KR101845739B1 - Novel Oncolytic Virus, Method for Preparing, and Uses thereof - Google Patents

Abstract

The present invention relates to the use of a novel avian influenza virus, and more particularly, to a dual-use shuttle virus containing a sequence encoding a TRAIL gene which is a cancer suicide gene and Ang1 which is a normal angiogenesis gene, A recombinant virus produced using the vector, and an anticancer vaccine composition containing the recombinant virus as an active ingredient. According to the present invention, an Ang1 or Comp-Ang1 gene that regenerates a normal blood vessel that is not TRAIL gene and / or tumor blood vessel that causes TK and / or VGF to be removed to improve cancer specificity and cause cell apoptosis in cancer So that it can be used for various chemotherapeutic treatments.

Description

Novel Oncological Virus, Method for Preparing, and Uses thereof < RTI ID = 0.0 >

The present invention relates to a novel avian influenza virus, a method for producing the same and a use thereof.

To date, a variety of therapies such as surgical, radiation, and chemotherapy have been studied to treat cancer, but they have yet to be fully treated.

The dual virus is one of the biotherapeutic agents and has the concept of a targeted therapeutic agent that attacks using genetic mutations in tumor cells. Cancerous or oncolytic viruses selectively proliferate in cancer cells and induce tumor necrosis and death.

Oncolytic virus is a virus used for the treatment of cancer, and its use is called oncolytic viral therapy (OV). Research on cancer therapy using wild-type oncolytic virus is based on gene therapy (Gene therapy), in which a therapeutic gene is inserted into a virus and the tumor killing effect is expressed by the expression of the therapeutic gene Differently, studies on wild-type tumor-destroying viruses began with the discovery that some wild-type viruses have inherently strong tumor-destructive capacity.

It has long been known that cancer is treated naturally due to natural infection caused by various kinds of viruses. Since the study on tumor-specific killing mechanism by wild-type virus has been started in earnest, it has been reported that cancer caused by wild type Reovirus Therapeutic studies have reached clinical trials. Other adenoviruses, polioviruses, herpes zoster viruses, and vesicular stomatitis viruses have been developed and methods for increasing the efficacy and stability of viruses are being studied.

These wild-type tumor-killing viruses are viruses already existing in nature and thus have excellent stability and clinical applications. In addition, wild-type tumor killing virus is characterized in that it specifically induces proliferation and cell destruction only in cancer cells, and is applied to clinical studies using various wild-type tumor-destroying viruses.

It is necessary to develop a new "target type cancer treatment drug" which can distinguish cancer cells not normal cells from other cancer cells by raising the cancer risk more in view of the lack of selectivity of the current chemotherapeutic drugs and side effects such as normal cytotoxicity. In addition to the side effects caused by conventional chemotherapy, anticancer drug resistance due to treatment has emerged as a secondary problem, and there has been a demand for an anticancer treatment method which is more stable and highly selective for cancer.

The present inventors have made an effort to develop a novel oncolytic virus. As a result, it is possible to remove the TK (Thymidine Kinase) or VGF (Vaccinia Growth Factor) of the virus, thereby increasing the cancer affinity and enhancing the cancer selectivity. In addition, TRAIL gene which causes cell apoptosis in cancer and normal blood vessels A shuttle vector containing sequences encoding the Ang1 gene to be reconstructed was constructed, and the avian influenza virus produced using the vector would play an important role in the induction of cancer cell death and normal vascularization.

Accordingly, it is an object of the present invention to provide a novel shuttle vector and a novel avian influenza virus.

Another object of the present invention is to provide a pharmaceutical composition for preventing and treating cancer.

According to one aspect of the present invention, there is provided a recombinant shuttle vector into which a foreign gene for treating cancer is introduced.

A feature of the present invention is to provide a method for inhibiting the growth of cancer cells and specifically destroying cancer cells by causing an oncolytic activity of specifically infecting cancer cells and affecting cancer cells, It is possible to provide a novel anti-cancer virus and a therapeutic agent for a target cancer using the same.

