CN110577585A

CN110577585A - Vesicular stomatitis virus envelope glycoprotein variant, and construction method and application thereof

Info

Publication number: CN110577585A
Application number: CN201910497491.8A
Authority: CN
Inventors: 石磊; 慈雅丽
Original assignee: Institute of Basic Medical Sciences of CAMS
Current assignee: Institute of Basic Medical Sciences of CAMS
Priority date: 2018-06-07
Filing date: 2019-06-10
Publication date: 2019-12-17

Abstract

The invention relates to a vesicular stomatitis virus envelope glycoprotein (VSV-G) variant, a construction method thereof and application thereof in preparation of a Vesicular Stomatitis Virus (VSV) vaccine.

Description

Vesicular stomatitis virus envelope glycoprotein variant, and construction method and application thereof

Technical Field

The invention relates to a vesicular stomatitis virus envelope glycoprotein (VSV-G) function deletion variant, and a construction method and application thereof.

background

Vesicular Stomatitis Virus/Vesicular Stomatitis Virus (VSV) is a member of the rhabdovirus family, belonging to enveloped viruses, which are named for their bullet-like shape, and which family members include rabies and passenger viruses in addition to VSV.

VSV infects a variety of animals and insects. Animals such as horses, cattle, sheep, and pigs are naturally infected with VSV, and wild animals (wild boars, raccoons, and deer, etc.) can be naturally infected with VSV. Infected animals exhibit fever, lethargy, decreased appetite, and the appearance of vesicular lesions and canulate rings in the legs in the mouth, nipples, spaces between toes and hoof crowns. The blister is easy to break, the granulation tissue is exposed, red erosion is formed, and scraped epithelium is arranged around the blister, so that the blister can be self-healed within 7 to 10 days. Infection of livestock by vesicular stomatitis virus is not usually fatal, but can cause local secondary bacterial and fungal infections, resulting in lameness, weight loss, reduced milk production, mastitis, and other symptoms, with significant economic loss. The infection of horses, cattle and pigs in the United states with this disease was reported as early as the beginning of the nineteenth century. During the united states civil war, the disease thus resulted in 4000 war horses not being able to fight. The united states has since developed almost every 10 years. Later on Mexico, Central America, Panama, Venezuela, Columbia, Peru, Argentina, Brazil, France and south Africa, etc. reported the disease in succession. Currently, large-area popularity in China has not emerged. According to the world animal health organization, countries in south america, middle america, and nearly all countries and regions in north america, and the united states in north america have developed a large VSV epidemic at the end of the twentieth century, causing serious economic losses. Thus, antiviral studies against VSV are of veterinary, livestock farming and economic importance.

Currently, there is no effective VSV vaccine on the market for the prevention and treatment of VSV infection. The research on virus vaccines mainly focuses on subunit vaccines, inactivated vaccines, attenuated vaccines and genetically engineered vaccines. Research shows that the subunit vaccine based on protein macromolecules has low immunogenicity, and needs to be combined with an adjuvant for multiple injections. Attenuated inactivated vaccines generally elicit only humoral immunity, do not elicit important cellular immunity, and require several vaccinations. The attenuated vaccine as live virus can induce both humoral immunity and cellular immunity after entering body, and has small inoculation amount, long immune maintaining time and excellent immune effect. Attenuated vaccines, however, have certain drawbacks, such as the need for extensive long-term screening of strains, the possibility of reversion of virulence, and the possibility of virus propagation in vivo, and may not be suitable for immunodeficient individuals.

Because of the wide prevalence, high infectivity, variability, specificity of antibody protection of VSV and huge economic loss brought by the prevalence, the development and utilization of a safe and efficient VSV vaccine have important practical significance and wide market prospect in the world.

Disclosure of Invention

The viral envelope protein is an important protein exposed on the surface of enveloped viruses, and plays a key role in mediating the fusion of the viral envelope with the cell membrane and then invading cells. The vesicular stomatitis virus is a good vaccine carrier because it is exposed on the surface of the virus, has good antigenicity, can be recognized by a host system and produces antibodies, can stimulate the organism to produce high-titer neutralizing antibodies and induce the production of humoral immunity (J Virol.2000 Dec; 74(23): 10903-.

The inventors have unexpectedly found in their studies that when the VSV-G transmembrane region is replaced with another sequence or the extracellular membrane-proximal region thereof is extended, the VSV-G mediated viral-cell fusion ability is significantly reduced, thereby inhibiting VSV-G mediated viral entry. After entering into the body, the virus carrying the VSV-G variant can not be fused with a cell membrane to invade cells for replication and proliferation, so that cell damage and necrosis are avoided; meanwhile, the complete virus particles can be fully contacted with the immune system of an organism to induce the generation of specific cellular immunity and humoral immunity, and can be used as a safe and effective genetic engineering attenuated vaccine. The attenuated vaccine of VSV gene engineering of the present invention can maintain the advantages of traditional attenuated vaccine, avoid the possibility of virulence reversion and meet the unmet health requirement.

The inventors have completed the present invention on this basis and have provided the following technical solutions.

In one aspect, the invention provides a VSV-G protein variant having a mutation in the transmembrane region.

Preferably, the transmembrane region is mutated to a substitution, i.e., the VSV-G transmembrane region is replaced by the transmembrane region of another transmembrane protein.

in one embodiment of the invention, the VSV-G gene sequence used is from Vesicular Stomatitis Indiana virus (GenBank accession number: J02428.1) as shown in SEQ ID NO: 1; the corresponding VSV-G protein sequence is shown in SEQ ID NO 2.

SEQ ID NO:2

In one embodiment, the invention constructs a series of VSV-G transmembrane region replacement variants.

wherein the transmembrane region is located at position 465 to 490 of the VSV-G amino acid sequence, and the sequence is shown as SEQ ID NO 3,

SEQ ID NO:3：IASFFFIIGLIIGLFLVLRVGIHLCI。

Wherein the transmembrane sequences of the non-VSV-G transmembrane region useful for replacing the VSV-G transmembrane region are selected from the group consisting of:

1) A transmembrane region sequence of a viral fusion protein, wherein the virus is not VSV;

2) A transmembrane region sequence of a membrane protein that is not a viral membrane protein; and

3) a transmembrane region sequence which is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of the transmembrane region of the proteins of 1) and 2).

