CN112941039A

CN112941039A - Novel vesicular oncolytic virus and application thereof in preparation of antitumor drugs

Info

Publication number: CN112941039A
Application number: CN202110140752.8A
Authority: CN
Inventors: 吴俊华; 魏继武; 张海林; 张永辉; 吴俊艺; 刘淑雯; 马丁
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2021-02-01
Filing date: 2021-02-01
Publication date: 2021-06-11
Also published as: WO2022160475A1

Abstract

The invention relates to the field of tumor treatment, in particular to application of a novel vesicular oncolytic virus in preparation of an anti-tumor drug. The invention discloses: the virus Ad5 sPGRCD 137L infects cells capable of expressing specific membrane protein VSV-G, and then uses centrifugal extrusion/shearing method to make these cells pass through membrane pores to form vesicles, so as to make the oncolytic virus covered by vesicle-like, the outer surface of vesicle retains the membrane protein VSV-G on the cell surface in the preparation process. The prepared novel vesicular oncolytic virus can realize retargeting through a membrane protein VSV-G, increase the gene transduction efficiency of the virus, escape the neutralization effect of an anti-virus antibody, improve the virus yield, and play the dual functions of blocking an immunity detection point and activating immunity co-stimulation by the soluble fusion protein sVRCD 137L expressed by the virus. The novel vesicular oncolytic virus can obviously activate anti-tumor immunity and finally obviously prolong the survival of mice.

Description

Novel vesicular oncolytic virus and application thereof in preparation of antitumor drugs

Technical Field

The invention relates to the field of tumor treatment, in particular to the field of tumor immunotherapy, and specifically relates to application of a novel vesicular oncolytic virus-like EM/VSV-G Ad5 sVRCD 137L in preparation of an antitumor drug.

Background

Cancer is one of the most harmful diseases to human life health, and millions of people die of cancer every year. Many patients are diagnosed at the middle and advanced stage, so that the chance of surgical treatment is lost, the traditional radiotherapy and chemotherapy does not bring breakthrough development to the tumor, and even small molecule targeted drugs face the huge challenge of relapse in a short period.

The exciting results of clinical studies on antitumor immunotherapy have renewed promise in recent decades. Simultaneous blockade of the electronegative regulation and activation of the costimulatory pathway is a proven effective method for treating tumors, and several immune checkpoint blockers have been approved as drugs for clinical tumor treatment to date. TIGIT (T cell immunoglobulin and immunoreceptor systemic inhibition motif) is another immune checkpoint regulatory molecule expressed on lymphocytes discovered in recent years, and is involved in negative regulation of activated T cells and NK cells. Blocking TIGIT to restore the immune co-stimulation pathway of CD226 is a clever and effective anti-tumor immunization strategy.

How to effectively induce I-type interferon mediated immune activation in tumor local and enhance infiltration of tumor microenvironment immune cells can make tumors more sensitive to treatment of targeted immune check points, which is probably one of effective means for solving the problem of low universality (efficiency) of immune check point treatment. However, how to solve the problem of immune checkpoint treatment "off-target" effects?

The virus can be used as a foreign invasion particle and can effectively activate the natural immunity and the adaptive immunity of the body. As oncolytic virus T-Vec was approved by the FDA for marketing by the end of 2015, oncolytic virus-mediated anti-tumor immunotherapy received increasing attention. Whether oncolytic virus immunotherapy can make tumors more sensitive to treatment targeting immune checkpoints solves the problem of not high universality (efficiency) of immune checkpoint treatment (the 1 st problem encountered with immune checkpoint treatment).

In addition, due to the advantage that the oncolytic virus has selective replication in tumor cells, the oncolytic adenovirus expressing the immune checkpoint blocker is utilized to replicate locally in the tumor, so that the immune activation is limited to the local microenvironment of the tumor to the maximum extent, and the phenomenon that the blocked immune checkpoint is off-target to cause the false injury of other normal tissues is effectively avoided (the 2 nd problem encountered by immune checkpoint treatment).

Currently, there is a lack of a replicative oncolytic adenovirus that has both a blockade of immune checkpoints and activation of immune co-stimulatory pathways.

In addition, although the strategy of expressing tumor immunotherapy proteins in tumor local by oncolytic adenovirus has great advantages, native tropism of adenovirus limits the application of oncolytic adenovirus. Ad5 enters cells by binding of the viral fiber knob to the CAR (the coxsackie and adenovirus receptor) on the target cell membrane. A large number of researches show that Ad5 has poor efficiency of infecting tumors with low expression of CAR on the cell surface, which also limits the clinical curative effect of oncolytic adenovirus Ad5 mediated tumor gene therapy, and because most tumor cells have low expression of CAR, the development of a method for enhancing the infection efficiency of adenovirus on CAR-low expression tumor cells is very important. In addition, the widely-existing adenovirus neutralizing antibodies in human are also important factors limiting the clinical efficacy of adenovirus therapy.

Methods are available to increase the efficiency of infection of CAR-low expressing cells by Ad 5. The first approach was to modify the capsid of Ad5 using covalent bonds, primarily by attaching artificial polymers, including polyethylene glycol (PEG), polylactic acid (PLGA), polyethylene imine (PEI), or lipids, to the Ad5 capsid. The second approach is to achieve retargeting of adenovirus by genetic engineering of Ad5, such as: ad5-RGD with Arg-Gly-Asp (RGD) polypeptide inserted in the HI loop region of Ad5 fiber knob domain; fiber chimeric Ad5/35 of Ad5 and Ad 35. These approaches do increase the efficiency of viral infection to some extent, but are relatively cumbersome to operate.

In recent years artificial vesicle technology has been used to load drugs instead of natural exosome technology. The artificial vesicle technology can overcome the problem of low yield of natural exosomes, is only used for coating drugs to realize targeted delivery at present, and has not been reported so far when the artificial vesicle technology is used for coating viruses. Moreover, it is widely believed that this technique is unlikely to be used to package such large therapeutic entities of viruses.

Therefore, the invention hopes to realize breakthrough through our research, constructs replication type oncolytic adenovirus Ad5 sVRCD 137L expressing sVRCD 137L, and wraps the replication type oncolytic adenovirus Ad5 sVRCD 137L by combining a centrifugal extrusion/shearing vesicle preparation technology, thereby preparing EM/VSV-G Ad5 sVRCD 137L. Thus, the prepared novel vesicular oncolytic virus EM/VSV-G Ad5 sPGRCD 137L can not only express a soluble protein sPGRCD 137L through oncolytic adenovirus, one end of the protein is PVR protein capable of being combined with TIGIT, the other end of the protein is CD137L protein capable of being combined with CD137, and the sPGRCD 137L can block a PVR-TIGIT immunodetection point passage after being combined with TIGIT; sPRVCD 137L is capable of activating CD 137-mediated immune co-stimulatory pathways upon binding to CD 137.

Furthermore, VSV-G protein is introduced to the vesicle wrapping the virus, so that the retargeting of the virus can be realized, the novel vesicular oncolytic virus EM/VSV-G Ad5 sPGRCD 137L can infect cells which cannot be infected or are low-infected by naked virus, and the prepared EM/VSV-G Ad5 sPGRCD 137L can resist anti-virus neutralizing antibody. Then is this novel oncolytic virus EM/VSV-G Ad5 sPGRCD 137L not able to exhibit unexpected properties in vitro and in vivo?

Disclosure of Invention

In the present invention, we show that the vesicle-like virus is prepared by preparing vesicle-like using a centrifugal extrusion/shearing method to encapsulate oncolytic virus. Specifically, 293T cells (293T-VSV-G cells) expressing a specific protein Vesicular stock Indiana virus G protein (VSV-G) were used to infect 293T-VSV-G cells with replication-competent oncolytic adenovirus Ad5 sVRCD 137L, and vesicle-like preparation was performed by centrifugal extrusion/shearing to produce vesicle-like encapsulated virus EM/VSV-G Ad5 sVRCD 137L. We refer to viruses produced by the technique of preparing vesicle-like viruses by centrifugal extrusion/shearing as vesicular-like viruses.

The key technical characteristics of the method for preparing the novel vesicular virus are as follows: the cells infected with the virus are sequentially filtered by a filter membrane with the pore diameter from large to small in a centrifugal extrusion/shearing mode. This extrusion step allows the virus to be surface coated with a membrane similar to an extracellular vesicle. As long as the pore size of the filter is selected appropriately, all cells infected with the virus can be prepared in such a way as to provide such extrusion.

It is because the extruded virus is coated with a membrane similar to the extracellular vesicles, which is derived from cells, and thus if the original cell membrane has extracellular membrane proteins, the vesicle-like membrane after the centrifugal extrusion/shearing step can be embedded with such extracellular membrane proteins. Thus we can select the proteins that ultimately appear on the membrane of the vesicle-like: allowing the cells to express the desired outer membrane proteins prior to infection with the virus.

The embodiments of the invention which follow show the method with simple process and clear principle, so that the claims of the patent claim section can be supported by the results shown by the embodiments.

The novel vesicular virus prepared by encapsulating the virus by the vesicular-like technology has the following prominent substantive characteristics and remarkable progress:

1. during the process of packaging viruses by the vesicle-like technology, VSV-G which is expected to be introduced and can bring targeting is introduced into the outer vesicle-like membrane of the packaged viruses. The targeting protein VSV-G on the outer membrane of the vesicle-like bodies can realize the retargeting of the virus, and the VSV-G can target most tumor cells.