In the present invention, a recombinant shuttle vector into which a foreign gene for cancer treatment is introduced is used.

As used herein, the term "heterologous gene" means that it accepts a gene that is not found in the viral genome.

The foreign gene may be a wild-type gene or an allelic variant thereof or a mutant gene. Preferably, the exogenous gene is operably linked to a control sequence that allows expression of the exogenous gene in cells of the in vivo condition. Thus, the virus of the present invention can be used to deliver foreign genes / genes to cells in vivo conditions in which the foreign gene can be expressed. The gene typically encodes a protein capable of promoting the tumor-destroying properties of the virus. The gene may itself encode a protein that is cytotoxin, activates prodrugs, or can promote / enhance antitumor immune responses.

The foreign gene may include all genes that insert into a vector to treat cancer.

According to a preferred embodiment of the present invention, the foreign gene for cancer therapy is a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a cancer angiogenesis gene, Ang1 (Angiopoietin 1) And at least one selected from the group consisting of

The variants refer to COMP-Angl, a soluble, stable and potent Angl variant, for in vivo use of Ang1.

That is, the vector of the present invention comprises at least one sequence encoding a therapeutic gene. The therapeutic gene has pharmacological or prophylactic activity upon proper administration to a patient, particularly a patient suffering from a disease or disease state, or a patient to be protected from such disease or condition. Such pharmacological or prophylactic activity is meant to relate to a beneficial effect on the disease or on the course or symptom of the condition. The sequence of interest may be homologous or heterologous to the target cell into which it is introduced, and it encodes all or part of a polypeptide, in particular a therapeutic or prophylactic polypeptide conferring therapeutic or prophylactic properties. Polypeptides are understood as any translational product of a polynucleotide, regardless of size and glycosylation, and include peptides and proteins. Therapeutic polypeptides include polypeptides that can compensate for deficient or deficient proteins in an animal or human organism, or those that act through toxic effects to limit or eliminate harmful cells from the body. They may also be immune-granting polypeptides that act as endogenous antigens to induce a humoral or cellular response, or both, and may be, for example, a drug susceptibility gene, a cell death gene, a cell proliferation inhibiting gene, a cytotoxic gene, Factor genes, antigenic genes, anti-angiogenic genes, cytokine genes, and the like.

According to a preferred embodiment of the present invention, the vector of the present invention is deficient in a sequence encoding a TK (Thymidine Kinase) gene or a VGF (Vaccinia Growth Factor) gene.

The vector of the present invention is preferably capable of introducing a reporter gene which can confirm the expression of the gene of interest and / or its protein, and can be linked to the foreign gene for cancer treatment.

According to a preferred embodiment of the present invention, the reporter gene is selected from the group consisting of LacZ, mCherry, tdTomato, mStrawberry, J-red, DsRed, chloramphenicol acetyltransferase (CAT), Renila luciferase, firefly ) Genes encoding luciferase, red fluorescent protein (RFP), green fluorescent protein (GFP), secreted placental alkaline phosphatase (SEAP) or HSV-tk (Herpes simplex virus-thymidine kinase) And more preferably LacZ, mCherry, tdTomato, mStrawberry, J-red, DsRed, chloramphenicol acetyltransferase (CAT), Renila luciferase, firefly luciferase, Protein (RFP), most preferably LacZ or mCherry.