The term "viral fusion protein" as used herein refers to a viral protein that mediates fusion of a virus to a host cell.

in some embodiments, the transmembrane sequence is selected from the following protein transmembrane region amino acid sequences:

A) A transmembrane region of the Syntaxin1A protein or a fragment thereof, for example, the transmembrane region of the Syntaxin1A protein comprises or consists of an amino acid sequence shown in SEQ ID NO. 4; 4, IMIIICCVILGIIIASTIGGIFG;

B) A transmembrane region of the VAMP2 protein, or a fragment thereof, e.g., the transmembrane region of the VAMP2 protein comprises or consists of the amino acid sequence shown in SEQ ID NO. 5; 5, MMIILGVICAIILIIIIVYFST;

C) A transmembrane region of an influenza virus HA protein, for example comprising or consisting of the amino acid sequence shown in SEQ ID No. 6; 6, ILSIYSTVASSLALAIMVAGLSLW SEQ ID NO.

The SNARE protein is a kind of membrane fusion-mediated protein on the cell membrane (J Pept Sci.2015Aug; 21(8):621-9), and Syntaxin1A and VAMP2 belong to the protein.

the inventor unexpectedly discovers in research that the replacement of the VSV-G transmembrane region by the Syntaxin1A or VAMP2 transmembrane region results in about 30-40% reduction of the VSV-G mediated cell fusion capacity in a cell experiment, and the replacement of the VSV-G transmembrane region by the influenza virus HA transmembrane region results in about 80% reduction of the VSV-G mediated cell fusion capacity in the cell experiment.

In another aspect, the present invention provides a VSV-G protein variant having a mutation in the extracellular membrane proximal region located in the extracellular region of the VSV-G protein near the transmembrane region, comprising the sequence shown in SEQ ID NO. 7,

SEQ ID NO:7，TGLSKNPIELVEGWFSSWKSS。

Preferably, the extracellular membrane proximal region is mutated to an insertion, i.e. to insert one or more additional amino acids in this region.

In one embodiment, the invention constructs a series of VSV-G extracellular membrane proximal region insertion variants, preferably with 3 to 20 amino acids, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids, more preferably 6 amino acids. In the present invention, the kind of the inserted amino acid is not particularly limited.

The inventors have found that insertion of 1, 2, 3, 4, 5 and 6 amino acids into the extracellular membrane proximal region of VSV-G results in 7%, 18%, 26%, 39%, 79% and 90% decrease in the ability of VSV-G mediated cell fusion in cell experiments, respectively.

In another aspect, the invention provides a vaccine composition comprising a mutation of the VSV-G transmembrane region and/or extracellular membrane proximal region as described above.

preferably, the VSV-G protein variant is located on the surface of a viral particle that does not have the ability to cause VSV-induced disease.

Specifically, the virus particle may be VSV itself or may be another virus not containing a pathogenic gene.

Further, the vaccine composition further comprises an immunologically and pharmaceutically acceptable carrier or adjuvant.

In one embodiment of the invention, the VSV-G transmembrane variant is packaged onto the surface of a lentivirus and infects Hela cells, and it was found that a Syntaxin1A or VAMP2 transmembrane replacement results in about a 50% reduction in the infection efficiency of the lentivirus, while an influenza virus HA transmembrane replacement results in about a 90% reduction in the infection efficiency of the lentivirus. The replacement of the transmembrane region was shown to reduce the VSV-G mediated ability to fuse virus to cells.

In another embodiment of the invention, the VSV-G extracellular membrane proximal variant is packaged onto the surface of a lentivirus and infects Hela cells, and insertion of 1 to 6 amino acids of the VSV-G membrane proximal domain is found to result in a reduction in the infection efficiency of the lentivirus by 26%, 45%, 68%, 80%, 93% and 98%, respectively. The insertion of amino acids into the extracellular membrane-proximal region of VSV-G was shown to decrease the VSV-G mediated fusion ability of the virus to cells, and with a corresponding increase in the number of amino acid insertions, the fusion ability decreased accordingly. When the inserted amino acid is 6, the fusion ability of the virus to the cell is only 2% of that of wild-type VSV-G.

In yet another aspect, the invention provides VSV-G variants having both transmembrane and extracellular juxtamembrane region mutations.

It has been shown that the vesicular stomatitis virus, which is a virus that undergoes massive replication by invading cells to cause necrosis, does not have a specific pathogenic gene. VSV-G mediates fusion of the viral envelope with the cell membrane, which in turn allows the virus to invade the cell, and thus, the VSV-G mediated fusion function determines the virulence of the virus. Meanwhile, VSV-G is also the main surface antigen of vesicular stomatitis virus and can stimulate the body to produce neutralizing antibodies. The VSV-G variants provided by the invention have significantly reduced fusion capacity, thereby significantly reducing the infectivity of the virus. In addition, since vesicular stomatitis virus infection is not lethal, the infected animal can heal itself after a period of time without complications. Therefore, the virus containing the VSV-G variant provided by the invention has good safety; meanwhile, the VSV-G variant has mutation only in a transmembrane region and/or an extracellular membrane-proximal region, while the whole extracellular region exposed on the membrane surface is kept intact, so that the antigenicity of VSV-G is completely retained, and the VSV-G variant can be used for preparing a safe and efficient VSV genetic engineering vaccine.

in another aspect, the present invention also provides a method for constructing a VSV genetically engineered vaccine, comprising the steps of:

(1) Replacing in vitro a nucleic acid sequence encoding a transmembrane region of the VSV-G nucleic acid sequence using genetic engineering techniques; and/or

(2) inserting a sequence encoding one or more amino acids into a nucleic acid sequence encoding an extracellular membrane proximal region of a VSV-G nucleic acid sequence in vitro using genetic engineering techniques;

(3) introducing the sequence obtained in the step (1) or (2) into a packaging cell together with a virus packaging system to obtain the virus.

In some embodiments, the sequence used to replace the VSV-G transmembrane region in step 1) is selected from the amino acid sequences of the transmembrane regions of proteins selected from the group consisting of:

A) a transmembrane region of the Syntaxin1A protein or a fragment thereof, for example, the transmembrane region of the Syntaxin1A protein has an amino acid sequence shown in SEQ ID NO. 4;

B) A transmembrane region of the VAMP2 protein or a fragment thereof, for example, the transmembrane region of the VAMP2 protein has the amino acid sequence shown in SEQ ID NO. 5;

C) A transmembrane region of an influenza virus HA protein, for example, having the amino acid sequence of SEQ ID NO 6, or a fragment thereof.

In other embodiments, the amino acid inserted in step 2) is 3 to 20 additional amino acids, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional amino acids, more preferably at least 6 additional amino acids.

In another aspect, the invention provides a vector comprising a nucleotide sequence encoding a VSV-G protein variant described above.