2. The prepared novel vesiculovirus can escape from an anti-virus neutralizing antibody. After use, the virus normally produces antibodies in vivo, with the high probability of producing neutralizing antibodies (antibodies that prevent re-infection of the virus). In particular, some viruses may have been exposed or infected prior to treatment with the virus and have produced neutralizing antibodies. In either case, the production of neutralizing antibodies appears to be unavoidable. The problem then arises that a longer treatment window is required for a good therapeutic effect. The vesicle-like virus prepared by the vesicle-like technology changes the possible action chance of a neutralizing antibody due to the existence of the vesicle-like membrane, prolongs the window period of treatment and can obviously improve the treatment effect.

3. The vesicle-like technology is simple in preparing the vesicle-like virus, the yield of the vesicle-like virus prepared by the vesicle-like technology is improved by multiple times compared with that of the traditional method, and the corresponding preparation cost is also obviously reduced.

4. The novel vesiculooncolytic virus EM/VSV-G Ad5 sVRCD 137L has high infection efficiency on tumor cells with low expression of various CAR;

5. the oncolytic capacity of the novel vesicular oncolytic virus EM/VSV-G Ad5 sVRCD 137L on various tumor cells;

6. the novel vesiculooncolytic virus EM/VSV-G Ad5 sPGRCD 137L has high expression level of sPGRCD 137L in tumor cells and tissues, is continuous and stable, and can ensure the anti-tumor treatment effect of the virus;

7. the novel vesiculooncolytic-like virus EM/VSV-G Ad5 sPGRCD 137L is capable of resisting neutralization of Ad5 antibody, prolonging the viral therapeutic window, and continuously expressing sPGRCD 137L.

8. The novel vesiculooncolytic-like virus EM/VSV-G Ad5 sVRCD 137L has a remarkable anti-tumor immune activation effect, can remarkably prolong the survival time of a mouse, and has a remarkable tumor treatment effect.

Drawings

FIG. 1 relationship of CAR expression to Ad5-GFP infection efficiency. Non-replicating adenovirus Ad5-GFP expressing GFP was constructed, and 293T, A549, HCC-LM3, Hepa1-6, B16-F10, CT26.WT, H22, K562 and Jurkat cells were infected with Ad5-GFP for 72 hours and then used for fluorescent microscopy analysis or flow analysis. a. The same number of 293T, A549, HCC-LM3, Hepa1-6, B16-F10, ct26.wt, H22, K562 and Jurkat cells were stained with monoclonal antibody anti-CAR-PE for flow analysis of the expression level of CAR of each cell line; isotype is a control in which cells are treated with homologous IgG-PE antibodies.

b. Schematic diagram of genome structure of adenovirus Ad 5-GFP. And c, typical fluorescence photographs of Ad5-GFP virus infected 293T, A549, HCC-LM3, Hepa1-6, B16-F10 and CT26.WT cells after 72h, wherein histograms are flow statistics of GFP positive cell ratios, and the infection efficiency of the Ad5 in other cell strains in each group is calculated by taking the 293T infection efficiency as 100%. A typical scatter diagram is analyzed in a flow mode after Ad5-GFP virus infects suspension cells H22/K562/Jurkat, and a histogram is a flow result statistical chart. Data are presented as mean ± SD.

FIG. 2 preparation of novel vesiculoid Virus EM/VSV-G Ad5 sPGRCD 137L with VSV-G. Schematic of a centrifugal extrusion/shearing apparatus. After loading the syringe with cells suspended in a liquid, the piston will press the liquid in the syringe below by applying a centrifugal force (5000 rpm), and the liquid together with the cells will be pressed/sheared through the PCTE membranes with different pore sizes, and vesicles will be produced when the cells pass through the pores of these PCTE membranes. Finally, the nano vesicles can be produced through the extrusion passing of membrane pores from large size to small size.

FIG. 3 schematic diagram of genome structure of oncolytic adenovirus Ad5 sVRCD 137L

Figure 4EM/VSV-G Ad5sPVRCD137L vs Ad5sPVRCD137L infection efficiency comparison: p <0.01 FIG. 5EM/VSV-G Ad5 sVRCD 137L vs. Ad5 sVRCD 137L replication capacity comparison ns: without significant difference

Figure 6EM/VSV-G Ad5sPVRCD137L vs Ad5sPVRCD137L oncolytic capacity x: p <0.01

Figure 7EM/VSV-G Ad5sPVRCD137L vs Ad5sPVRCD137L comparison of soluble PVRCD137L expression capacity: p <0.01

Figure 8EM/VSV-G Ad5sPVRCD137L in comparison to Ad5sPVRCD137L effect on mouse survival on H22 ascites carcinoma model: p <0.01

Figure 9EM/VSV-G Ad5sPVRCD137L in comparison to Ad5sPVRCD137L effect on mouse survival on B16 mouse solid tumor model: p <0.05, ns: without significant difference

Figure 10EM/VSV-G Ad5sPVRCD137L in comparison to Ad5sPVRCD137L effect on mouse survival on CT26 mouse solid tumor model: p <0.05

Detailed Description

All experimental materials and methods referred to in the examples section

1. Cell lines and cell culture methods

Human and murine hepatocellular carcinoma cell lines HCC-LM3 and H22 were purchased from the cell bank of the culture collection committee of the chinese academy of sciences and were identified by STR and detected by mycoplasma. The mouse liver cell cancer cell line Hepa1-6 human embryonic kidney cell line 293T, human T cell leukemia cell line Jurkat, human chronic myelocytic leukemia cell line K562, human alveolar adenocarcinoma cell line A549, mouse melanoma B16-F10, and Lewis colon cancer cell line CT26.WT were from ATCC company of America. 293T-VSV-G is an engineered 293T cell line expressing VSV-G. Jurkat K562 and H22 were cultured using RPMI 1640 medium containing 10% fetal bovine serum 2mM glutamine, 100units/mL penicillin, and 0.1mg/mL streptomycin. HCC-LM3 Hepa 1-6A 549B16-F10 and 293 were cultured using DMEM medium containing 10% fetal bovine serum 2mM glutamine, 100units/mL penicillin, and 0.1mg/mL streptomycin. All cell cultures were incubated in a thermostatted cell incubator containing 5% carbon dioxide at 37 ℃.

Construction of 293T-VSV-G cell line

Eukaryotic expression plasmid pCMV-VSV-G (Kinry Synthesis and construction) for expressing VSV-G, eukaryotic expression vector pCMV-REV (Kinry Synthesis and construction) for expressing REV, eukaryotic plasmid pCMV-gag-pol (Kinry Synthesis and construction) for expressing gag-pol lentivirus backbone protein, and eukaryotic expression plasmid pCMV-CAG-VSV-G-2A-puro (Kinry Synthesis and construction) for expressing lentivirus genome carrying VSV-G gene were co-transfected into 293T cells, and the supernatant was collected after 72 hours and filtered using a 0.45 μm syringe filter. 293T cells were infected with viral supernatant and maintained in culture after 48 hours using puromycin containing 1 microgram per ml. The sequence of CAG-VSV-G-2A-PURO is shown in SEQ ID NO: 11.

2. Construction of recombinant adenovirus Ad5 sPGRCD 137L

(1) Gene cloning of soluble protein sVRCD 137L and construction of adenovirus shuttle plasmid carrying sVRCD 137L gene

Mouse PVR and CD137L belong to membrane proteins, and the structures of the membrane proteins are sequentially an N-terminal signal peptide-extracellular region-transmembrane region-intracellular region C-terminal. The functional unit for binding PVR and TIGIT is an extracellular region, and the functional unit for binding CD137L and CD137 is also an extracellular region, so that sPGRCD 137L fuses the extracellular region of PVR and the extracellular region of CD137L, a connecting peptide GGGSGGGS is used for connection, and a signal peptide is retained at the N-terminal.

Gene cloning of the soluble protein sPVRCD 137L: respectively designing synthetic primers PVR-F, PVR-R, CD137L-F, CD 137L-R; PVR-F and PVR-R primers are used, and cDNA of mouse hepatoma cell Hep1-6 is used as a template to be amplified to obtain a segment EXO-PVR; using CD137L-F and CD137L-R primers and taking cDNA of mouse hepatoma cell Hep1-6 as a template to amplify to obtain a fragment EXO-CD 137L; synthesizing Linker DNA in vitro; the primers PVR-R and CD137L-F have about 16bp respectively and completely accord with the 5 'and 3' of the linker sequence. And (3) splicing EXO-PVR, linker and EXO-CD137L fragments according to design by using a PCR technology and taking PVR-R and CD137L-F as primers to finish the cloning of the sPVRCD137L gene. The protein sequences of sVRCD 137L, EXO-PVR, EXO-CD137L, Linker and signal peptide (CD33) are shown in sequence tables SEQ ID NO 1-5; the DNA sequences of sVRCD 137L, EXO-PVR, EXO-CD137L and Linker are shown in the sequence table SEQ ID NO. 6-10. Gene template construction related primers are shown in Table 1:

TABLE 1

Construction of the adenovirus shuttle plasmid Ad5 sVRCD 137 LShuttle-sVRCD 137L vector carrying soluble protein genes: the full-length sequence of sVRCD 137L was synthesized, and a HIS tag sequence was added to the C-terminus, and then the HIS tag-containing sVRCD 137L fragment was ligated to Ad5 sVRCD 137LShuttle (pZD55) using Infusion technology. The method comprises the following specific steps: ad5sPVRCD137LShuttle (pZD55) was first linearized with the restriction enzyme BglII and the purified fragment was as per sPVRCD 137L: the 2:1 ratio of Ad5 sVRCD 137LShuttle was ligated using the Infusion kit (clontech lab. Inc.), followed by transformation amplification verification to obtain the adenovirus shuttle plasmid Ad5 sVRCD 137 LShuttle-sVRCD 137L carrying the sVRCD 137L gene.