According to the present invention, the shuttle vector of the present invention comprises:

(i) a gene comprising the nucleotide sequence of SEQ ID NO: 1, more specifically, TRAIL, which is a cancer suicide gene and a lacZ gene operably linked thereto, as a foreign gene, and wherein the TK (Thymidine Kinase) The recombinant vector pYSYTRAIL-LacZ, represented by cleaved map;

(ii) a nucleotide sequence of SEQ ID NO: 2, and more specifically, an angiogenesis gene Ang1 (Angiopoietin 1) as a foreign gene and an mCherry gene operably linked thereto, wherein the VGF (Vaccinia Growth Factor) gene The deleted recombinant vector pYSY-Angl-mcherry represented by the cleaved map of Fig. 3A; And

(iii) the sequence of SEQ ID NO: 3, and more particularly, the sequence of CompA-Ang1 as a foreign gene and the mCherry gene operably linked thereto, wherein the VGF (Vaccinia Growth Factor) It is the recombinant vector pYSY-Comp-Ang1-mcherry which is represented by the map.

According to another aspect of the present invention, the present invention provides an oncolytic virus transformed with said vector.

According to the present invention, the anti-

(a) a vaccinia virus which is transformed by the recombinant vector pYSY-TRAIL-LacZ described above and can not produce TK, and has LacZ activity when TRAIL gene is introduced from the outside, vYSYTRAILLACZ;

(b) a vaccinia virus which is transformed by the recombinant vector pYSY-Angl-mcherry and can not produce VGF, and has an mCherry activity when an Ang1 gene is introduced from the outside, and vYSYAng1 mCherry;

(c) Vaccinia virus which is transformed by the above recombinant vector pYSY-Comp-Angl-mcherry and can not produce VGF, and has mCherry activity when the Comp-Angl gene is introduced from the outside, vYSYCompAng1mCherry; And

(d) Vaccinia viros which are transformed using the two recombinant vectors and are unable to produce TK and VGF, and which contain TRAIL and Angl as foreign genes and have lacZ and mCherry activity operatively linked to each other It is a characteristic anti-virus virus vYSYTRAILAng1.

In other words, a shuttle vector is needed to create an antidote virus to increase cancer risk, and the function of the antiretroviral virus is enhanced according to the design of the shuttle vector used and the functionality of the gene. In the present invention, the TRAIL gene, which causes apoptosis of cells in cancer cells, enhances cancer affinity and cancer selectivity by eliminating TK and VGF, and has a gene encoding Ang1 for the reconstruction of normal blood vessels (PYSYTRAIL-LacZ, pYSYCol-1-mCherry, pYSYComp-Ang1-mCherry) and the combination thereof to produce an anti-cancer virus that helps reconstitute normal cells with normal vascular reconstruction with the death of cancer cells have.

According to still another aspect of the present invention, there is provided a pharmaceutical composition for preventing and treating cancer comprising the above-described avian influenza virus as an active ingredient.

As used herein, the term "prophylactic " means any act that inhibits cancer or delays progression by administration of the composition of the present invention.

The term "treatment ", as used herein, refers to any action in which the administration of a composition of the present invention improves or alleviates cancer and refers to an attempt to obtain beneficial or desired results, including clinical results. Useful or desirable clinical results may include, but are not limited to, the reduction or amelioration of one or more symptoms or conditions, the reduction of disease extent, the stabilization of disease states, the inhibition of disease outbreaks, the inhibition of disease spread, , Delay or delayed onset of the disease, improvement or relief of the disease state, and decline (partial or total).

Also, " treatment "may mean prolonging the survival of the patient beyond that predicted in the absence of treatment. It may also mean inhibiting disease progression, temporarily delaying disease progression, As is understood by those skilled in the art, the treatment results in the opposite outcome, i.e. greater than all the benefits that are affected by the treatment, in the treated patient, while improving the particular disease state If the results are not beneficial or undesirable.