In yet another aspect, the invention also provides the use of a VSV-G protein variant, polynucleotide and/or vaccine composition as described above in the preparation of a medicament for the prevention and/or treatment of a disease caused by VSV.

In yet another aspect, the present invention also provides a method for preventing and/or treating a VSV-caused disease, comprising administering the VSV-G protein variant, polynucleotide and/or vaccine composition to a subject in need thereof. The administration is selected from the group consisting of intramuscular injection, intradermal injection, subcutaneous injection, intravenous injection, mucosal administration, and any combination thereof. The subject is selected from the group consisting of horses, cattle, sheep and pigs.

the modified gene engineering virus is identified by the immune system of the organism and induces specific immune response after entering blood/body fluid, so that the organism can obtain the immunity to the same kind of virus. Meanwhile, because the complete live virus is used for immunization, the required inoculation weight is small, and the titer of the obtained antibody is high. The defect of fusion ability can also slow down the process of VSV-G degradation by cells, prolong the time of antigen activation effect and enhance immune response. These were not achievable in the past VSV vaccine design.

in one embodiment, the inventors load the VSV-G protein with the fusion ability defect but retaining the active epitope onto the surface of a lentivirus without self-replication ability, and utilize a viral vector to ensure the high-efficiency transportation in vivo, avoid the additional degradation during the transportation process, and ensure the recognition and phagocytosis of the VSV-G protein by host cells to activate the immune response in vivo, thereby increasing the safety of the vaccine and enhancing the application potential of the system as a viral vaccine.

The above aspects show that the virus system which is built by the inventor and depends on VSV-G fusion ability deficiency variant assembly is a safe and effective genetic engineering vaccine system and has wide application prospect.

Drawings

FIG. 1 shows a schematic diagram of a VSV-G mediated cell-cell fusion assay. The cytoplasm expresses red fluorescent protein, simultaneously, the VSV-G carrying cell and the nucleus expresses cyan fluorescent protein are fused under the condition of low pH, and the fused cell contains red cytoplasm and a plurality of cyan nuclei.

FIG. 2 shows a HeLa cell fusion model to test the ability of VSV-G fusion. FIG. 2A shows that HeLa cells that do not express VSV-G do not have fusion events occurring at low pH; FIG. 2B shows that Hela cells expressing VSV-G can induce the occurrence of a number of fusion events under low pH conditions.

FIG. 3 shows that transmembrane region replacement significantly reduced VSV-G mediated cell-cell fusion ability. The giant cells with multiple nuclei in the figure are fused cells, scale 40 μm.

FIG. 4 is a statistical analysis of the experiment of FIG. 3. Denotes P < 0.001.

FIG. 5 shows that amino acid insertion in the juxtamembrane domain significantly reduced VSV-G mediated cell-cell fusion ability, and that the reduction in fusion ability is inversely proportional to the number of inserted amino acids. The giant cells with multiple nuclei in the figure are cells after fusion. Scale, 50 μm.

FIG. 6 the results of statistical analysis of the FIG. 5 experiment.

FIG. 7 shows that transmembrane region replacement significantly reduced the efficiency of VSV-G mediated lentiviral infection of cells. Chimeric VSV-G, replaced by wild-type or transmembrane region, was used to construct recombinant lentiviruses to infect HeLa cells, and the virus-infected cells expressed GFP as a marker and observed by confocal microscopy. Scale, 30 μm.

figure 8 flow cytometry analysis of the figure 7 experiment. Fig. 8A shows flow cytometry recordings and fig. 8B shows statistical analysis, P < 0.001.

FIG. 9 shows that amino acid insertion in the juxtamembrane domain significantly reduced the efficiency of VSV-G mediated lentiviral infection of cells. Confocal microscopy images were observed after infection of cells with viruses expressing different VSV-G variants. Scale, 50 μm.

FIG. 10 shows the statistical results of the FIG. 9 experiment.

Detailed Description

the present invention is further described below, but is not limited in any way, and any variations based on the teachings of the present invention are within the scope of the present invention.

examples

The following examples, taken in conjunction with the accompanying drawings, are illustrative of the invention disclosed herein and should not be construed as limiting the scope of the appended claims in any way.

Example 1 Experimental related methods and procedures

1.1. Cell culture

HEK293T(CRL-11268) and Hela (CRM-CCL-2) cell culture at 37 deg.C, saturated humidity, 5% CO₂In a cell culture incubator.

Western blot

1) discarding the culture medium, and washing with PBS buffer solution for 2 times;

2) adding pre-cooled whole cell protein lysate (50mM Tris (pH 7.4), 150mM NaCl, 1% NP-40, 0.5% sodium deoxyholate), about 30 μ L/mL cells, pipetting, mixing, and lysing on ice for 10 min;

3) centrifuging the cell lysis suspension at 4 deg.C 13300rpm for 10min, transferring the supernatant to a new EP tube to obtain cell whole protein extract;

4) Adding 6x SDS sample buffer (6x loading buffer:3.75mL 1M Tris pH 6.8, 1.2g SDS, 6mL glycerol, 0.006g bromopenol blue, 0.462g DTT) in proportion, and boiling water bath at 100 deg.C for 5-10 min;

5) Loading the sample on SDS-PAGE gel, carrying out electrophoresis at 80V when the sample is in the concentrated gel, and carrying out electrophoresis at 120V when the sample is migrated into the separation gel;

6) An NC membrane (nitrocellulose membrane) of appropriate size was soaked in an electrotransfer buffer (3.03g Tris +14.42g Gly +200mL CH)₃OH is prepared into 1L) of neutral ion strength;

7) After the electrophoresis is stopped, transferring the gel after the electrophoresis to an electrotransfer buffer solution;

8) A membrane transfer system was prepared in the following order (from bottom to top) in the electrotransfer buffer: filter paper, NC membrane, gel with protein sample, filter paper;

9) Putting the membrane transferring system into a spongy cushion soaked in an electrotransformation buffer solution in advance, wherein one side of the gel faces to a negative electrode, and one side of the NC membrane faces to a positive electrode;

10) pouring an electric conversion buffer solution into an electric conversion tank, and placing the electric conversion tank in an ice water bath for electric conversion for 1.5-2h at a constant current of 200 mA;

11) And (3) sealing: placing NC membrane in 5% skimmed milk powder solution, incubating for 0.5-1 hr at room temperature under slow shaking, and washing with TBST (Tris-HCl (1M, pH7.5):50 Ml; NaCl:8 g; KCL:0.2 g; Tween: 0.5 Ml; adding distilled water to constant volume of 1L) for 3 times under shaking;

