBOV purified immunoglobulin and EBOV subunit vaccine (EBOV GP delta muc) were evaluated in a golden-yellow mice model, which demonstrated that equine anti-EBOV purified immunoglobulin and EBOV GP delta muc could protect golden-yellow mice 100% against the lethal infection of VSV-EBOV/GP, and that golden-yellow mice model could be an effective tool for preclinical screening of anti-EBOV drugs.
Example 1 construction and identification of EBOV GP recombinant VSV and its use in neutralizing antibody detection
1. Materials and methods
1. Construction and identification of full-length plasmid of EBOV GP recombinant VSV
The recombinant nucleic acid molecule shown in SEQ ID NO.2 (1-204 bp of SEQ ID NO.2 is CMV promoter, 205-223bp is T7 promoter, 224-1419bp is N gene, 1420-3450bp is EBOV GP gene, 3451-4248bp is P gene, 4249-4938bp is M gene, 4939-5658bp is eGFP gene, 5659-11932bp is L gene, 11933-12008bp is hepatitis delta virus ribozyme sequence) is connected into pcDNA3.1 plasmid, the recombinant full-length plasmid is obtained, the recombinant full-length plasmid is named P3.1-VSV delta G-eGFP-EBOV/GP, asc I and PvuI double enzyme digestion identification is carried out on the recombinant full-length plasmid, and the recombinant full-length plasmid is sent to vincristocene biotechnology limited company for sequencing.
2. Rescue and subculture of recombinant viruses
BSR cells were cultured with DMEM containing 100mL/L fetal bovine serum, transferred to six well cell plates, transfected with calcium phosphate transfection reagent when cell density was as high as about 80% of the area, and co-transfected BSR cells with recombinant full-length plasmid p3.1-vsvΔg-eGFP-EBOV/GP and 4 helper plasmids p3.1-VSV-N, p 3.1.1-VSV-P, p 3.1.1-VSV-L, p3.1-VSV-G, see kit instructions for specific steps. After the transfection, the cells were incubated in a 50mL/L CO 2 incubator at 37℃for 18 hours, and then shocked for 2.5 minutes by adding 100mL/L DMSO-containing PBS, the supernatant was discarded, and further incubated for 24 hours by adding 50mL/L fetal calf serum-containing DMEM, and then observed under a fluorescence microscope. The successfully constructed recombinant virus is named as VSV-EBOV/GP (the genome of the VSV-EBOV/GP is obtained by deleting G genes in the genome of vesicular stomatitis virus, inserting Zael ebola virus envelope glycoprotein GP genes between N genes and P genes, inserting marker protein coding genes between M genes and L genes, wherein the genome sequence is shown as 224-11932bp of SEQ ID NO. 2), and when in passage, virus supernatant is repeatedly frozen and thawed for 3 times and inoculated into Vero E6 cells, and after the genome is sensed as 1h in a 50mL/L CO 2 incubator, DMEM containing 20mL/L fetal bovine serum is supplemented for continuous culture for 48h. Passaging on Vero E6 cells for 5 times, collecting recombinant virus supernatant, and freezing into a refrigerator at-80deg.C for preservation.
3. RT-PCR identification of recombinant viruses
The RNA of the recombinant virus and the 5 th generation virus of the maternal VSV (WT-VSV) is extracted by using a virus RNA extraction kit, and the main steps are as follows: (1) 140. Mu.L of the virus supernatant was added to a 1.5mL EP tube, 560. Mu L CARRIER RNA of working solution was added, and the mixture was shaken for 15s and incubated at room temperature for 10min; (2) adding 500 mu L of precooled absolute ethyl alcohol, and vibrating for 15s; (3) Transferring the liquid to an RNA adsorption column, centrifuging at 8000rpm for 1min, and adsorbing RNA onto the column; (4) washing 1 time by adding 500. Mu.L GD solution; (5) washing 2 times with 500. Mu.L RW solution; (6) 13000rpm for 3min; (7) The RNA adsorption column was placed in a 1.5mL centrifuge tube, and RNase-Free ddH 2 O was added to elute the RNA. Using the RNA as a template, using GP gene primer, carrying out RT-PCR amplification according to a reverse transcription kit, and setting a maternal VSV extraction product as a negative control group. And then carrying out agarose gel electrophoresis identification and sequencing identification on the PCR amplification product.