1.2.1.2Ad5 sPGRCD 137L Virus construction (plasmid construction, Virus rescue and amplification)

A.ad5spvrcd137l full-length plasmid construction: the constructed shuttle vector Ad5 sPGRCD 137 LShuttle-sPGRCD 137L is linearized by PmeI and then transferred into competent pAdEasy-BJ5183, screening is carried out by using LB plate containing 50ug/ml kanamycin, positive clone is picked for culture and identification, the correct clone plasmid is identified, DH5a competence is retransformed for secondary screening and identification, and plasmid is extracted after the correct identification to obtain the full-length plasmid AD5 sPGRCD 137L.

Ad5sPGRCD137L virus rescue: the Ad5 sVRCD 137L full-length plasmid was linearized using PacI and after purification 293T cells were transfected at 1. mu.g/well in 6-well plates, 5% CO₂Culturing at 37 deg.C, digesting cells after 2 days, transferring to 10cm plate, changing liquid for 2-3 days until 80% cells have pathological changes, collecting cells into 15ml centrifuge tube by blowing down 10ml culture medium, repeatedly freezing and thawing for 2 times, centrifuging at 3000rpm/min for 15min, collecting virus supernatant, and storing at-80 deg.C as virus seed.

C. And (3) virus amplification: adding 50 μ l of virus liquid into 60% 293T cell 10cm dish, and adding 5% CO₂Culturing at 37 deg.C, with cell density above 90%, according to 1-pass3, passage at a ratio of 3 until 80% of cells have lesions, approximately 10 cells on a plate, collecting the virus according to the method, and purifying the virus by using cesium chloride density gradient centrifugation; titre determinations were performed using the TCID50 method.

Using 293T-VSV cells for Ad5 sVRCD 137L adenovirus amplification and purification, infecting the cells for 72 hours by using viruses with MOI of 5, repeatedly freezing and thawing the cells, centrifuging the cells for 20min at 4000 Xg to remove cell debris, and collecting supernatant to obtain virus suspension; purification of the virus was performed using iodixanol density gradient centrifugation. Virus titer was determined by inoculating 96-Wells plates with 293T cells, 10000 cells/well; diluting the virus by 10 times; 100ul/Well, cultured for 4 days, observed using fluorescence microscopy, wells with green fluorescent cells were defined as positive, each positive Well amounting to 0.1, and all positive wells were summed as S. Virus TCID50 ═ 10² ^+(S/10-0.5)/ml,

pfu/ml＝0.7×TCID50/ml。

3. Preparation of novel vesiculooncolytic-like adenovirus EM/VSV-G Ad5 sPGRCD 137L

6 seeds of 10cm were seeded with 293T-VSV-G cells²Culture dish (1X 10)⁷cells/plate), after overnight incubation, inoculated at 5X 10⁷pfu of Ad5 sPGRCD 137L virus, was cultured for 72 hours, and the cell-depleted cell culture supernatant was collected and resuspended in 30ml DMEM.

The cell suspension was extruded 3 times through a 10 μm polycarbonate track-etched (PCTE) membrane (Whatman) using a centrifugal extrusion/shearing device as shown in FIG. 2; the collected liquid was then used to squeeze the cells 3 times through a5 μm polycarbonate track-etched (PCTE) membrane (Whatman) using a centrifugal squeeze/shear device as shown in FIG. 2; the collected liquid was further passed through a1 μm polycarbonate track-etched (PCTE) membrane (Whatman) 3 times by squeezing/shearing the cells using a centrifugal squeezing/shearing apparatus shown in FIG. 2. The squeezed virus suspension was collected, added to a centrifuge tube with 15% 20% and 40% iodixanol (iodixanol) cushions, centrifuged at 100000 Xg for 90min, and the lesions between 15% and 20% and between 20% and 40% iodixanol were collected, respectively. Dialyzing with 5% glycerol, 1mM MgCl2, 150mM NaCl, 10mM Tris-HCl (pH7.6) for 2 times, determining virus titer, and storing in-80 deg.C refrigerator.

4. Detection of viral infection efficiency

The virus infection efficiency test firstly uses 1 × 10⁵cells/well to be detected cells are inoculated on a 24-well plate, corresponding viruses are added according to the specified virus amount, the cells are cultured for 48 hours at 37 ℃ under 5 percent CO2, and the cells are observed and photographed by a fluorescence microscope; the cells were collected and the rate of GFP positive cells was counted by flow counting, and the rate of infection of 293T GFP positive cells with Ad5 sVRCD 137L virus was set to 1, to calculate the infection efficiency of other cell viruses.

5. Neutralization test

Adenovirus neutralizing antibodies were given to adenovirus immune rabbit serum by the king world soldier (university of chef, zhejiang). VSV-G neutralizing antibodies were from verified volunteer sera. Animal Experimental ascites sample treatment cells and cell debris were removed by centrifugation for 10min at 400 Xg and 12000 Xg respectively. Neutralization test serum and ascites were both inactivated at 56 ℃ for 30 min. 293T inoculation with 24well-plate (1X 10)⁵cells/well), cultured at 37 ℃ for 4 hours, and then used in 100ul of 1X 10-containing medium⁵DMEM of pfu virus was incubated with the indicated dilution of neutralizing serum or ascites fluid at 37 ℃ for 30min, and the virus antibody mixture was added to pre-inoculated 293T cells (1X 10)⁵cells/well) at 37 ℃ for 1 hour, removing the virus supernatant, adding complete medium for another 48 hours, observing fluorescence, performing flow statistics on the ratio of GFP positive cells, and calculating the ratio of antibody neutralization in each treatment group with the ratio of GFP positive cells in the group not treated with the antibody as 100%.

6. MTT cell viability assay

Inoculating a cell strain to be detected to 96-well plate, culturing 1 ten thousand cells in each hole at 37 ℃ for 4 hours, adding corresponding viruses according to the specified virus amount, removing virus suspension after 4 hours, replacing complete culture medium, and continuously culturing for 72 hours. 100ul of MTT dilution (1mg/ml) per well was incubated for an additional 4 hours. The supernatant was removed, 150ul isopropanol was added, shaking was carried out for 15min, and absorbance was measured using 570 wavelengths. The cell force of the other treatment groups was calculated by taking the absorbance of the untreated cell culture wells as 100% of the cell viability.

7. Q-PCR detection of replication function of virus

Vaccination with the virus Ad5 sVRCD 137L or EM/VSV-G Ad5 sVRCD 137L at MOI 5After the Hepa1-6, B16/F10, CT26.WT and H22 cells are treated, the cells are collected at 6H, 24H, 36H, 48H, 60H and 72H respectively, and total DNA is extracted by using a genome extraction kit (Tiangen Biochemical technology). Using PowerUp^TM SYBR^TMAnd (3) Green Master Mix, taking the quantified shuttle-Ad5 sPRVCD 137L plasmid as a standard substance and Q-E1A F and Q-EIA R as primers, and carrying out quantitative PCR (polymerase chain reaction) detection on the genome copies of the virus at each time point. The number of 6h virus copies was 1 for each treatment group, and the fold increase of virus copies at each time was calculated.

8. Quantitative detection of Enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay ELISA)

Content determination of soluble PVRCD 137L: ELISA 96-well plates were coated with 2. mu.g/ml Anti-His-tag monoclonal antibody (purchased from Nanjing King Shirui Biotech Co., Ltd., Nanjing, China). Adding 100 mul of cell supernatant or ascites, and incubating at 37 ℃ for 2 hours; washing away unbound soluble protein with phosphate buffer solution (PBS-T) containing 0.1% Tween 20, adding rabbit anti-mouse PVR monoclonal antibody as detection antibody, and incubating at 37 deg.C for 1 hr; PBS-T washing for 5 times, adding 100 μ l biotin-labeled goat anti-rabbit IgG antibody and HRP-labeled avidin diluted solution, and incubating for 1 hour at room temperature. After washing with PBS-T for 10 times, substrate TMB was added thereto and reacted at room temperature for 30 minutes, 50. mu.l of stop solution (2N sulfuric acid) was added thereto, and OD450 was detected using a microplate reader.

ELISA detection of other proteins was performed in a similar manner as described above.