12) Primary antibody incubation: diluting primary antibody with 5% BSA (prepared by TBST) solution according to a proper proportion, putting the NC membrane into a sealed bag, adding the primary antibody, and incubating at 4 ℃ overnight or at room temperature for 2 h;

13) washing of the NC membrane: after the primary antibody incubation is finished, taking out the NC membrane, adding a proper amount of TBST solution, and slowly shaking and washing for 3 times, 5min each time;

14) And (3) secondary antibody incubation: diluting the secondary antibody with 5% BSA (prepared in TBST) at a ratio of 1:5000, placing the NC membrane in a sealed bag, adding the secondary antibody, and incubating at room temperature for 1 h;

15) washing of the NC membrane: after the incubation of the secondary antibody is finished, taking out the NC membrane by using a small forceps, adding a proper amount of TBST solution, and slowly shaking and washing for 3 times, 5min each time;

16) ECL exposure: the NC film is properly drained on filter paper, laid on a plastic film, and added with ECL reaction solution (PerkinElmer, USA) dropwise according to the instruction, after the reaction is finished, the signal image is obtained by exposure with an X-ray film in a dark room, development and fixation.

The antibody sources used in the examples are as follows: anti-VSV G antibody (1:12,000; Santa Cruz, sc-66180), anti-p24antibody (1:30,000; Sino Biological Inc.), anti- β -actinobody (1:10,000; Sigma-Aldrich, A5441).

1.3. membrane protein biotin labeling assay

Principle of experiment

cell surface proteins are an important group of cellular components that are localized to the plasma membrane surface of cells. In order to facilitate the study of the functions of these membrane surface proteins, it is often necessary to isolate the membrane surface proteins. Cell surface proteins can be biotinylated by forming a stable covalent linkage with amino groups of the cell surface protein using a membrane-impermeable, amino-reactive biotinylation reagent, sulfo-NHS-Biotin (Thermo scientific, cat. 21217). The proteins on the membrane surface can then be separated by the interaction between streptavidin (streptavidin) and biotin using streptavidin agarose resin (Thermo fisher scientific, cat # 20349).

Experimental procedure

1) Inoculation of 4X 10 into 12-well plates⁵Hela cells in the hole, and the next day transfects target plasmids;

2) After transfection for 48h, the cell culture medium was discarded and washed 3 times with precooled hbss (hyclone);

3) Adding EZ-Link sulfo-NHS-Biotin at 0.25mg/ml, and incubating at 4 ℃ for 15 min;

4) Washing with pre-cooled HBSS for 3 times, adding 200mM glycine, and incubating at 4 deg.C for 15 min;

5) washing with pre-cooled HBSS for 3 times, adding 400 μ L cell lysate, and performing lysis on ice for 10 min;

6) collecting cell lysate into 1.5ml EP tube, and performing ultrasonic treatment at 200W power for 3 times at intervals of 3s for 5s each time;

7) Centrifuging the cell lysate in a 4 ℃ centrifuge at 13300rpm for 10 min;

8) The supernatant was transferred to a new tube, 40. mu.L was taken as a control, 30. mu.L of streptavidin agarose resin was added to the remaining cell lysate, and rotary incubation was performed at 4 ℃ for 4 h;

9) Centrifuging to remove supernatant, washing resin with precooled cell lysate for 3 times, and adding protein sample buffer solution to boil for 7 min;

10) Western blotting was performed to detect the level of the protein of interest.

1.4 site-directed mutagenesis of the Gene of interest

PCR amplification of DNA of interest

reaction system (all formula gold):

5×buffer	4μL
		Plasmid	50-100ng
dNTP	2μL
		Upstream and downstream primers (10. mu.M)	1.5μL
DNA polymerase	0.8μL
		ddH₂O	Make up to 20. mu.L

the PCR primers used to construct the VSV-G transmembrane variants are shown in the following table (each pair of primers carries a mutation corresponding to 3-5 amino acids, and each transmembrane variant is constructed by 5 rounds of PCR):

The PCR primers used to construct the VSV-G extracellular membrane proximal region variants are shown in the following table:

And (3) PCR reaction conditions:

Adding 1 μ L of DpnI enzyme into the PCR product, digesting for 3h at 37 ℃;

Taking a proper amount of PCR product to transform escherichia coli;

and respectively inoculating the grown single colonies into a liquid culture medium for amplification on the next day, and extracting plasmids for sequencing verification.

EXAMPLE 2VSV-G mediated cell fusion

VSV-G has been demonstrated to have fusion ability as a viral fusion protein. In order to quantify their fusion function and monitor the kinetics of fusion in real time, the inventors constructed a cell-cell fusion system. Co-transfecting cells with red fluorescent protein (dsRED-nes) with nuclear output signals and VSV-G, wherein the cytoplasm of the transfected cells is red under a fluorescent microscope, and the VSV-G is expressed on the surface of cell membranes; in addition, cyan fluorescent protein (CFP-nls) with nuclear localization signal was transfected into cells, and the nuclei of transfected cells were cyan under a fluorescent microscope. The two cells were co-cultured and VSV-G mediated fusion was induced using low pH, and cells that had fused if the cytoplasm was red and contained more than one cyan nucleus were present, as shown in FIGS. 1 and 2.

1. Cell-cell fusion experimental procedure

1) In 24-well plates at 2X 10⁵The density of the wells was inoculated with Hela cells;

2) the next day, a plasmid expressing dsRED-nes and a plasmid expressing VSV-G were transfected;

3) Put the slide in another 24-well plate, and put it on the slide at 7X 10⁴density of wells HeLa cells stably expressing CFP in the nucleus were seeded;

4) The Hela cells expressing VSV-G obtained in step 2) were digested with sodium citrate buffer (11G KCl,4.4G sodium citrate, water to 1 liter) at 1X 10⁵The density of each well is inoculated into the 24-well plate in the step 3) and is co-cultured with the CFP stable cells;

5) After 16h, MES buffer (MES, cat No.18886, pH 5.0-6.0; USB, USA) for 1 min;

6) Replacing with fresh complete culture solution, and culturing for 3 h;

7) Discarding the culture solution, fixing the cells with 4% Paraformaldehyde (PFA) for 10min, washing with PBS for 3 times, 5min each time;

8) And taking out the slide, sealing, observing under a laser confocal microscope, and recording an image.