4. Western-blot identification of recombinant viruses
Taking supernatant of 5 th generation virus of recombinant virus and maternal VSV, adding SDS-PAGE loading buffer, boiling in boiling water for 10min after sample preparation to denature protein, performing SDS-PAGE electrophoresis, transferring protein in gel onto NC membrane by using a transfer membrane instrument, sealing NC membrane by using 50g/L skimmed milk powder solution at room temperature for 2H, incubating horse anti-EBOV-GP polyclonal antibody (diluted by PBST according to 1:1000) at room temperature for 2H, washing the membrane by PBST for 3 times, incubating rabbit anti-horse IgG (H+L) HRP secondary antibody (diluted by PBST according to 1:25000) at room temperature for 1H, washing the membrane by PBST for 3 times, and then placing the membrane in Tanon full-automatic fluorescence chemiluminescence image analysis system for development.
5. Immunofluorescence identification of recombinant viruses
Vero E6 cells were passaged to 24 well plates, recombinant virus and maternal VSV were inoculated into Vero E6 cells respectively at 37 ℃,50 mL/L CO 2 incubator, cultured for 24H, fixed with 4% paraformaldehyde at room temperature for 30min, pbst washed 5 times, blocked with 3% BSA solution for 1H, pbst washed 5 times, mouse anti-EBOV-GP polyclonal antibody (200-fold dilution with 3% BSA solution), incubated for 2H at 37 ℃, pbst washed 5 times, cy 3-labeled goat anti-mouse IgG (h+l) secondary antibody (1000-fold dilution with 3% BSA solution) for 1H, pbst washed 5 times, nuclei were stained with DAPI for 10min, pbst washed 5 times, and then observed under a fluorescence microscope.
6. Electron microscopic observation of recombinant viruses
Taking 100 mu L of the supernatant of the recombinant virus and the maternal VSV 5 th generation virus, and performing transmission electron microscope observation after phosphotungstic acid negative staining.
7. Titer determination and growth kinetics curve plotting of recombinant viruses
Recombinant virus titer assay: vero E6 cells were passaged one night in advance into 96 well plates, cultured in a 37 ℃ cell incubator for 12h, 100 μl of the 5 th generation recombinant virus supernatant was taken, serial 10-fold-ratio dilutions were performed with DMEM broth until dilution was 10 -12, the cell broth in 96 well plates was discarded, 10 -1～10-12 dilutions of virus dilutions were inoculated into 96 well plates in order from front to back, and 100 μl of virus dilutions were inoculated into each well. After 1 hour of incubation in a 37℃incubator, the virus solution was discarded, 100. Mu.L of DMEM containing 2% serum was added, and the mixture was incubated in a 5% CO 2 incubator at 37℃for 48 hours, followed by observation of the results.
Growth kinetics detection: vero E6 cells were passaged into T75 cell culture flasks, when the cells were confluent with monolayers, the 5 th generation viral supernatants of recombinant virus and maternal VSV were inoculated with Vero E6 cells at moi=1, moi=0.1, moi=0.01, respectively, incubated in a 5% CO 2 incubator at 37 ℃,200 μl supernatant was collected at 0h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, respectively, and after 3 freeze thawing, virus titer was determined as described above. The TCID 50 of the recombinant VSV virus at each time point is calculated by using a Reed-Muench method, and a growth dynamics curve is drawn.
8. Application of recombinant virus in neutralizing antibody detection
(1) Antibody dilution: the initial concentration of 20mg/mL of the equine anti-EBOV purified immunoglobulin was diluted to a first well concentration of 50. Mu.g/mL, added to the first row of 96 well cell culture plates, and subjected to successive 3-fold gradient dilutions with DMEM diluent. Neutralizing antibody detection was performed using recombinant virus VSV-EBOV/GP. In row 2-8 of 96 well cell culture plates, 100 μl of DMEM was added to each well, the antibody dilutions of the first row were blown and mixed 20 times with a row gun, and 100 μl was aspirated and added to row 2, followed by dilution down the fold ratio.
(2) Adding recombinant virus: diluting the recombinant virus VSV-EBOV/GP to 100TCID 50/100 mu L by using DMEM, correspondingly adding 100 mu L of diluted recombinant virus into a 96-well cell culture plate, shaking and mixing uniformly, and then placing in a cell culture box at 37 ℃ for standing for 1h.
(3) Preparing cells: during the rest, a Vero E6 cell suspension was prepared and the cell suspension was diluted to 2X 10 5 cells/mL.