9. Flow cytometry analysis of immune cell subpopulation detection in ascites

Ascites from mice were extracted according to the protocol, centrifuged at 400 Xg for 5 minutes to collect cells, and washed twice with PBS. The antibodies were incubated for 30min at room temperature in the dark according to the cell marker combination. PBS wash twice using 500 u l PBS heavy suspension, using flow cytometry detection analysis. T cells were transfected with CD3-APC, CD4-FITC, and CD8-PerCP-Cy^TM5.5 two T cell subsets of labeled CD3+ CD4+ or CD3+ CD8 +. NK cells CD3-APC and NK1.1-FITC labeled CD3-NK1.1+ cells were used. PD-L1 positive cells were labeled with CD274 PE. CD3 APC, CD8a PerCP-Cy^TM5.5, CD4 FITC, NK1.1 FITC and CD274 PE (available from BD Biosciences, Franklin Lakes, NJ, USA).

Expression detection of CAR on tumor cell surface

100 million cells were collected from each cell line by centrifugation and washed 2 times with PBS. Incubate with anti-CAR-PE antibody for 30min at room temperature (protected from light). Washed 2 times with PBS and resuspended in 500. mu.l of PBS. Analysis was detected using a flow cytometer.

10. Activated lymphocyte detection in ascites by IFN-gamma enzyme linked spot assay (IFN-gamma EILSpot assay)

Preparation of IFN- γ EILSpot assay plate: IFN-. gamma.EILSpot detection plates were prepared as per the instructions of the Mouse IFN-. gamma.EILSpot PLUS kit (available from 3321-2 AW-PLUS; Mabtech, Nacka Strand, Sweden). Ascites of the mice are extracted at the appointed time point according to the experimental scheme, and cells in the ascites are collected by removing the ascites through a centrifugal method. After washing twice with PBS, the cells were stained with placental blue and counted. Preparation of 1X 10⁶cells/ml cell suspension. The cell suspension was added to a previously prepared IFN- γ EILSpot assay plate in a volume of 200. mu.l per well. The cells were incubated overnight at 37 ℃ in a cell incubator containing 5% carbon dioxide for 20 hours. The cell suspension was removed, treated as per the instructions and developed. Detection assays were performed using an ELISpot plate reader.

Example 1 adenovirus Ad5 infection of cells with low expression of CAR was inefficient

CAR (the coxsackie and adenovirus receptor) is the main receptor for adenovirus Ad5 infected cells. Early studies have demonstrated that adenovirus Ad5 infects cells with low expression of CAR with low efficiency.^13,19We found by flow analysis that 293T, A549, HCC-LM3 and Hepa1-6 cells stained with anti-CAR-PE showed a significant increase in fluorescence intensity compared to the isotype treated group, indicating high expression of 293T, A549, HCC-LM3 and Hepa1-6 cell surface CARs (fig. 1 a); and after the surfaces of B16-F10, CT26.WT and H22 cells are stained by using the CAR antibody, the fluorescence intensity is not obviously changed compared with that of an isotype treatment group; indicating that these cell lines do not express CAR; whereas, the fluorescence intensity of K562 and Jurkat cells stained with the CAR antibody was only slightly increased compared to the isotype treated group, much lower than that of 293T cell lines, indicating that these cell lines underexpress CAR (FIG. 1 a). Next, to validate the relationship between CAR expression and Ad5 infection efficiency, we constructed a tableNon-replicative adenovirus Ad5-GFP with GFP expression, (FIG. 1B), 293T, A549, HCC-LM3, Hepa1-6, B16-F10, CT26.WT and H22 cells are treated by Ad5-GFP with MOI of 1, 50-60% of the cells of 293T, A549, HCC-LM3 and Hepa1-6 with CAR high expression are infected by Ad5-GFP and express GFP after 72H, and the infection efficiency of the Ad5-GFP is not obviously different among the cells; however, the efficiency of infection of adherent cells B16-F10, CT26.WT, H22 with Ad5-GFP, which are low in expression of CAR, is less than 5%, and is very significantly lower than 293T and other CAR-highly expressing cell strains (P < 0.001) (FIG. 1c, d). Suspension cell lines K562 and Jurkat infected only 8.26% + -0.64% and 12.08% + -0.81% of cells with Ad5-GFP virus at MOI of 100 for 72h, respectively, and very significantly less than the infection efficiency (52.3% + -2.61%) of Ad5-GFP virus at MOI of 1 for 293T (P < 0.001) (FIG. 1 d). The results prove that the infection efficiency of the adenovirus Ad5 on cells is related to the amount of the CAR expressed on the cell surface, the infection efficiency of the adenovirus Ad5 on the cells with high expression of the CAR is high, and the infection efficiency of the adenovirus Ad5 on the cells with low expression of the CAR is low.

FIG. 1 relationship of CAR expression to Ad5-GFP infection efficiency. Non-replicating adenovirus Ad5-GFP expressing GFP was constructed, and 293T, A549, HCC-LM3, Hepa1-6, B16-F10, CT26.WT, H22, K562 and Jurkat cells were infected with Ad5-GFP for 72 hours and then used for fluorescent microscopy analysis or flow analysis. a. The same number of 293T, A549, HCC-LM3, Hepa1-6, B16-F10, ct26.wt, H22, K562 and Jurkat cells were stained with monoclonal antibody anti-CAR-PE for flow analysis of the expression level of CAR of each cell line; isotype is a control in which cells are treated with homologous IgG-PE antibodies. b. Schematic diagram of genome structure of adenovirus Ad 5-GFP. And c, typical fluorescence photographs of Ad5-GFP virus infected 293T, A549, HCC-LM3, Hepa1-6, B16-F10 and CT26.WT cells after 72h, wherein histograms are flow statistics of GFP positive cell ratios, and the infection efficiency of the Ad5 in other cell strains in each group is calculated by taking the 293T infection efficiency as 100%. A typical scatter diagram is analyzed in a flow mode after Ad5-GFP virus infects suspension cells H22/K562/Jurkat, and a histogram is a flow result statistical chart. Data are presented as mean ± SD.

EXAMPLE 2 vesicle-like technique for preparing novel vesicle-like oncolytic Virus EM/VSV-G Ad5 sVRCD 137L carrying VSV-G

Vesicular stock Indiana virus G protein (VSV-G) is capable of mediating viral entry into all cell types tested to date and is widely used for gene transduction and gene therapy²⁶. The research hopes that the wide tropism of VSV-G to cells is utilized to realize the retargeting of Ad5 and increase the infection efficiency of Ad5 to CAR low-expression cell lines by virtue of a vesicle-like technology.

Firstly, 293T cells (293T-VSV-G) for expressing VSV-G are constructed, Ad5 sVRCD 137L with MOI of 5 is used for infecting the 293T-VSV-G cells, the cells are collected after culturing for 72h at 37 ℃, the collected cells are divided into two parts, one part is frozen and thawed 3 times by using a traditional scheme, and the virus is purified by iodixanol density gradient centrifugation; another part is prepared into a novel vesiculooncolytic virus EM/VSV-G Ad5 sPGRCD 137L with VSV-G, and the components between 20% and 40% of density gradient are collected by repeated centrifugal extrusion and sequentially passing through polycarbonate membranes with the pore diameter of 10 mu m, 5 mu m and 1 mu m, and using 15%, 20% and 40% iodixanol density gradient for centrifugation.

We performed characterization analyses of naked Ad5 and novel vesiculooncolytic-like virus EM/VSV-G Ad5 sPGRCD 137L using the DLS method (Zetasizer, Malvern). Naked virus Ad5 with size of 60-90 nm; the diameter of the novel vesiculooncolytic virus EM/VSV-G Ad5 sVRCD 137L is between 100 nm and 200nm, and the size of the novel vesiculooncolytic virus is similar to that of an extracellular vesicle, so that the novel vesiculooncolytic virus is larger than that of naked virus Ad5 due to the fact that the virus is wrapped by a double-layer membrane.

We have also examined the VSV-G protein of the virosome novel vesiculooncolytic virus EM/VSV-G Ad5 sVRCD 137L by a conventional ELISA method (Anti-VSV-G tag antibody, ab3861), confirming that the virosome novel vesiculooncolytic virus EM/VSV-G Ad5 sVRCD 137L contains VSV-G protein.

In order to further study the influence of the vesicle-like technological method on the virus recovery yield, the virus recovery yield of the vesicle-like technological method and the virus recovery yield of the traditional freeze-thaw scheme are detected, and the vesicle-like technological method can improve the virus yield by 8.4 +/-1.43 times (P <0.01) compared with the traditional freeze-thaw scheme.

The results show that the vesicle-like technology can load adenovirus, and the novel vesicle-like oncolytic virus EM/VSV-G Ad5 sVRCD 137L prepared by the technology is wrapped by a layer of vesicle-like membrane at the outer layer, so the size of the vesicle-like oncolytic virus is larger than that of naked virus; presence of VSV-G protein on EM/VSV-G Ad5 sVRCD 137L; and the recovery yield of the virus is obviously increased by using the technology to prepare the virus.

FIG. 2 preparation of novel vesiculoid Virus EM/VSV-G Ad5 sPGRCD 137L with VSV-G. Schematic of a centrifugal extrusion/shearing apparatus. After the cells suspended in the liquid are filled into the syringe, centrifugal force is applied, the piston presses the liquid in the syringe below, and the liquid together with the cells is pressed/sheared through the PCTE membranes with different pore sizes, and vesicles are generated when the cells pass through the pores of the PCTE membranes. Finally, the nano vesicles can be produced through the extrusion passing of membrane pores from large size to small size.