2. Live cell imaging assay

1) in 12-well plate at 4X 10⁵The density of the wells was inoculated with Hela cells;

3) Stable cells with nuclei expressing CFP at 7X 10⁴the density of the culture medium is inoculated in a special glass-bottom culture dish of a 35mm microscope;

4) Digesting the VSV-G-expressing Hela cells obtained in step 2) with a sodium citrate buffer at 4X 10⁵The density of the/hole is inoculated into the culture dish in the step 3) to be co-cultured with the CFP stable cells;

5) After 16h, the cells were treated with MES buffer pH6.0 for 1min and then replaced with fresh complete medium;

6) The culture dish was placed in a mini-culture device of a confocal laser microscope, and under the Time Series function of the microscope, one image was captured every 15s, and the dynamic change process of cell fusion was recorded.

Detection of fusion Capacity of VSV-G transmembrane Domain Displacement variants

The VSV-G transmembrane region replacement variants were constructed using conventional molecular cloning techniques, with PCR primers as described in example 1, using an expression vector pMD2.G (from Addgene), with the transmembrane regions located at positions 465 to 490 of the VSV-G amino acid sequence (IASFFFIIGLIIGLFLVLRVGIHLCI, SEQ ID NO: 3).

replacing the transmembrane region of VSV-G with the transmembrane region of Syntaxin1A protein (IMIIICCVILGIIIASTIGGIFG, SEQ ID NO:4) to obtain a Stx1A transmembrane region replacement variant (abbreviated as Stx 1A);

Replacing the VSV-G transmembrane region with the transmembrane region of VAMP2 protein (MMIILGVICAIILIIIIVYFST, SEQ ID NO:5) to obtain a VAMP2 transmembrane region replacement variant (abbreviated as VAMP 2);

The VSV-G transmembrane region was replaced with the transmembrane region of the influenza virus HA protein (ILSIYSTVASSLALAIMVAGLSLW, SEQ ID NO:6), yielding an HA transmembrane region replacement variant (abbreviated HA).

the expression plasmids constructed above are respectively introduced into a cell-cell fusion system, and the ability of the VSV-G transmembrane region replacement variant to mediate cell fusion is detected. The experiment was set up in 4 groups: wild type control group (VSV-G), Stx1A transmembrane region replacement variant (Stx1A), VAMP2 transmembrane region replacement variant (VAMP2), HA transmembrane region replacement variant (HA). The results are shown in FIGS. 3 and 4. As can be seen in FIG. 3, wild-type VSV-G mediated massive cell fusion, forming a large fused cell with multiple nuclei. Each VSV-G variant results in a different reduction in the number of cells undergoing fusion. Statistical analysis of FIG. 4 shows that Syntaxin1A or VAMP2 transmembrane region replacement results in about a 30-40% reduction in VSV-G mediated cell fusion capacity in the experiment, while influenza virus HA transmembrane region replacement results in about an 80% reduction in VSV-G mediated cell fusion capacity in the experiment. This result indicates that replacement of the wild-type VSV-G transmembrane region with that of another protein results in a reduction in VSV-G mediated cell fusion ability to varying degrees.

Fusion ability test of insertion variants of the extracellular membrane proximal region of VSV-G

The VSV-G extracellular membrane-proximal region insert was constructed using conventional molecular cloning techniques, with PCR primers as described in example 1, using an expression vector pMD2.G, with the extracellular membrane-proximal region being located between positions 444 and 464 (TGLSKNPIELVEGWFSSWKSS, SEQ ID NO:7) of the VSV-G amino acid sequence, and the insert being located between positions 459 and 460.

Cell fusion experiments were established in 7 groups: wild type control (WT), 1 amino acid inserted variant (linker1, one serine inserted), 2 amino acid inserted variant (linker 2, glycine and serine inserted), 3 amino acid inserted variant (linker 3, glycine-serine inserted), 4 amino acid inserted variant (linker 4, glycine-serine-glycine inserted), 5 amino acid inserted variant (linker 5, glycine-serine-glycine inserted), 6 amino acid inserted variant (linker 6, glycine-serine-glycine-serine inserted). The results are shown in FIGS. 5 and 6. As can be seen in FIG. 5, wild-type VSV-G mediated massive cell fusion, forming a large fused cell with multiple nuclei. And the VSV-G mediated cell fusion capacity is reduced along with the increase of the number of the inserted amino acids of the extracellular membrane proximal region. Statistical analysis of FIG. 6 shows that insertion of 1-6 amino acids into the VSV-G juxtamembrane domain resulted in 7%, 18%, 26%, 39%, 79% and 90% decrease in the ability of VSV-G mediated cell fusion in the experiment, respectively.

EXAMPLE 3 preparation of lentivirus expressing VSV-G variant and cell infection experiment

VSV-G, a viral fusion protein, mediates fusion of the viral envelope with the membrane of the target cell endocyton, and thus invades the cell. The host range of VSV-G mediated fusion is very wide, VSV-G can be expressed on the surface of a genetically engineered lentivirus and used as a fusion source of a lentivirus transfection vector, and the genetically engineered lentivirus has no self-replication capacity and high safety, so that the system is widely applied to research. In a lentivirus packaging system consisting of three plasmids, namely pLVTHM, pMD2.G and psPAX2 (all purchased from Addgene), pMD2.G carries VSV-G gene, so that VSV-G is expressed on the surface of the obtained virus, and the fusion of the virus and cells can be mediated. The inventors have modified VSV-G by using conventional molecular cloning techniques, with reference to example 2, to replace its transmembrane region or to insert amino acids into the extracellular membrane region. In the lentivirus packaging system, pLVTHM plasmid carries GFP gene, and after virus invades cells, green fluorescent protein can be expressed to serve as an observation marker, and whether VSV-G can mediate fusion of the virus and the cells or not is judged by observing the green fluorescent protein.

1. Virus preparation

1) At 1 × 10⁶Density of wells 293T cells were seeded into 6-well plates;

2) The following day, 293T cells per well were transfected with 1.17. mu.g pLVTHM, 0.8. mu.g psPAX2, and 0.53. mu.g pMD2.G (expressing VSV-G or a variant thereof);

3) after 24h of transfection, the cell culture medium was discarded and replaced with fresh Opti-MEM culture medium (Invitrogen), and the culture was continued;

4) After 24-48h, the virus-containing culture medium was collected, centrifuged at 4000rpm for 10min, and the supernatant was transferred to a new tube as virus stock for immediate use or stored at-80 ℃ for further use.