Cancers which can be treated in the present invention include cancer of the blood, colorectal cancer, brain cancer, glioma, neurocytoma, neurocytoma, retrograde astrocytoma, hematoblastoma, stomach cancer, lung cancer, small cell lung carcinoma, cervical carcinoma, Cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, cholangiocarcinoma, papillary carcinoma, testicular tumor, endometrial carcinoma, endometrial carcinoma, endometrial carcinoma, endometrial carcinoma The present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the treatment or prophylaxis of a disease selected from the group consisting of Wilm's tumor, Ewing's tumor, bladder carcinoma, angiosarcoma, endothelium, adenocarcinoma, adenocarcinoma, adenocarcinoma, papillary carcinoma, Neuroblastoma, neuroblastoma, retinoblastoma, oligodendroglioma, neuroblastoma, angioblastoma, meningioma, pineal gland, ependymoma, craniopharyngioma, epithelial carcinoma, embryonal carcinoma, squamous cell carcinoma, basal cell carcinoma, , And a myxoma, mucus sarcoma, liposarcoma, chondrosarcoma, such as bone or immunogenic six kinds of leukemia, but is not limited to this.

The virus of the present invention may be administered alone or in combination with other therapies, including chemotherapy, radiotherapy or other anti-viral therapy. For example, the virus may be administered prior to or after removal of the primary tumor via surgery, or prior to, concurrently with, or subsequent to treatment, such as with radiotherapy or conventional chemotherapy drugs. The virus can be administered with other tumor killing viruses that are specific for various tumor cell types, or in a sequential manner. Treatments that have been routinely used to treat, prevent or treat cancer include, but are not limited to, surgery, chemotherapy, radiotherapy, hormone therapy, biological therapy and immunotherapy.

The composition of the present invention can be administered orally or parenterally at the time of clinical administration and can be used in the form of a general pharmaceutical preparation. Dosage forms may be oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenterally (e.g., subcutaneously, intravenously, bolus injected, intramuscularly or intraarterially), topical , Eye), transdermal, or transcutaneous manner. Examples of dosage forms include tablets; Caplets; A capsule such as a soft elastic gelatin capsule; Cachets; Troches; Lozenges; Dispersing agent; Suppository; powder; Aerosols (e. G., Nasal sprays or inhalers); Gel; Liquid formulations suitable for oral or mucosal administration to a patient, including suspensions (e. G., Aqueous or non-aqueous liquid suspensions, water-in-oil emulsions, or water-in-oil liquid emulsions) solutions and elixirs; A liquid dosage formulation suitable for administration to a patient; Eye drops or other ophthalmic formulations suitable for topical administration; And sterile solid preparations (e. G., Crystalline or amorphous solids) that can be reconstituted to provide the patient with a liquid dosage form suitable for injectable administration. The types, shapes, and types of dosage forms of the present invention generally vary widely depending on their use. For example, the dosage form used for the acute treatment of the disease may comprise a greater amount of the active ingredient than the dosage form used for the chronic treatment of the same disease. In addition, parenteral dosage forms may contain less active ingredients than the oral dosage forms used to treat the same disease. The modes of administration and modes of administration encompassed by the present invention are very varied and will be apparent to those skilled in the art (see Remington ' s Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA .)

The compositions of the present invention may comprise one or more excipients. The excipient can be in a wide variety of forms depending on the form of preparation desired for administration. For example, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring ingredients, preservatives, and coloring ingredients. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules and caplets) include starches, sugars, microcrystalline cellulose, excipients, granulating agents, lubricants, binders and disintegrating agents, But is not limited thereto. For convenience of administration, tablets and capsules are the most useful form of oral administration unit, in which case solid excipients are applied. Preferably the tablets may be coated using standard aqueous or nonaqueous techniques. Examples of excipients that can be used in the oral dosage forms of the present invention include, but are not limited to, binders, fillers, disintegrants, and glidants.

Suitable binding agents for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch or other starches, natural and synthetic gums such as gelatin, acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth ), Guar gum, cellulose and derivatives thereof (e.g., ethyl cellulose, cellulose acetate, carboxymethylcellulose calcium, sodium carboxymethylcellulose), polyvinylpyrrolidone, methylcellulose, pregelatinized starch, (For example, Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

The active ingredient of the present invention may be administered by controlled release means or by delivery devices well known to those of ordinary skill in the art.

As used herein, the term "administering" means providing a composition of the invention to a subject in any suitable manner.