Example 3EM/VSV-G technology the EM/VSV-G Ad5 sPGRCCD 137L has higher infection efficiency, ability to express and secrete soluble PVRCD137L and oncolytic ability in vitro

The oncolytic adenovirus Ad5 sVRCD 137L is an oncolytic adenovirus Ad5 (named Ad5 sVRCD 137L) expressing soluble PVRCD137L, and the genome structure schematic diagram of the oncolytic adenovirus Ad5 sVRCD 137L is shown in FIG. 3. The low infection rate of oncolytic adenovirus Ad5sPVRCD137L on CAR low expressing tumor types severely limits the transformation utility of Ad5sPVRCD 137L. The EM/VSV-G technology is applied to the oncolytic adenovirus Ad5 sVRCD 137L, the infection efficiency of the oncolytic adenovirus Ad5 sVRCD 137L on a CAR low-expression tumor cell line is expected to be remarkably increased through the EM/VSV-G technology, the treatment effect of the oncolytic adenovirus Ad5 sVRCD 137L is improved, and the application range of the oncolytic adenovirus Ad5 sVRCD 137L is expanded. The virus obtained by preparing oncolytic adenovirus Ad5 sVRCD 137L by EM/VSV-G technology is named as EM/VSV-G Ad5 sVRCD 137L.

Compared with Ad5 sPGRCD 137L virus (abbreviated as Ad5PC), the EM/VSV-G Ad5 sPGRCD 137L (abbreviated as EM/VSV-G Ad5PC) has a very significant improvement in the infection efficiency of CAR-low expression tumor cell lines, and the infection efficiency of the CAR-low expression tumor cell lines is improved to 3.2 +/-0.38, 4.1 +/-0.47, 5.3 +/-0.71, 3.6 +/-0.43 and 9.8 +/-1.12 on K562, Jurkat, B16/F10, CT26.WT and H22 cell strains respectively (FIG. 4).

Figure 4EM/VSV-G Ad5sPVRCD137L vs Ad5sPVRCD137L infection efficiency comparison: p <0.01

We compared the replication efficiencies of EM/VSV-G Ad5 sVRCD 137L and Ad5 sVRCD 137L in different cell lines and found that there was no significant difference in the replication efficiencies of EM/VSV-G Ad5 sVRCD 137L and Ad5 sVRCD 137L on K562, Jurkat, B16/F10, CT26.WT and H22 cell lines (FIG. 5). The results indicate that this EM/VSV-G technique only increases the efficiency of viral infection and does not affect viral replication.

FIG. 5EM/VSV-G comparison ns of Ad5 sVRCD 137L with Ad5 sVRCD 137L replication capacity: without significant difference

Next, we analyzed the oncolytic effect of EM/VSV-G Ad5 sVRCD 137L and Ad5 sVRCD 137L on K562, Jurkat, B16/F10, CT26.WT and H22 cell lines at a viral MOI value of 50, and the results showed that the oncolytic effect of EM/VSV-G Ad5 sVRCD 137L was significantly increased (FIG. 6), perhaps due to the fact that the infection efficiency of EM/VSV-G Ad5 sVRCD 137L was significantly higher than that of Ad5 sVRCD 137L, and the amount of virus entering the cells was significantly increased although the replication efficiency was not increased.

Figure 6EM/VSV-G Ad5sPVRCD137L vs Ad5sPVRCD137L oncolytic capacity x: p <0.01

Next, we infected cells with EM/VSV-G Ad5sPVRCD137L and Ad5sPVRCD137L of the same virus titer (MOI value of 10), and then examined the content of soluble PVRCD137L secreted in the supernatant of the virus-infected cells by the conventional Elisa method, and the results showed that the expression of soluble PVRCD137L in the supernatant of the culture medium of K562, Jurkat, B16/F10, CT26.WT and H22 cells showed a very significant increase after EM/VSV-G Ad5sPVRCD137L infection, compared with Ad5 sVRCD 137L (FIG. 7).

In summary, EM/VSV-G Ad5 sPGRCD 137L prepared by EM/VSV-G technology can improve the infection efficiency of oncolytic adenovirus Ad5 sPGRCD 137L in low CAR-expressing cell strains, increase the oncolytic effect and the expression level of soluble PVRCD137L, and the results of these in vitro studies suggest that our EM/VSV-G technology of centrifugal extrusion/shearing may be capable of improving the anti-tumor effect of oncolytic adenovirus Ad5 sPGRCD 137L in vivo.

Example 4EM/VSV-G Ad5 sPGRCD 137L induced a stronger anti-tumor effect, with a very significant extension of mouse survival.

We established a mouse ascites tumor model using the H22 cell line, a mouse solid tumor model using the B16 and CT26 cell lines, and studied the anti-tumor effect of EM/VSV-G Ad5 sVRCD 137L in vivo. The H22 cell line establishes a mouse ascites tumor model: intraperitoneal injection at 0 day 5X 10⁶H22 cells, injected intraperitoneally 5X 10/mouse on days 3, 9, 15, and 21⁸pfu virus, observed for survival. The B16 and CT26 cell lines established a mouse solid tumor model: subcutaneous injection at 0 day 5X 10⁶B16 or CT26 cells, injected intratumorally 5X 10/mouse on

days

11, 14, 17, and 20⁸pfu virus, observed for survival.

As a result of a mouse ascites tumor model, the survival time of mice in an EM/VSV-G Ad5 sVRCD 137L-treated group and an Ad5 sVRCD 137L-treated group is remarkably prolonged compared with a saline control group, and 4 mice and 1 mouse in an EM/VSV-G Ad5 sVRCD 137L and an Ad5 sVRCD 137L group are respectively cured and respectively account for 40% and 10% (FIG. 8); the survival of mice was also significantly prolonged in the EM/VSV-G Ad5 sVRCD 137L treated group compared to the Ad5 sVRCD 137L treated group (FIG. 8).

B16 mouse solid tumor model results found that EM/VSV-G Ad5 sPGRCD 137L treated group had significantly prolonged survival although no mice were cured compared to saline control group (FIG. 9); the survival of mice was increased in Ad5sPVRCD 137L-treated group compared to saline control group (fig. 9); the survival of mice was also significantly prolonged in the EM/VSV-G Ad5 sVRCD 137L treated group compared to the Ad5 sVRCD 137L treated group (FIG. 9).

The same results in the CT26 mouse solid tumor model found that the EM/VSV-G Ad5sPVRCD 137L-treated group and Ad5sPVRCD 137L-treated group had significantly prolonged survival of mice, although no mice were cured, compared to the salene control group (fig. 10); the survival of mice was also significantly prolonged in the EM/VSV-G Ad5 sVRCD 137L treated group compared to the Ad5 sVRCD 137L treated group (FIG. 10).

In conclusion, the novel vesicular virus prepared by encapsulating the virus by the vesicular-like technology has the following prominent substantive characteristics and remarkable progress:

Sequence listing

<110> Nanjing university

<120> a novel vesicular oncolytic virus and application thereof in preparing antitumor drugs

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Met Asp Val Val Val Gln Ala Pro Thr Gln Val Pro Gly Phe Leu Gly

20 25 30

Asp Ser Val Thr Leu Pro Cys Tyr Leu Gln Val Pro Asn Met Glu Val

35 40 45

Thr His Val Ser Gln Leu Thr Trp Ala Arg His Gly Glu Ser Gly Ser

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Met Ala Val Phe His Gln Thr Gln Gly Pro Ser Tyr Ser Glu Ser Lys

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Arg Leu Glu Phe Val Ala Ala Arg Leu Gly Ala Glu Leu Arg Asn Ala

85 90 95

Ser Leu Arg Met Phe Gly Leu Arg Val Glu Asp Glu Gly Asn Tyr Thr

100 105 110

Cys Leu Phe Val Thr Phe Pro Gln Gly Ser Arg Ser Val Asp Ile Trp

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Leu Arg Val Leu Ala Lys Pro Gln Asn Thr Ala Glu Val Gln Lys Val

130 135 140

Gln Leu Thr Gly Glu Pro Val Pro Met Ala Arg Cys Val Ser Thr Gly

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Gly Arg Pro Pro Ala Gln Ile Thr Trp His Ser Asp Leu Gly Gly Met

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Pro Asn Thr Ser Gln Val Pro Gly Phe Leu Ser Gly Thr Val Thr Val