2. Experiment of viral infection

1) Adding the virus stock solution into a 6-well plate inoculated with the same number of Hela cells according to the proportion of 1:5, 1:25, 1:125 and 1: 625;

2) After culturing for 24h, replacing the culture solution with a fresh complete culture medium, and continuing culturing;

3) After 12h, removing the culture solution, fixing the cells by using 4% PFA, observing under a confocal microscope, and recording images; or after digesting the cells with pancreatin, the ratio of virus-infected cells (GFP-positive cells) was quantitatively analyzed by flow cytometry.

3. Determination of infectivity of variant viruses with replacement of the VSV-G transmembrane region

the wild VSV-G gene carried by pMD2.G plasmid is modified by conventional molecular cloning technique to construct VSV-G transmembrane region replacement variant, and cell infection experiment is carried out after virus preparation. The experiment was set up in 4 groups: a viral control group expressing wild-type VSV-G (VSV-G), a virus expressing a Stx1A transmembrane region replacement variant (Stx1A), a virus expressing a VAMP2 transmembrane region replacement variant (VAMP2), a virus expressing a HA transmembrane region replacement variant (HA). The results after infection of the cells are shown in FIGS. 7 and 8. Under confocal fluorescence microscopy, infected cells appear green at a specific excitation wavelength due to the expression of the GFP reporter. As can be seen in FIG. 7, the virus expressing wild type VSV-G was able to fuse with cells, infect into cells, express the GFP reporter gene, and the cells were green under a fluorescent microscope after being added to the cell culture dish. After the virus expressing the transmembrane region displacement variant is added into a cell culture dish, only a small number of cells are green, which indicates that the infection capacity of the virus is obviously reduced. The infected cells (GFP positive cells) were counted by flow cytometry, and the results are shown in FIG. 8A. Statistical analysis of fig. 8B shows that the Syntaxin1A or VAMP2 transmembrane region replacement results in about 50% reduction in the infection efficiency of the virus (p <0.01), while influenza HA transmembrane region replacement results in about 90% reduction in the infection efficiency of the lentivirus (p < 0.001).

4. Determination of the infectivity of a Virus having an insertion variant of the extracellular membrane proximal region of VSV-G

the wild VSV-G gene carried by pMD2.G plasmid is modified by conventional molecular cloning technique to construct VSV-G extracellular membrane insertion variant, and cell infection experiment is carried out after virus preparation. The experiment was performed in 7 groups: wild type control (WT), 1 amino acid inserted variant (linker1), 2 amino acid inserted variant (linker 2), 3 amino acid inserted variant (linker 3), 4 amino acid inserted variant (linker 4), 5 amino acid inserted variant (linker 5), 6 amino acid inserted variant (linker 6). The results are shown in FIGS. 9 and 10. As can be seen in FIG. 9, the virus expressing wild type VSV-G was fused to the cells after addition to the cell culture dish, infected into the cells, expressed the GFP reporter gene and the cells were green under a fluorescent microscope. And with the increase of the number of the amino acids inserted into the extracellular membrane proximal region, the number of the cells expressing the green fluorescent protein is obviously reduced, which indicates that the fusion capacity of the virus and the cells is reduced, and the number of infected cells is reduced. Statistical analysis of FIG. 10 shows that insertion of VSV-G at 1-6 amino acids in the membrane-proximal domain reduces the infection efficiency of the virus by 26%, 45%, 68%, 80%, 93% and 98%, respectively (p < 0.01).

The results show that the vesicular stomatitis virus fusion protein (VSV-G) function deletion variants (including transmembrane region replacement variants and membrane-proximal domain insertion variants) constructed by the invention can significantly reduce VSV-G mediated fusion of virus and cells, and block cell damage or necrosis caused by virus invasion of cells; the VSV-G constructed by the invention does not change the original sequence and structure of the VSV-G extracellular region, fully retains the antigenicity thereof, can be used for manufacturing safe and effective VSV genetic engineering vaccines, and achieves effective prevention and treatment effects.

SEQUENCE LISTING

<110> vesicular stomatitis virus envelope glycoprotein variant, and construction method and application thereof