The term "individual" as used herein means any animal such as a human, a monkey, a dog, a goat, a pig, or a mouse having a disease in which cancer can be improved by administering the composition of the present invention.

The term " pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit or risk rate applicable to medical treatment, including the type of disease, severity, activity of the drug, The time of administration, the route and rate of excretion of the drug, the duration of the treatment, factors including drugs used simultaneously and other factors well known in the medical arts.

The effective amount of the virus may be sufficient to alleviate, improve, mitigate, ameliorate, stabilize, inhibit the spread of the disease, delay or delay the progression of the disease, Respectively.

For example, an effective amount is an amount sufficient to achieve the effect of reducing the number of cancer cells or reducing or destroying the number of cells chronically infected with the virus or virus, or inhibiting the growth and / or proliferation of such cells .

The effective amount may vary depending on a number of factors such as the pharmacokinetic characteristics of the virus, the method of administration, the age, the health and weight of the patient, the nature and extent of the disease state, the number of treatments and the current mode of treatment, It can also vary depending on the activity. Those skilled in the art will be able to adjust the titration based on these factors. The virus may initially be administered in an appropriate amount depending on the clinical response of the patient, as needed. The effective amount of the virus can be determined empirically and can depend on the maximum amount of virus that can be safely administered and the minimum amount of virus that produces the desired result.

When the virus is administered systemically, very high doses of the virus may be required to produce a clinical effect similar to that realized by infecting the disease site with the virus. However, a suitable capacity level should be the minimum amount to realize the desired result.

The concentration of the virus to be administered will depend on the virulence of the virus to be administered and the characteristics of the target cell. An effective amount of the virus can be repeatedly administered according to the effect of the first therapeutic regimen. In general, administration is periodically administered during monitoring of all reactions. Those skilled in the art will readily be able to ascertain that doses lower or higher than those indicated above may be administered depending on the dosing schedule and the selected route.

According to still another aspect of the present invention, the present invention provides a method for producing a novel avian influenza virus comprising the steps of:

(a) preparing a vector of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; And

(b) transforming the vaccinia virus with the vector prepared in the step (a).

Since the method of the present invention is a method for producing the above-mentioned main vector, the common description thereof is omitted in order to avoid the excessive complexity of the present specification according to the repetitive description.

The present invention relates to a novel avian influenza virus and its use. According to the present invention, since it can exert an excellent effect not only specifically killing cancer cells but also reconstructing normal blood vessels, it can be applied to prevention or treatment of cancer have.

Fig. 1 shows the cloning conditions (Fig. 1a) of the present invention, the shuttle vector of the present invention and the schematic diagram of the virus prepared using the shuttle vector (Fig. 1b and Fig. 1c).
FIG. 2 shows the production (FIG. 2A), expression (FIG. 2B) of the shuttle vector pYSYTRAIL-LacZ and confirmation result (FIG. 2C) of the virus vYSYTRAILLACZ prepared using the vector.
Fig. 3 shows the production (Fig. 3a), expression (Fig. 3b) and viruses vYSYAng1mCherry and vYSYTRAILAng1 (Fig. 3c) of the shuttle vector pYSY-Ang1-mcherry.
Fig. 4 shows the production (Fig. 4A), expression (Fig. 4B) of the shuttle vector pYSY-Comp-Ang1-mcherry and confirmation result (Fig. 4C) of the virus vYSYAng1 mCherry prepared using the vector.
Figures 5A-5C show the ability of TK-deleted virus (VVD) to kill in cancer cells.
FIG. 6 shows the assassination effect of a virus (VVDD +) in which a functional gene is inserted into a virus (VVDD) or VVDD in which a deletion of VGF is concurrent with deletion of TK.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Materials and Methods

Cloning  Way

Cloning was carried out under the conditions of Figure 1A below using methods known in the art.