(4) Adding cells: after 1h of standing, the cell suspension was added to a 96-well cell culture plate at 50. Mu.L per well, and the 96-well cell culture plate was placed in a 5% CO 2 cell incubator at 37 ℃.
(5) Reading the result: after 48h incubation, the neutralization titers were read under a fluorescent microscope to give the highest dilution capable of 100% neutralization of the recombinant virus as the neutralization titer of the antibody.
2. Experimental results
1. Construction and identification of full-length plasmid for EBOV GP recombinant VSV infectious clone
To identify whether the recombinant full-length plasmid was constructed correctly, double restriction identification was performed on the recombinant full-length plasmid. The recombinant plasmid is identified by AscI and PvuI double enzyme digestion, and specific target bands (figure 1) can be observed at about 15000bp and 2031bp respectively, and the sequencing result of the plasmid shows that the gene fragment of the recombinant full-length plasmid connected with the vector is consistent with the EBOV GP gene sequence, and has no mutation site. The results showed that the recombinant full-length plasmid was constructed correctly.
2. Rescue and passaging results of recombinant viruses
To rescue EBOV GP recombinant VSV virus, BSR cells were co-transfected with the recombinant full-length plasmid and VSV helper plasmid for virus rescue, and about 60 hours after transfection, BSR cells were observed to develop syncytial lesions with green fluorescence under a fluorescence microscope, and the rescued recombinant virus was passaged on Vero E6 cells, and cells were observed to develop round-shrinkage lesions with green fluorescence under a fluorescence microscope (fig. 2). The results show that: recombinant viruses carrying the eGFP tag were successfully rescued and can be successfully passaged on Vero E6 cells.
3. RT-PCR identification of recombinant viruses
To identify whether the rescued recombinant viruses were correct, RT-PCR identification of the recombinant viruses was performed, and the results showed that: the RT-PCR product of the recombinant virus RNA extract found specific target band at 2031bp (FIG. 3), and the result shows that the EBOV GP gene can be amplified in the genome extract of the recombinant virus.
4. Western-blot identification of recombinant viruses
To further verify whether the rescued recombinant viruses can successfully express the GP protein of the EBOV, the 5 th generation virus supernatant of the recombinant viruses is taken for Western-blot identification, and the Western-blot result shows that: the recombinant virus showed a specific target band around 130kDa (FIG. 4), which shows that: after VSV-EBOV/GP infection of Vero E6 cells, EBOV GP protein can be successfully expressed.
5. IF identification of recombinant viruses
To determine the site of expression of EBOV GP protein after infection of Vero E6 cells with recombinant virus, indirect immunofluorescence assay was performed on recombinant virus, and the results showed (fig. 5): the EBOV-GP protein is combined with the mouse anti-EBOV-GP polyclonal antibody, then is dyed red by the secondary antibody, the cell nucleus dyed by DAPI is blue, the vector expresses green fluorescent protein, and lesions appear after Vero E6 cells are infected with recombinant viruses under natural light. The IF results indicate that: after the recombinant virus infects Vero E6 cells, the EBOV GP protein can be expressed.
6. Electron microscopic observation of recombinant viruses
In order to explore whether the original form of the virus is affected after the EBOV GP protein is recombined on the surface of the VSV, transmission electron microscopy observation is carried out on the recombined virus, and the result shows that the recombined virus is basically consistent with the form of the VSV of the female parent, and is in a typical bullet shape, and fibers are arranged on the surface (figure 6), so that the normal assembly of the virus is not affected after the EBOV GP protein is recombined on the surface of the VSV.
7. Growth kinetics detection of recombinant viruses
To explore the growth kinetics of recombinant virus, the growth kinetics curves of recombinant virus and maternal VSV were compared (fig. 7), and the results showed that: when moi=0.1 was measured according to the different multiplicity of infection, the recombinant virus had a highest growth titer of about 10 7TCID50/mL at about 60h, whereas the maternal VSV had a highest virulence of about 10 8.5TCID50/mL at about 36h, and the results showed that: substitution of the foreign glycoprotein for the VSV glycoprotein results in a reduction in virulence of the recombinant VSV virus to some extent, as well as a reduction in the rate of infection of the cell.