<120> institute of basic medicine of Chinese academy of medical sciences

<130> MP1910835

<160> 49

<170> PatentIn version 3.2

<210> 1

<211> 1536

<212> DNA

<213> Vesicular stomatitis virus

<400> 1

atgaagtgcc ttttgtactt agccttttta ttcattgggg tgaattgcaa gttcaccata 60

gtttttccac acaaccaaaa aggaaactgg aaaaatgttc cttctaatta ccattattgc 120

ccgtcaagct cagatttaaa ttggcataat gacttaatag gcacagccat acaagtcaaa 180

atgcccaaga gtcacaaggc tattcaagca gacggttgga tgtgtcatgc ttccaaatgg 240

gtcactactt gtgatttccg ctggtatgga ccgaagtata taacacagtc catccgatcc 300

ttcactccat ctgtagaaca atgcaaggaa agcattgaac aaacgaaaca aggaacttgg 360

ctgaatccag gcttccctcc tcaaagttgt ggatatgcaa ctgtgacgga tgccgaagca 420

gtgattgtcc aggtgactcc tcaccatgtg ctggttgatg aatacacagg agaatgggtt 480

gattcacagt tcatcaacgg aaaatgcagc aattacatat gccccactgt ccataactct 540

acaacctggc attctgacta taaggtcaaa gggctatgtg attctaacct catttccatg 600

gacatcacct tcttctcaga ggacggagag ctatcatccc tgggaaagga gggcacaggg 660

ttcagaagta actactttgc ttatgaaact ggaggcaagg cctgcaaaat gcaatactgc 720

aagcattggg gagtcagact cccatcaggt gtctggttcg agatggctga taaggatctc 780

tttgctgcag ccagattccc tgaatgccca gaagggtcaa gtatctctgc tccatctcag 840

acctcagtgg atgtaagtct aattcaggac gttgagagga tcttggatta ttccctctgc 900

caagaaacct ggagcaaaat cagagcgggt cttccaatct ctccagtgga tctcagctat 960

cttgctccta aaaacccagg aaccggtcct gctttcacca taatcaatgg taccctaaaa 1020

tactttgaga ccagatacat cagagtcgat attgctgctc caatcctctc aagaatggtc 1080

ggaatgatca gtggaactac cacagaaagg gaactgtggg atgactgggc accatatgaa 1140

gacgtggaaa ttggacccaa tggagttctg aggaccagtt caggatataa gtttccttta 1200

tacatgattg gacatggtat gttggactcc gatcttcatc ttagctcaaa ggctcaggtg 1260

ttcgaacatc ctcacattca agacgctgct tcgcaacttc ctgatgatga gagtttattt 1320

tttggtgata ctgggctatc caaaaatcca atcgagcttg tagaaggttg gttcagtagt 1380

tggaaaagct ctattgcctc ttttttcttt atcatagggt taatcattgg actattcttg 1440

gttctccgag ttggtatcca tctttgcatt aaattaaagc acaccaagaa aagacagatt 1500

tatacagaca tagagatgaa ccgacttgga aagtaa 1536

<210> 2

<211> 511

<212> PRT

<213> Vesicular stomatitis virus

<400> 2

Met Lys Cys Leu Leu Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys

1 5 10 15

Lys Phe Thr Ile Val Phe Pro His Asn Gln Lys Gly Asn Trp Lys Asn

20 25 30

Val Pro Ser Asn Tyr His Tyr Cys Pro Ser Ser Ser Asp Leu Asn Trp

35 40 45

His Asn Asp Leu Ile Gly Thr Ala Ile Gln Val Lys Met Pro Lys Ser

50 55 60

His Lys Ala Ile Gln Ala Asp Gly Trp Met Cys His Ala Ser Lys Trp

65 70 75 80

Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr Ile Thr Gln

85 90 95

Ser Ile Arg Ser Phe Thr Pro Ser Val Glu Gln Cys Lys Glu Ser Ile

100 105 110

Glu Gln Thr Lys Gln Gly Thr Trp Leu Asn Pro Gly Phe Pro Pro Gln

115 120 125

Ser Cys Gly Tyr Ala Thr Val Thr Asp Ala Glu Ala Val Ile Val Gln

130 135 140

Val Thr Pro His His Val Leu Val Asp Glu Tyr Thr Gly Glu Trp Val

145 150 155 160

Asp Ser Gln Phe Ile Asn Gly Lys Cys Ser Asn Tyr Ile Cys Pro Thr

165 170 175

Val His Asn Ser Thr Thr Trp His Ser Asp Tyr Lys Val Lys Gly Leu

180 185 190

Cys Asp Ser Asn Leu Ile Ser Met Asp Ile Thr Phe Phe Ser Glu Asp

195 200 205

Gly Glu Leu Ser Ser Leu Gly Lys Glu Gly Thr Gly Phe Arg Ser Asn

210 215 220

Tyr Phe Ala Tyr Glu Thr Gly Gly Lys Ala Cys Lys Met Gln Tyr Cys

225 230 235 240

Lys His Trp Gly Val Arg Leu Pro Ser Gly Val Trp Phe Glu Met Ala

245 250 255

Asp Lys Asp Leu Phe Ala Ala Ala Arg Phe Pro Glu Cys Pro Glu Gly

260 265 270

Ser Ser Ile Ser Ala Pro Ser Gln Thr Ser Val Asp Val Ser Leu Ile

275 280 285

Gln Asp Val Glu Arg Ile Leu Asp Tyr Ser Leu Cys Gln Glu Thr Trp

290 295 300

Ser Lys Ile Arg Ala Gly Leu Pro Ile Ser Pro Val Asp Leu Ser Tyr

305 310 315 320

Leu Ala Pro Lys Asn Pro Gly Thr Gly Pro Ala Phe Thr Ile Ile Asn

325 330 335

Gly Thr Leu Lys Tyr Phe Glu Thr Arg Tyr Ile Arg Val Asp Ile Ala

340 345 350

Ala Pro Ile Leu Ser Arg Met Val Gly Met Ile Ser Gly Thr Thr Thr

355 360 365

Glu Arg Glu Leu Trp Asp Asp Trp Ala Pro Tyr Glu Asp Val Glu Ile

370 375 380

Gly Pro Asn Gly Val Leu Arg Thr Ser Ser Gly Tyr Lys Phe Pro Leu

385 390 395 400

Tyr Met Ile Gly His Gly Met Leu Asp Ser Asp Leu His Leu Ser Ser

405 410 415

Lys Ala Gln Val Phe Glu His Pro His Ile Gln Asp Ala Ala Ser Gln

420 425 430

Leu Pro Asp Asp Glu Ser Leu Phe Phe Gly Asp Thr Gly Leu Ser Lys

435 440 445

Asn Pro Ile Glu Leu Val Glu Gly Trp Phe Ser Ser Trp Lys Ser Ser

450 455 460

Ile Ala Ser Phe Phe Phe Ile Ile Gly Leu Ile Ile Gly Leu Phe Leu

465 470 475 480

Val Leu Arg Val Gly Ile His Leu Cys Ile Lys Leu Lys His Thr Lys

485 490 495

Lys Arg Gln Ile Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys

500 505 510

<210> 3

<211> 26

<212> PRT

<213> Artificial sequence

<220>

<223> VSV-G protein transmembrane region

<400> 3

Ile Ala Ser Phe Phe Phe Ile Ile Gly Leu Ile Ile Gly Leu Phe Leu

1 5 10 15

Val Leu Arg Val Gly Ile His Leu Cys Ile

20 25

<210> 4

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> Syntaxin1A protein transmembrane region

<400> 4

Ile Met Ile Ile Ile Cys Cys Val Ile Leu Gly Ile Ile Ile Ala Ser

1 5 10 15

Thr Ile Gly Gly Ile Phe Gly

20

<210> 5

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> protein transmembrane region of VAMP2

<400> 5

Met Met Ile Ile Leu Gly Val Ile Cys Ala Ile Ile Leu Ile Ile Ile

1 5 10 15

Ile Val Tyr Phe Ser Thr

20

<210> 6

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<223> HA protein transmembrane region

<400> 6

Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu Ala Ile

1 5 10 15

Met Val Ala Gly Leu Ser Leu Trp

20

<210> 7

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> extracellular membrane region of VSV-G