180 185 190

Thr Ser Leu Trp Ile Leu Val Pro Ser Ser Gln Val Asp Gly Lys Asn

195 200 205

Val Thr Cys Lys Val Glu His Glu Ser Phe Glu Lys Pro Gln Leu Leu

210 215 220

Thr Val Asn Leu Thr Val Tyr Tyr Pro Pro Glu Val Ser Ile Ser Gly

225 230 235 240

Tyr Asp Asn Asn Trp Tyr Leu Gly Gln Asn Glu Ala Thr Leu Thr Cys

245 250 255

Asp Ala Arg Ser Asn Pro Glu Pro Thr Gly Tyr Asn Trp Ser Thr Thr

260 265 270

Met Gly Pro Leu Pro Pro Phe Ala Val Ala Gln Gly Ala Gln Leu Leu

275 280 285

Ile Arg Pro Val Asp Lys Pro Ile Asn Thr Thr Leu Ile Cys Asn Val

290 295 300

Thr Asn Ala Leu Gly Ala Arg Gln Ala Glu Leu Thr Val Gln Val Lys

305 310 315 320

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala

325 330 335

Leu Thr Ile Thr Thr Ser Pro Asn Leu Gly Thr Arg Glu Asn Asn Ala

340 345 350

Asp Gln Val Thr Pro Val Ser His Ile Gly Cys Pro Asn Thr Thr Gln

355 360 365

Gln Gly Ser Pro Val Phe Ala Lys Leu Leu Ala Lys Asn Gln Ala Ser

370 375 380

Leu Cys Asn Thr Thr Leu Asn Trp His Ser Gln Asp Gly Ala Gly Ser

385 390 395 400

Ser Tyr Leu Ser Gln Gly Leu Arg Tyr Glu Glu Asp Lys Lys Glu Leu

405 410 415

Val Val Asp Ser Pro Gly Leu Tyr Tyr Val Phe Leu Glu Leu Lys Leu

420 425 430

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

435 440 445

Leu Val Leu Gln Ala Lys Pro Gln Val Asp Asp Phe Asp Asn Leu Ala

450 455 460

Leu Thr Val Glu Leu Phe Pro Cys Ser Met Glu Asn Lys Leu Val Asp

465 470 475 480

Arg Ser Trp Ser Gln Leu Leu Leu Leu Lys Ala Gly His Arg Leu Ser

485 490 495

Val Gly Leu Arg Ala Tyr Leu His Gly Ala Gln Asp Ala Tyr Arg Asp

500 505 510

Trp Glu Leu Ser Tyr Pro Asn Thr Thr Ser Phe Gly Leu Phe Leu Val

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Lys Pro Asp Asn Pro Trp Glu His His His His His His

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Asp Val Val Val Gln Ala Pro Thr Gln Val Pro Gly Phe Leu Gly Asp

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Ser Val Thr Leu Pro Cys Tyr Leu Gln Val Pro Asn Met Glu Val Thr

20 25 30

His Val Ser Gln Leu Thr Trp Ala Arg His Gly Glu Ser Gly Ser Met

35 40 45

Ala Val Phe His Gln Thr Gln Gly Pro Ser Tyr Ser Glu Ser Lys Arg

50 55 60

Leu Glu Phe Val Ala Ala Arg Leu Gly Ala Glu Leu Arg Asn Ala Ser

65 70 75 80

Leu Arg Met Phe Gly Leu Arg Val Glu Asp Glu Gly Asn Tyr Thr Cys

85 90 95

Leu Phe Val Thr Phe Pro Gln Gly Ser Arg Ser Val Asp Ile Trp Leu

100 105 110

Arg Val Leu Ala Lys Pro Gln Asn Thr Ala Glu Val Gln Lys Val Gln

115 120 125

Leu Thr Gly Glu Pro Val Pro Met Ala Arg Cys Val Ser Thr Gly Gly

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Arg Pro Pro Ala Gln Ile Thr Trp His Ser Asp Leu Gly Gly Met Pro

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Asn Thr Ser Gln Val Pro Gly Phe Leu Ser Gly Thr Val Thr Val Thr

165 170 175

Ser Leu Trp Ile Leu Val Pro Ser Ser Gln Val Asp Gly Lys Asn Val

180 185 190

Thr Cys Lys Val Glu His Glu Ser Phe Glu Lys Pro Gln Leu Leu Thr

195 200 205

Val Asn Leu Thr Val Tyr Tyr Pro Pro Glu Val Ser Ile Ser Gly Tyr

210 215 220

Asp Asn Asn Trp Tyr Leu Gly Gln Asn Glu Ala Thr Leu Thr Cys Asp

225 230 235 240

Ala Arg Ser Asn Pro Glu Pro Thr Gly Tyr Asn Trp Ser Thr Thr Met

245 250 255

Gly Pro Leu Pro Pro Phe Ala Val Ala Gln Gly Ala Gln Leu Leu Ile

260 265 270

Arg Pro Val Asp Lys Pro Ile Asn Thr Thr Leu Ile Cys Asn Val Thr

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Asn Ala Leu Gly Ala Arg Gln Ala Glu Leu Thr Val Gln Val Lys

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Ala Leu Thr Ile Thr Thr Ser Pro Asn Leu Gly Thr Arg Glu Asn Asn

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Ala Asp Gln Val Thr Pro Val Ser His Ile Gly Cys Pro Asn Thr Thr

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Gln Gln Gly Ser Pro Val Phe Ala Lys Leu Leu Ala Lys Asn Gln Ala

35 40 45

Ser Leu Cys Asn Thr Thr Leu Asn Trp His Ser Gln Asp Gly Ala Gly

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Ser Ser Tyr Leu Ser Gln Gly Leu Arg Tyr Glu Glu Asp Lys Lys Glu

65 70 75 80

Leu Val Val Asp Ser Pro Gly Leu Tyr Tyr Val Phe Leu Glu Leu Lys

85 90 95

Leu Ser Pro Thr Phe Thr Asn Thr Gly His Lys Val Gln Gly Trp Val

100 105 110

Ser Leu Val Leu Gln Ala Lys Pro Gln Val Asp Asp Phe Asp Asn Leu

115 120 125

Ala Leu Thr Val Glu Leu Phe Pro Cys Ser Met Glu Asn Lys Leu Val

130 135 140

Asp Arg Ser Trp Ser Gln Leu Leu Leu Leu Lys Ala Gly His Arg Leu

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Ser Val Gly Leu Arg Ala Tyr Leu His Gly Ala Gln Asp Ala Tyr Arg

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Asp Trp Glu Leu Ser Tyr Pro Asn Thr Thr Ser Phe Gly Leu Phe Leu

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Val Lys Pro Asp Asn Pro Trp Glu

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

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Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala

1 5 10 15

Met

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atgccgctgc tgctactgct gcccctgctg tgggcagggg ccctggctat ggacgtcgtc 60

gtgcaggcgc ccacccaggt gcccggcttc ttgggcgact ccgtgacgct gccctgctac 120

ctacaggtgc ccaacatgga ggtgacgcat gtgtcacagc tgacttgggc gcggcatggt 180

gaatctggca gcatggccgt cttccaccaa acgcagggcc ccagctattc ggagtccaaa 240

cggctggaat tcgtggcagc cagactgggc gcggagctgc ggaatgcctc gctgaggatg 300

ttcgggttgc gcgtagagga tgaaggcaac tacacctgcc tgttcgtcac gttcccgcag 360

ggcagcagga gcgtggatat ctggctccga gtgcttgcca agccccagaa cacagctgag 420

gttcagaagg tccagctcac tggagagcca gtgcccatgg cccgctgcgt ctccacaggg 480

ggtcgcccgc cagcccaaat cacctggcac tcagacctgg gcgggatgcc caatacgagc 540

caggtgccag ggttcctgtc tggcacagtc actgtcacca gcctctggat attggtgccc 600

tcaagccagg tggacggcaa gaatgtgacc tgcaaggtgg agcacgagag ctttgagaag 660

cctcagctgc tgactgtgaa cctcaccgtg tactaccccc cagaggtatc catctctggc 720

tatgataaca actggtacct tggccagaat gaggccaccc tgacctgcga tgctcgcagc 780

aacccagagc ccacaggcta taattggagc acgaccatgg gtcccctgcc accctttgct 840

gtggcccagg gcgcccagct cctgatccgt cctgtggaca aaccaatcaa cacaacttta 900

atctgcaacg tcaccaatgc cctaggagct cgccaggcag aactgaccgt ccaggtcaaa 960

ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcggcgct cacaatcacc 1020

acctcgccca acctgggtac ccgagagaat aatgcagacc aggtcacccc tgtttcccac 1080

attggctgcc ccaacactac acaacagggc tctcctgtgt tcgccaagct actggctaaa 1140

aaccaagcat cgttgtgcaa tacaactctg aactggcaca gccaagatgg agctgggagc 1200

tcatacctat ctcaaggtct gaggtacgaa gaagacaaaa aggagttggt ggtagacagt 1260

cccgggctct actacgtatt tttggaactg aagctcagtc caacattcac aaacacaggc 1320

cacaaggtgc agggctgggt ctctcttgtt ttgcaagcaa agcctcaggt agatgacttt 1380

gacaacttgg ccctgacagt ggaactgttc ccttgctcca tggagaacaa gttagtggac 1440

cgttcctgga gtcaactgtt gctcctgaag gctggccacc gcctcagtgt gggtctgagg 1500

gcttatctgc atggagccca ggatgcatac agagactggg agctgtctta tcccaacacc 1560

accagctttg gactctttct tgtgaaaccc gacaacccat gggaa 1605

<210> 7

<211> 909

<212> DNA

<213> Artificial sequence (.)