8. Application of recombinant virus in neutralizing antibody detection
To evaluate the neutralizing potency of the equine anti-EBOV purified immunoglobulin, virus neutralizing antibody detection was performed using VSV-EBOV/GP recombinant virus instead of EBOV solid virus, and the results showed that: horse anti-EBOV refined immunoglobulin the complete neutralization titers were 1:64 as measured with recombinant virus VSV-EBOV/GP.
EXAMPLE 2 establishment of recombinant Virus golden yellow mice infection model
An animal infection model was constructed using the recombinant virus VSV-EBOV/GP expressing the zaire ebola virus envelope glycoprotein constructed in example 1, and specifically as follows:
1. Material and method 1, susceptibility of recombinant virus VSV-EBOV/GP expressing zaire Ebola virus envelope glycoprotein in rodents
5 Balb/c mice, SD-rats, guinea pigs and golden mice each of 3 weeks of age were taken, and an experimental group and a control group were set. Animals of the experimental group were each observed for weight change and survival rate by intraperitoneal injection of 1mL of recombinant virus VSV-EBOV/GP (10 7TCID50).
2. Glycoprotein correlation of VSV-EBOV/GP pathogenicity
20 Golden yellow mice of 3 weeks of age were randomly divided into 4 groups, each group of 5 groups was respectively intraperitoneally injected with Vesicular Stomatitis Virus (VSV), recombinant virus VSV-EBOV/GP expressing zaire-type ebola virus envelope glycoprotein, recombinant virus VSV-SUDV/GP expressing Sudan-type ebola virus envelope glycoprotein (construction method referenced VSV-EBOV/GP, the difference was only that zaire-type ebola virus envelope glycoprotein gene was replaced with Sudan-type ebola virus envelope glycoprotein gene), and recombinant virus VSV-LASV/GP expressing Lasal-type ebola virus envelope glycoprotein (construction method referenced VSV-EBOV/GP, the difference was only that zaire-type ebola virus envelope glycoprotein gene was replaced with Lasalsa-heat virus envelope glycoprotein gene), each 1mL, and weight change and survival rate were observed. 3. Comparing the characterization changes of golden yellow mice with different sexes after toxicity attack
10 Female and male golden mice at 3 weeks of age are respectively arranged into an experimental group and a control group, 5 golden mice in each group are subjected to intraperitoneal injection of 1mL of recombinant virus VSV-EBOV/GP (10 7TCID50), clinical characterization is observed, and weight change and survival rate are determined.
5 Female and male golden-yellow mice at 3 weeks of age are respectively taken, each golden-yellow mouse is injected with 1mL of recombinant virus VSV-EBOV/GP (10 7TCID50) in the abdominal cavity, after 36 hours, the blood change is measured, the blood biochemistry and blood routine are detected, the euthanasia is carried out, and the tissues such as heart, liver, spleen, lung, kidney, stomach, intestine, brain and the like are taken, so as to measure the virus load, immunohistochemistry and pathological change of the tissues.
4. Comparison of sensitivity of golden yellow rats of different ages to VSV-EBOV/GP
16 Female and male golden yellow mice of 3 weeks old, 3 months old and 1 year old are randomly divided into an experimental group and a control group respectively, 8 golden yellow mice in each group, and the clinical characterization of the golden yellow mice is observed by intraperitoneal injection of 1mL of recombinant virus VSV-EBOV/GP (10 7TCID50) to determine the weight change and survival rate of the golden yellow mice. 3 days after infection, 3 groups were euthanized and liver tissue was taken to determine its tissue viral load.
5. Comparing sensitivity of golden mice to VSV-EBOV/GP under different toxin attacking modes
32 Female golden mice of 3 weeks of age are divided into 4 groups, 8 groups are respectively arranged into an intraperitoneal injection group, a subcutaneous injection group and an intramuscular injection group, each group is injected with 1mL of recombinant virus VSV-EBOV/GP (10 7TCID50), clinical characterization is observed, weight change and survival rate are determined, blood is collected by a retroorbital venous plexus blood collection method after 36 hours, and blood routine and blood biochemical change are determined. 3 animals were euthanized from each group, and liver, spleen, lung and kidney were taken and tested for tissue viral load, immunohistochemistry and pathological changes.
2. Experimental results
1. Susceptibility of recombinant virus VSV-EBOV/GP in rodents
The recombinant virus can not produce lethal infection on BALB/c mice, SD-rats and guinea pigs, the body weight of an experimental group is gradually decreased and then gradually increased after infection, and the body weight of a control group is gradually increased. Recombinant viruses produced a fatal infection to golden mice and decreased body weight (fig. 8, 9).