<400> 7

Thr Gly Leu Ser Lys Asn Pro Ile Glu Leu Val Glu Gly Trp Phe Ser

1 5 10 15

Ser Trp Lys Ser Ser

20

<210> 8

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 8

ttcagtagtt ggaaaagctc tatactgtct atttatttta tcatagggtt aatcatt 57

<210> 9

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 9

aatgattaac cctatgataa aataaataga cagtatagag cttttccaac tactgaa 57

<210> 10

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 10

agctctatac tgtctattta ttcaacagtg gcgagtatca ttggactatt cttggtt 57

<210> 11

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 11

aaccaagaat agtccaatga tactcgccac tgttgaataa atagacagta tagagct 57

<210> 12

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 12

tattcaacag tggcgagttc cctagcactg gcattggttc tccgagttgg t 51

<210> 13

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 13

accaactcgg agaaccaatg ccagtgctag ggaactcgcc actgttgaat a 51

<210> 14

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 14

agttccctag cactggcaat catggtagct ggtggtatcc atctttgcat t 51

<210> 15

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 15

aatgcaaaga tggataccac cagctaccat gattgccagt gctagggaac t 51

<210> 16

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 16

gcaatcatgg tagctggtct atctttatgg aaattaaagc acaccaag 48

<210> 17

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 17

cttggtgtgc tttaatttcc ataaagatag accagctacc atgattgc 48

<210> 18

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 18

ttcagtagtt ggaaaagctc tatcatgatc atcattttta tcatagggtt aatcatt 57

<210> 19

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 19

aatgattaac cctatgataa aaatgatgat catgatagag cttttccaac tactgaa 57

<210> 20

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 20

agctctatca tgatcatcat ttgctgtgtg attctgatca ttggactatt cttggtt 57

<210> 21

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 21

aaccaagaat agtccaatga tcagaatcac acagcaaatg atgatcatga tagagct 57

<210> 22

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 22

atttgctgtg tgattctggg catcatcatc gccttggttc tccgagttgg t 51

<210> 23

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 23

accaactcgg agaaccaagg cgatgatgat gcccagaatc acacagcaaa t 51

<210> 24

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 24

ctgggcatca tcatcgcctc caccatcggg ggcggtatcc atctttgcat t 51

<210> 25

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 25

aatgcaaaga tggataccgc ccccgatggt ggaggcgatg atgatgccca g 51

<210> 26

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 26

gcctccacca tcgggggcat ctttggaaaa ttaaagcaca ccaag 45

<210> 27

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 27

cttggtgtgc tttaattttc caaagatgcc cccgatggtg gaggc 45

<210> 28

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 28

ttcagtagtt ggaaaagctc tatgatgatc atcttgttta tcatagggtt aatcatt 57

<210> 29

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 29

aatgattaac cctatgataa acaagatgat catcatagag cttttccaac tactgaa 57

<210> 30

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 30

agctctatga tgatcatctt gggagtgatc tgcgccatca ttggactatt cttggtt 57

<210> 31

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 31

aaccaagaat agtccaatga tggcgcagat cactcccaag atgatcatca tagagct 57

<210> 32

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 32

gtgatctgcg ccatcattct catcatcttg gttctccgag ttggt 45

<210> 33

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 33

accaactcgg agaaccaaga tgatgagaat gatggcgcag atcac 45

<210> 34

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 34

gccatcattc tcatcatcat catcgtttac ttcggtatcc atctttgcat t 51

<210> 35

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 35

aatgcaaaga tggataccga agtaaacgat gatgatgatg agaatgatgg c 51

<210> 36

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 36

atcatcatcg tttacttcag cactaaatta aagcacacca ag 42

<210> 37

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 37

cttggtgtgc tttaatttag tgctgaagta aacgatgatg at 42

<210> 38

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 38

gaaggttggt tcagtagcag ttggaaaagc tct 33

<210> 39

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 39

agagcttttc caactgctac tgaaccaacc ttc 33

<210> 40

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 40

gtagaaggtt ggttcagtgg aagcagttgg aaaagctcta tt 42

<210> 41

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 41

aatagagctt ttccaactgc ttccactgaa ccaaccttct ac 42

<210> 42

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 42

gtagaaggtt ggttcagtgg aggaagcagt tggaaaagct ctatt 45

<210> 43

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 43

aatagagctt ttccaactgc ttcctccact gaaccaacct tctac 45

<210> 44

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 44

ttcagtggag gaagcggaag ttggaaaagc tctat 35

<210> 45

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 45

atagagcttt tccaacttcc gcttcctcca ctgaa 35

<210> 46

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 46

tggttcagtg gaggaagcgg aggaagttgg aaaagctcta tt 42

<210> 47

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 47

aatagagctt ttccaacttc ctccgcttcc tccactgaac ca 42

<210> 48

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 48

tggttcagtg gaggaagcgg aggaagcagt tggaaaagct ctatt 45

<210> 49

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 49

aatagagctt ttccaactgc ttcctccgct tcctccactg aacca 45

Claims

1. A VSV-G protein variant having an amino acid sequence at a transmembrane region and/or an extracellular membrane proximal region that differs from a wild-type VSV-G protein, said variant having reduced/lost ability to mediate viral fusion with a cell while retaining its ability to form a transmembrane protein.

2. The VSV-G protein variant of claim 1, wherein the transmembrane region is replaced with a transmembrane sequence that is not the VSV-G transmembrane region.

3. The VSV-G protein variant of claim 2, wherein the transmembrane sequence is selected from the group consisting of the following protein transmembrane region amino acid sequences:

4. The VSV-G protein variant of claim 2 or 3, the transmembrane sequence selected from the group consisting of the following protein transmembrane region amino acid sequences:

A) a transmembrane region of the Syntaxin1A protein or a fragment thereof, for example, the transmembrane region of the Syntaxin1A protein comprises an amino acid sequence shown as SEQ ID NO. 4;

B) A transmembrane region of the VAMP2 protein, or a fragment thereof, e.g., the transmembrane region of the VAMP2 protein comprises an amino acid sequence as set forth in SEQ ID NO. 5; and

C) A transmembrane region of an influenza virus HA protein, for example comprising an amino acid sequence as set forth in SEQ ID No. 6, or a fragment thereof.

5. The VSV-G protein variant of any of claims 1 to 4, having one or more additional amino acids for an extracellular membrane proximal region comprising an amino acid sequence set forth as SEQ ID NO 7.

6. the VSV-G protein variant of claim 5, having 2 or more additional amino acids, such as 3 to 20 additional amino acids, for example 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 additional amino acids, more preferably at least 6 additional amino acids, for the extracellular membrane proximal region.

7. a polynucleotide comprising a nucleotide sequence encoding the VSV-G protein variant of any of claims 1-6.

8. A vaccine composition comprising the VSV-G protein variant of any one of claims 1 to 6 and/or the polynucleotide of claim 7.

9. The vaccine composition of claim 8, wherein the VSV-G protein variant is located on the surface of a viral particle, preferably the viral particle does not have the ability to cause VSV-induced disease.

10. The vaccine composition of claim 8 or 9, further comprising an immunologically and pharmaceutically acceptable carrier or adjuvant.

11. Use of the VSV-G protein variant of any of claims 1-6, the polynucleotide of claim 7, and/or the vaccine composition of any of claims 8-10 in the manufacture of a medicament for preventing and/or treating a VSV infection in a host.

12. the use of claim 11, said host being selected from the group consisting of horses, cattle, sheep and pigs.