<400> 7

gacgtcgtcg tgcaggcgcc cacccaggtg cccggcttct tgggcgactc cgtgacgctg 60

ccctgctacc tacaggtgcc caacatggag gtgacgcatg tgtcacagct gacttgggcg 120

cggcatggtg aatctggcag catggccgtc ttccaccaaa cgcagggccc cagctattcg 180

gagtccaaac ggctggaatt cgtggcagcc agactgggcg cggagctgcg gaatgcctcg 240

ctgaggatgt tcgggttgcg cgtagaggat gaaggcaact acacctgcct gttcgtcacg 300

ttcccgcagg gcagcaggag cgtggatatc tggctccgag tgcttgccaa gccccagaac 360

acagctgagg ttcagaaggt ccagctcact ggagagccag tgcccatggc ccgctgcgtc 420

tccacagggg gtcgcccgcc agcccaaatc acctggcact cagacctggg cgggatgccc 480

aatacgagcc aggtgccagg gttcctgtct ggcacagtca ctgtcaccag cctctggata 540

ttggtgccct caagccaggt ggacggcaag aatgtgacct gcaaggtgga gcacgagagc 600

tttgagaagc ctcagctgct gactgtgaac ctcaccgtgt actacccccc agaggtatcc 660

atctctggct atgataacaa ctggtacctt ggccagaatg aggccaccct gacctgcgat 720

gctcgcagca acccagagcc cacaggctat aattggagca cgaccatggg tcccctgcca 780

ccctttgctg tggcccaggg cgcccagctc ctgatccgtc ctgtggacaa accaatcaac 840

acaactttaa tctgcaacgt caccaatgcc ctaggagctc gccaggcaga actgaccgtc 900

caggtcaaa 909

<210> 8

<211> 600

<212> DNA

<213> Artificial sequence (.)

<400> 8

gcgctcacaa tcaccacctc gcccaacctg ggtacccgag agaataatgc agaccaggtc 60

acccctgttt cccacattgg ctgccccaac actacacaac agggctctcc tgtgttcgcc 120

aagctactgg ctaaaaacca agcatcgttg tgcaatacaa ctctgaactg gcacagccaa 180

gatggagctg ggagctcata cctatctcaa ggtctgaggt acgaagaaga caaaaaggag 240

ttggtggtag acagtcccgg gctctactac gtatttttgg aactgaagct cagtccaaca 300

ttcacaaaca caggccacaa ggtgcagggc tgggtctctc ttgttttgca agcaaagcct 360

caggtagatg actttgacaa cttggccctg acagtggaac tgttcccttg ctccatggag 420

aacaagttag tggaccgttc ctggagtcaa ctgttgctcc tgaaggctgg ccaccgcctc 480

agtgtgggtc tgagggctta tctgcatgga gcccaggatg catacagaga ctgggagctg 540

tcttatccca acaccaccag ctttggactc tttcttgtga aacccgacaa cccatgggaa 600

<210> 9

<211> 45

<212> DNA

<213> Artificial sequence (.)

<400> 9

ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcg 45

<210> 10

<211> 51

<212> DNA

<213> Artificial sequence (.)

<400> 10

atgccgctgc tgctactgct gcccctgctg tgggcagggg ccctggctat g 51

<210> 11

<211> 3136

<212> DNA

<213> Artificial sequence (.)

<400> 11

attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg 60

tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 120

gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca 180

gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 240

taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 300

acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 360

gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 420

gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 480

gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg 540

ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact 600

gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta 660

gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct 720

ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg gacggctgcc 780

ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag 840

cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg 900

ttattgtgct gtctcatcat tttggcaaag aattgccacc atgaagtgcc ttttgtactt 960

agccttttta ttcattgggg tgaattgcaa gttcaccata gtttttccac acaaccaaaa 1020

aggaaactgg aaaaatgttc cttctaatta ccattattgc ccgtcaagct cagatttaaa 1080

ttggcataat gacttaatag gcacagcctt acaagtcaaa atgcccaaga gtcacaaggc 1140

tattcaagca gacggttgga tgtgtcatgc ttccaaatgg gtcactactt gtgatttccg 1200

ctggtatgga ccgaagtata taacacattc catccgatcc ttcactccat ctgtagaaca 1260

atgcaaggaa agcattgaac aaacgaaaca aggaacttgg ctgaatccag gcttccctcc 1320

tcaaagttgt ggatatgcaa ctgtgacgga tgccgaagca gtgattgtcc aggtgactcc 1380

tcaccatgtg ctggttgatg aatacacagg agaatgggtt gattcacagt tcatcaacgg 1440

aaaatgcagc aattacatat gccccactgt ccataactct acaacctggc attctgacta 1500

taaggtcaaa gggctatgtg attctaacct catttccatg gacatcacct tcttctcaga 1560

ggacggagag ctatcatccc tgggaaagga gggcacaggg ttcagaagta actactttgc 1620

ttatgaaact ggaggcaagg cctgcaaaat gcaatactgc aagcattggg gagtcagact 1680

cccatcaggt gtctggttcg agatggctga taaggatctc tttgctgcag ccagattccc 1740

tgaatgccca gaagggtcaa gtatctctgc tccatctcag acctcagtgg atgtaagtct 1800

aattcaggac gttgagagga tcttggatta ttccctctgc caagaaacct ggagcaaaat 1860

cagagcgggt cttccaatct ctccagtgga tctcagctat cttgctccta aaaacccagg 1920

aaccggtcct gctttcacca taatcaatgg taccctaaaa tactttgaga ccagatacat 1980

cagagtcgat attgctgctc caatcctctc aagaatggtc ggaatgatca gtggaactac 2040

cacagaaagg gaactgtggg atgactgggc accatatgaa gacgtggaaa ttggacccaa 2100

tggagttctg aggaccagtt caggatataa gtttccttta tacatgattg gacatggtat 2160

gttggactcc gatcttcatc ttagctcaaa ggctcaggtg ttcgaacatc ctcacattca 2220

agacgctgct tcgcaacttc ctgatgatga gagtttattt tttggtgata ctgggctatc 2280

caaaaatcca atcgagcttg tagaaggttg gttcagtagt tggaaaagct ctattgcctc 2340

ttttttcttt atcatagggt taatcattgg actattcttg gttctccgag ttggtatcca 2400

tctttgcatt aaattaaagc acaccaagaa aagacagatt tatacagaca tagagatgaa 2460

ccgacttgga aagagcggcg ccaccaactt cagcctgctg aagcaggccg gcgacgtgga 2520

ggagaacccc ggccccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga 2580

cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca 2640

caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac 2700

gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgcgg ccgtggcggt 2760

ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat 2820

ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc 2880

gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc ccgaccacca 2940

gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg 3000

ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg 3060

cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg 3120

caagcccggt gcctga 3136

<210> 12

<211> 18

<212> DNA

<213> Artificial sequence (.)

<400> 12

caccaccacc accaccac 18

<210> 13

<211> 538

<212> DNA

<213> Artificial sequence (.)

<400> 13

ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 60

ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 120

ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 180

tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 240

tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 300

cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga 360

ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 420

aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg 480

taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatc 538

<210> 14

<211> 870

<212> DNA

<213> Artificial sequence (.)

<400> 14

atgagacata ttatctgcca cggaggtgtt attaccgaag aaatggccgc cagtcttttg 60

gaccagctga tcgaagaggt actggctgat aatcttccac ctcctagcca ttttgaacca 120

cctacccttc acgaactgta tgatttagac gtgacggccc ccgaagatcc caacgaggag 180

gcggtttcgc agatttttcc cgactctgta atgttggcgg tgcaggaagg gattgactta 240

ctcacttttc cgccggcgcc cggttctccg gagccgcctc acctttcccg gcagcccgag 300

cagccggagc agagagcctt gggtccggtt tctatgccaa accttgtacc ggaggtgatc 360

gatcttacct gccacgaggc tggctttcca cccagtgacg acgaggatga agagggtgag 420

gagtttgtgt tagattatgt ggagcacccc gggcacggtt gcaggtcttg tcattatcac 480

cggaggaata cgggggaccc agatattatg tgttcgcttt gctatatgag gacctgtggc 540

atgtttgtct acagtcctgt gtctgaacct gagcctgagc ccgagccaga accggagcct 600

gcaagaccta cccgccgtcc taaaatggcg cctgctatcc tgagacgccc gacatcacct 660

gtgtctagag aatgcaatag tagtacggat agctgtgact ccggtccttc taacacacct 720

cctgagatac acccggtggt cccgctgtgc cccattaaac cagttgccgt gagagttggt 780

gggcgtcgcc aggctgtgga atgtatcgag gacttgctta acgagcctgg gcaacctttg 840

gacttgagct gtaaacgccc caggccataa 870

<210> 15

<211> 54

<212> DNA

<213> A Artificial sequence (.)

<400> 15

gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccct 54

<210> 16

<211> 297

<212> DNA

<213> Artificial sequence (.)

<400> 16

ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc 60

cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 120

actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 180

attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg 240

catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctgccac 297

<210> 17

<211> 3739

<212> DNA

<213> Artificial sequence (.)