2. Glycoprotein correlation of recombinant virus VSV-EBOV/GP pathogenicity
Neither the recombinant viruses VSV-SUDV/GP nor VSV-LASV/GP could kill golden mice, but VSV could cause lethal infection to golden mice, and the recombinant viruses VSV-EBOV/GP were all lethal to golden mice, demonstrating that the pathogenicity of VSV-EBOV/GP was due to the EBOV-GP glycoprotein and not the VSV- ΔG vector (FIG. 10).
In addition, as a control, the insertion position of the zaire ebola virus envelope glycoprotein GP gene in the VSV genome is changed, and the obtained control recombinant virus is only different from the recombinant virus VSV-EBOV/GP in that: the zaire ebola virus envelope glycoprotein GP gene is inserted between the M and L genes, and eGFP is inserted between the N and P genes. The control recombinant virus and the recombinant virus VSV-EBOV/GP were intraperitoneally injected into golden mice at a dose of 1mL each. The results show (FIG. 10) that the recombinant virus VSV-EBOV/GP with the Zaeel ebola virus envelope glycoprotein GP gene inserted with the N and P genes was the highest in lethality to golden yellow mice (mortality was 100%), whereas the control recombinant virus with the Zaeel ebola virus envelope glycoprotein GP gene inserted with the M and L genes was significantly reduced in lethality (mortality was only 40%).
3. Comparing the characterization changes of golden yellow mice with different sexes after toxicity attack
Diseased animals are often isolated from other animals and may exhibit weight loss, reduced body temperature, bowed back, listlessness, coarse and messy hair, dyspnea, and loss of exploratory behavior. Female golden mice were one hundred percent killed, and had recovered from infection, body weight and body temperature tended to rise (FIG. 11).
Blood biochemistry and blood routine results cues (fig. 12, fig. 13): the number of White Blood Cells (WBC) of the golden yellow mice after infection is obviously increased, the number of Platelets (PLT) is reduced, the neutrophil ratio (GRAN%) is obviously increased, the lymphocyte ratio (LMY%) is obviously reduced, which indicates that the organism has serious virus infection and indicates coagulation disorder; the albumin content of golden yellow mice after infection is obviously reduced, alanine Aminotransferase (ALT) is increased, and alkaline phosphatase (ALP) is obviously increased, which indicates that the body has serious liver function damage.
The viral load results are known: live virus was detected in the heart, liver, spleen, lung, kidney, stomach, intestine, brain of golden mice, indicating systemic replication of the virus, with overall higher viral load in liver and spleen, and minimal viral load in brain tissue.
In female and male golden mice, liver develops multiple hepatocyte necrosis, multiple lymphocyte infiltration, and granulocyte infiltration; spleen has irregular white marrow shape, multiple lymphocyte necrosis, red marrow with multiple cell necrosis and nuclear fragmentation; a large number of neutrophil infiltration was seen; the diffuse alveolar wall is slightly thickened, and inflammatory cells infiltrate; the kidneys see tubular dilation. The results of immunohistochemistry showed that antigen positivity was detected in the liver, spleen, lung and kidney of both female and male golden mice, and was confirmed by the results of viral load in tissues and organs (fig. 14, 15 and 16).
Notably, ocular secretions were found in post-infection female golden mice, similar to those found in humans infected with EBOV postoptic nerve complications (fig. 17).
4. Comparison of sensitivity of golden yellow rats of different ages to VSV-EBOV/GP
The results show that female golden yellow rats at 3 weeks of age are more sensitive to the recombinant virus VSV-EBOV/GP than male golden yellow rats, and female golden yellow rats at 3 months of age do not produce deadly infection, the weight of the female golden yellow rats at 1 age does not produce deadly infection, and the weight recovery time is slower than that of female golden yellow rats at 3 weeks of age and 3 months of age. The results of viral load can be seen: overall liver viral load was higher than spleen, 3 week old tissue viral load was highest, 3 month old golden yellow mice tissue viral load was lowest, recovery from infection was fast, and was confirmed by fast body weight regain (fig. 18, 19).