<400> 17

cacctatcga taagcttggg agttccgcgt tacataactt acggtaaatg gcccgcctgg 60

ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac 120

gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt 180

ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa 240

atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta 300

catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg 360

gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg 420

gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc 480

attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt 540

agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca 600

ccgactctag aggatccgcc accatggtga gcaagggcga ggagctgttc accggggtgg 660

tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg 720

agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca 780

agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca 840

gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct 900

acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg 960

tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg 1020

aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata 1080

tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg 1140

aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc 1200

ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca 1260

acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg 1320

gcatggacga gctgtacaag gctagcgagg gcagaggaag tcttctaaca tgcggtgacg 1380

tggaggagaa tcccggccct accggaatga gacatattat ctgccacgga ggtgttatta 1440

ccgaagaaat ggccgccagt cttttggacc agctgatcga agaggtactg gctgataatc 1500

ttccacctcc tagccatttt gaaccaccta cccttcacga actgtatgat ttagacgtga 1560

cggcccccga agatcccaac gaggaggcgg tttcgcagat ttttcccgac tctgtaatgt 1620

tggcggtgca ggaagggatt gacttactca cttttccgcc ggcgcccggt tctccggagc 1680

cgcctcacct ttcccggcag cccgagcagc cggagcagag agccttgggt ccggtttcta 1740

tgccaaacct tgtaccggag gtgatcgatc ttacctgcca cgaggctggc tttccaccca 1800

gtgacgacga ggatgaagag ggtgaggagt ttgtgttaga ttatgtggag caccccgggc 1860

acggttgcag gtcttgtcat tatcaccgga ggaatacggg ggacccagat attatgtgtt 1920

cgctttgcta tatgaggacc tgtggcatgt ttgtctacag tcctgtgtct gaacctgagc 1980

ctgagcccga gccagaaccg gagcctgcaa gacctacccg ccgtcctaaa atggcgcctg 2040

ctatcctgag acgcccgaca tcacctgtgt ctagagaatg caatagtagt acggatagct 2100

gtgactccgg tccttctaac acacctcctg agatacaccc ggtggtcccg ctgtgcccca 2160

ttaaaccagt tgccgtgaga gttggtgggc gtcgccaggc tgtggaatgt atcgaggact 2220

tgcttaacga gcctgggcaa cctttggact tgagctgtaa acgccccagg ccataacacc 2280

tatcgataag cttgggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga 2340

ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca 2400

atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca 2460

gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg 2520

cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc 2580

tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt 2640

ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt 2700

ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg 2760

acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcgtttagtg 2820

aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccga 2880

ctctagagga tccgccacca tgaccggtat gtgggtccgg caggtaccct ggtcattcac 2940

ttgggctgtg ctgcagttga gctggcaatc agggtggctt ctagaggtcc ccaatgggcc 3000

ctggaggtcc ctcaccttct acccagcctg gctcacagtg tcagagggag caaatgccac 3060

cttcacctgc agcttgtcca actggtcgga ggatcttatg ctgaactgga accgcctgag 3120

tcccagcaac cagactgaaa aacaggccgc cttctgtaat ggtttgagcc aacccgtcca 3180

ggatgcccgc ttccagatca tacagctgcc caacaggcat gacttccaca tgaacatcct 3240

tgacacacgg cgcaatgaca gtggcatcta cctctgtggg gccatctccc tgcaccccaa 3300

ggcaaaaatc gaggagagcc ctggagcaga gctcgtggta acagagagaa tcctggagac 3360

ctcaacaaga tatcccagcc cctcgcccaa accagaaggc cggtttcaag gcatggtcca 3420

ccaccaccac caccaccact aactcgagtc tagagggccc gtttaaaccc gctgatcagc 3480

ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 3540

gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 3600

ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga 3660

ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc 3720

ggaaagaacc agctgccac 3739

<210> 18

<211> 1687

<212> DNA

<213> Artificial sequence (.)

<400> 18

ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 60

ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 120

ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 180

tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 240

tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 300

cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga 360

ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 420

aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg 480

taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcgc 540

ctggagacgc catccacgct gttttgacct ccatagaaga caccgactct agaggatccg 600

ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc 660

tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca 720

cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc 780

ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca 840

tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca 900

tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca 960

ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg 1020

ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga 1080

agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc 1140

tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca 1200

accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca 1260

tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca 1320

agtaatggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc agatatccag 1380

cacagtggcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg 1440

tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 1500

aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 1560

gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 1620

aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 1680

ccagctg 1687

<210> 19

<211> 1536

<212> DNA

<213> Artificial sequence (.)

<400> 19

atgaagtgcc ttttgtactt agccttttta ttcattgggg tgaattgcaa gttcaccata 60

gtttttccac acaaccaaaa aggaaactgg aaaaatgttc cttctaatta ccattattgc 120

ccgtcaagct cagatttaaa ttggcataat gacttaatag gcacagcctt acaagtcaaa 180

atgcccaaga gtcacaaggc tattcaagca gacggttgga tgtgtcatgc ttccaaatgg 240

gtcactactt gtgatttccg ctggtatgga ccgaagtata taacacattc 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> 20

<211> 511

<212> PRT

<213> Artificial sequence (.)

<400> 20

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 Leu 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 His

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> 21

<211> 934

<212> DNA

<213> Artificial sequence (.)

<400> 21

attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg 60

tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 120

gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca 180

gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 240

taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 300

acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 360

gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 420

gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 480

gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg 540

ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact 600

gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta 660

gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct 720

ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg gacggctgcc 780

ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag 840

cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg 900

ttattgtgct gtctcatcat tttggcaaag aatt 934

<210> 22

<211> 600

<212> DNA

<213> Artificial sequence (.)

<400> 22

atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta 60

cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac 120

cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac 180

atcggcaagg tgtgggtcgc ggacgacggc gcggccgtgg cggtctggac cacgccggag 240

agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt 300

tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag 360

cccgcgtggt tcctggccac cgtcggagtc tcgcccgacc accagggcaa gggtctgggc 420

agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg 480

gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc 540

gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga 600

Claims

1. A novel vesiculooncolytic-like virus EM/VSV-G Ad5 sPGRCD 137L, characterized by: the novel vesicular oncolytic virus EM/VSV-G Ad5 sVRCD 137L is an adenovirus capable of expressing a soluble protein PVRCD137L, the outer surface of the adenovirus is coated with a vesicular vesicle, and VSV-G protein is arranged on the membrane of the vesicular vesicle; the DNA sequence of the VSV-G protein is shown in SEQ ID NO. 19, and the amino acid sequence of the VSV-G protein is shown in SEQ ID NO. 20; the DNA sequence of the soluble protein PVRCD137L is shown in SEQ ID NO. 6, and the protein sequence of the soluble protein PVRCD137L is shown in SEQ ID NO. 1.

2. A novel vesiculooncolytic virus EM/VSV-G Ad5sPVRCD137L, according to claim 1, characterized by: the VSV-G protein can realize the targeting of tumor cells; and due to the existence of the vesicle-like nano vesicles, the novel vesicle-like oncolytic virus EM/VSV-G Ad5 sPGRCD 137L can not be acted by a neutralizing antibody of the anti-adenovirus, and has a remarkably prolonged administration window period; the novel vesiculooncolytic virus EM/VSV-G Ad5 sPGRCD 137L can more continuously express soluble PVRCD137L, continuously block a PVR/TIGIT immune checkpoint pathway and activate a CD137 immune co-stimulation pathway.

3. The novel vesiculooncolytic virus EM/VSV-G Ad5 sPGRCD 137L as claimed in claim 1, which is prepared by the following method:

(1) preparing a cell in which a VSV-G protein is inserted into the outer membrane of the cell, and designating the cell as a VSV-G cell;

(2) infecting VSV-G cells with adenovirus Ad5 sPVRCCD 137L, wherein the adenovirus Ad5 sPVRCCD 137L is adenovirus capable of expressing soluble PVRCD 137L;

(3) resuspending the collected VSV-G cells after adenovirus infection with culture medium, PBS or other buffer solution, and extruding and shearing the cell suspension through membrane filter paper with the aperture of 10 μm, 5 μm or 1 μm by using a centrifugal extrusion shearing method;

(4) collecting the virus suspension after centrifugal extrusion shearing, and enriching and collecting the novel vesicle-like oncolytic virus EM/VSV-G Ad5 sPGRCD 137L wrapped by the vesicle by a density gradient centrifugation method, wherein the novel vesicle-like oncolytic virus EM/VSV-G Ad5 sPGRCD 137L is wrapped by the vesicle-like, and VSV-G protein is arranged on the wrapped vesicle-like membrane, so that targeting can be realized.

4. The use of the novel vesicular oncolytic virus EM/VSV-G Ad5 sPGRCD 137L as claimed in claim 1 for the preparation of anti-tumor drugs, including but not limited to liver cancer, kidney cancer, leukemia, lung cancer, melanoma and colorectal cancer.

5. The use of a novel vesiculooncolytic virus EM/VSV-G Ad5 sPGRCD 137L as claimed in claim 1 for the manufacture of a medicament for immunotherapy of tumors including but not limited to liver, kidney, leukemia, lung, melanoma and colorectal cancer.

6. The use of the novel vesicular oncolytic virus EM/VSV-G Ad5 sPGRCD 137L in the preparation of a medicament for blocking PVR/TIGIT immunodetection site pathway and activating CD137 pathway in tumors including but not limited to liver cancer, kidney cancer, leukemia, lung cancer, melanoma and colorectal cancer according to claim 1, wherein the novel vesicular oncolytic virus EM/VSV-G Ad5 sPGRCD 137L can infect tumor cells and replicate and express soluble PVRCD137L, the expressed PVRCD137L binds with TIGIT to block PVR/TIGIT immunodetection site pathway, and the expressed PVRCD137L binds with CD137 to activate CD137 immune co-stimulatory pathway.