5. Comparing sensitivity of golden mice to VSV-EBOV/GP under different toxin attacking modes
The method can be known by comparing different toxin attacking modes: golden mice were all killed only by intraperitoneal injection. The weight of the golden yellow mice injected by intramuscular injection firstly drops and then rises, and the mice return at 48 hours; the weight of the golden yellow mice injected subcutaneously also tended to drop and rise firstly, and the weight of the golden yellow mice injected by nasal drops was slowly rising at 48h, and gradually recovered at 96 h. The change in tissue viral load is known: the highest viral load was found in the tissues of golden yellow rats injected intraperitoneally. The viral load of the liver is highest in liver, spleen, lung and kidney assays. By blood analysis of golden mice 36 hours after infection, compared with a control group, the change of the intraperitoneal injection group is most obvious, ALB is obviously reduced, ALT is increased, and ALP is obviously increased; WBC numbers increased significantly, PLT numbers decreased, GRAN% increased significantly, LMY% decreased significantly. The pathological and histochemical results also showed that the intraperitoneally injected golden mice were most severe (fig. 20, 21, 22, 23).
Example 3 application of golden yellow mice infection model in EBOV antibody and vaccine evaluation
1. Materials and methods
1. Application of golden mouse infection model in antibody evaluation
33 Female golden mice of 3 weeks of age were randomly divided into three groups, which were set as control, prophylactic and therapeutic groups, respectively. In the prophylaxis group and the treatment group, each 5mg of the equine anti-EBOV immunoglobulin was administered by intraperitoneal injection 24 hours before and after the infection of the golden mice with VSV-EBOV/GP, respectively, and the control group was given the same volume of PBS. 3 golden yellow mice were euthanized for liver tissue and their tissue viral load was determined for each group at 3 and 5 days post infection, respectively, and clinical signs of treated golden yellow mice were observed.
2. Application of golden-yellow-rat infection model in vaccine evaluation
22 Female syrian hamsters of 3 weeks of age were randomly divided into two groups, a vaccine immunization group and a control group were set. Immunized groups of golden mice were intraperitoneally injected with the vaccine EBOV GP delta muc (mixed with freund's adjuvant 1:1) following the immunization schedule of 0, 1,3 weeks. The control group of golden mice was given equal amounts of PBS. After immunization, blood samples were collected through the orbital venous plexus at various time points, serum was isolated, stored at-80 ℃, and antibodies in the serum were determined. 3 days and 5 days after infection, 3 golden mice were euthanized for liver tissue from each group, their tissue viral load was determined, and clinical signs of the golden mice after treatment were observed.
2. Experimental results
1. Application of golden mouse infection model in antibody evaluation
Female golden yellow mice of 3 weeks old are respectively provided with a prevention group, a treatment group and a control group, wherein the prevention group and the treatment group respectively administer intervention (5 mg/mouse) of the horse anti-EBOV refined immunoglobulin 24 hours before and after the virus attack, the control group kills after the virus attack, golden yellow mice with the dry prognosis of the horse anti-EBOV refined immunoglobulin do not die, the weight is in a trend of decreasing before increasing, the viral load is measured by taking liver tissues after 3 days and 5 days, and the gradual decrease of the viral load is observed, which indicates that the horse anti-EBOV refined immunoglobulin has prevention and treatment effects on golden yellow mice (figure 24).
2. Application of golden-yellow-rat infection model in vaccine evaluation
The female golden yellow mice of 3 weeks old are respectively provided with a vaccine immunization group and a control group, the vaccine immunization groups are respectively in 0 week, 1 week and 3 weeks, the intervention of the EBOV vaccine is given, the ages of the golden yellow mice are increased along with the prolongation of the virus attack time, the control group and the immunization group are subjected to virus attack after one week of three-free, death does not occur after the virus attack, the neutralization titers of serum antibodies are found after two-free and three-free, the in-vivo neutralization titers of the three-free are higher (the two-free is 1:512, the three-free is 1:1024), the body weight of the golden yellow mice of the control group is in a trend of decreasing first and then increasing, and the golden yellow mice of the immunized group are in a trend of increasing. Progressive reduction in viral load was observed with liver tissue taken 3 days and 5 days after challenge, and the immune group was overall lower than the control group. The EBOV vaccine was demonstrated to have a protective effect on golden mice (fig. 25).
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (14)

1. A recombinant nucleic acid molecule comprising a recombinant vesicular stomatitis virus genome;
The recombinant vesicular stomatitis virus genome is obtained by deleting a G gene in the vesicular stomatitis virus genome and inserting a zaire Ebola virus GP gene between an N gene and a P gene;
Or the recombinant vesicular stomatitis virus genome is obtained by deleting a G gene in the vesicular stomatitis virus genome, inserting a zaire Ebola virus GP gene between an N gene and a P gene, and inserting a marker protein coding gene between an M gene and an L gene;
the genome of the vesicular stomatitis virus is the genome of a vesicular stomatitis virus Indiana vaccine strain, and the Genbank accession number of the genome is J02428.1;
the nucleotide sequence of the Zaeel type ebola virus GP gene is shown as SEQ ID NO. 1.
2. The recombinant nucleic acid molecule of claim 1, further comprising a promoter located upstream of the recombinant vesicular stomatitis virus genome;
And/or the recombinant nucleic acid molecule further comprises a hepatitis delta virus ribozyme sequence downstream of the recombinant vesicular stomatitis virus genome.
3. The recombinant nucleic acid molecule of claim 2, wherein the promoters are a CMV promoter and a T7 promoter.
4. The recombinant nucleic acid molecule according to any one of claims 1 to 3, wherein the nucleotide sequence of the recombinant nucleic acid molecule is shown in SEQ ID No. 2.
5. A biological material, characterized in that the biological material is a recombinant vector or host cell;
the recombinant vector comprises the recombinant nucleic acid molecule of any one of claims 1-4;
the host cell comprises the recombinant nucleic acid molecule of any one of claims 1-4, or comprises the recombinant vector.
6. The biomaterial of claim 5, wherein the recombinant vector is a mammalian expression plasmid.
7. The recombinant vesicular stomatitis virus is characterized in that the genome of the recombinant vesicular stomatitis virus is obtained by deleting a G gene in a genome of a vesicular stomatitis virus Indiana vaccine strain and inserting a zaire Ebola virus GP gene between an N gene and a P gene;
or the recombinant vesicular stomatitis virus genome is obtained by deleting G genes in a vesicular stomatitis virus Indiana vaccine strain genome, inserting Zaeel Ebola virus GP genes between N genes and P genes, and inserting marker protein coding genes between M genes and L genes;
the genome of the vesicular stomatitis virus Indiana vaccine strain has Genbank accession number J02428.1;
the nucleotide sequence of the Zaeel type ebola virus GP gene is shown as SEQ ID NO. 1.
8. The recombinant vesicular stomatitis virus of claim 7, wherein the nucleotide sequence of the recombinant vesicular stomatitis virus genome is set forth in 224-11932bp of SEQ ID No. 2.
9. A method of preparing a recombinant vesicular stomatitis virus according to claim 7 or 8, characterized in that the method comprises: introducing a recombinant vector comprising the recombinant nucleic acid molecule of any one of claims 1-4 and a helper plasmid into a host cell to obtain a recombinant host cell, culturing the recombinant host cell and harvesting the released virus.
10. The method of claim 9, wherein the helper plasmid is four mammalian expression plasmids that express RNA polymerase, nucleoprotein, phosphoprotein and glycoprotein of vesicular stomatitis virus, respectively.
11. Use of the recombinant nucleic acid molecule of any one of claims 1 to 4 or the biomaterial of claim 5 or 6 or the recombinant vesicular stomatitis virus of claim 7 or 8, for any one of the following:
(1) The application in preparing recombinant vesicular stomatitis virus expressing ebola virus GP protein;
(2) The application in preparing an Ebola virus infection animal model;
(3) Use of a neutralizing antibody for detecting ebola virus in the preparation of a reagent for the presence or level of neutralizing antibody in a sample;
(4) Use of neutralizing antibodies to detect ebola virus in samples for the presence or level thereof for non-disease diagnosis and treatment purposes.
12. A method for constructing an animal model of ebola virus infection, the method comprising: the use of the recombinant vesicular stomatitis virus of claim 7 or 8 to detoxify female golden mice.
13. The method of claim 12, wherein the female golden mice are 2-4 weeks old;
and/or, the mode of attacking toxin is intraperitoneal injection.
14. Use of any one of the following ebola virus infection animal models constructed by the construction method of claim 12 or 13:
(1) Application in screening or effectiveness evaluation of Ebola virus antibodies;
(2) Application in screening or effectiveness evaluation of Ebola virus vaccines;
(3) Use in screening or efficacy evaluation of a drug for the prevention and/or treatment of ebola virus.
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