The gene to be inserted into the vector was amplified by polymerase chain reaction (PCR) using a pair of primers including a restriction enzyme in the method of FIG. 2, the gene amplification product and the vector to be inserted were cleaved with restriction enzymes (XhoI and NotI) at the site to be inserted at both sides. The restriction endonuclease-digested vector and the gene amplification product to be inserted were subjected to ligation to final insert the restriction endonuclease. The final vector made by the method of 4 was transformed into bacterial strains. It was confirmed that the final vector was inserted by the method 5.

Example  One. pYSYTRAIL - LacZ  Production of vectors and viruses expressing them

1-1. Produced pYSYTRAIL-LacZ vector

The present inventors produced a shuttle vector in which TRAIL and LacZ were inserted together at the Tk site of the avian influenza virus.

The sequence of the shuttle vector pYSYTRAIL-LacZ having a cleavage map in Fig. 2A is shown in SEQ ID NO: 1 and the expression of pYSYTRAIL-LacZ was confirmed as shown in Fig. 2B.

1-2. vYSYTRAILLACZ virus production and identification

The inventors of the present invention produced an avian influenza virus expressing LacZ, which expresses TRAIL, which is a cancer suicide gene, and can detect the presence or absence of the virus, by disrupting the Tk region with the pYSYTRAIL-LacZ vector prepared in Example 1-1.

The virus (vYSYTRAILLACZ) expressing the pYSYTRAIL-LacZ vector was prepared as follows.

In order to delete the thymidine kinase (Tk) in the virus, a vector pYSYTRAILLACZ having a homologous sequence with both of the genes of the thymidine kinase (left and right) and having the foreign genes TRAIL and LacZ in between, When the homologous portion of the Vaccinia virus sequence is exchanged, a foreign gene is inserted into the region of the thymidine kinase, thereby obtaining vishyya virus vYSYTRAILLACZ in which the thymidine kinase is deleted and the foreign genes TRAIL and LacZ are expressed. For homologous recombination, BSC-1 cells were used as host cells. Upon reaching 80% confluence, 0.01MOI wild type vaccinia virus (Wyeth strain, ATCC) was infected into the cells for 1 hour and then the medium was removed And transformed with the above vector using Lipofectamine 2000 (Life Technologies). The transformed mixture was prepared by adding Lipofectamine 2000 to a serum-free medium with 1-5 μg of the insertion vector so as to have a final volume of 150 μL, and then transfected into the virus-infected cells so that homologous recombination with the insertion vector Let it happen. Since the new virus expresses lacZ, it can be obtained by selecting only a single new virus plaque by purifying plaques expressing lacZ in three or more steps.

As a result, as shown in Fig. 2C, vYSYTRAILLACZ (red circle) was confirmed.

Example  2. pYSYComp - Ang1 - mcerry  With vector pYSY - Ang1 - mcerry  Production of vectors and viruses expressing them

2-1. Production of pYSY-Comp-Ang1-mcherry vector and pYSY-Ang1-mcherry vector

The present inventors constructed a shuttle vector in which Ang1 and mCherry, which are normal angiogenic genes, were inserted into the VGF region of the avian influenza virus and the mutant Ang-1 and mCherry were inserted into the VGF region of the avian influenza virus .

The sequence of the shuttle vector pYSY-Ang1-mcherry having the cleavage map in Fig. 3A is shown in SEQ ID NO: 2 and the expression of pYSY-Angl-mcherry was confirmed as shown in Figs. 3B and 3C.

The sequence of the shuttle vector pYSY-Comp-Ang1-mcherry having the cleaved map of Fig. 4A is shown in SEQ ID NO: 3, and the expression of pYSY-Comp-Angl-mcherry was confirmed as shown in Figs. 4B and 4C.

2-2. Manufacture of vYSYAng1mCherry and vYSYCompAng1mcherry virus

The inventors of the present invention found that the pYSY-Angl-mcherry vector prepared in Example 2-1 was able to express the angiogenic virus, pYSY-Angl-mcherry, which expresses the normal angiogenic gene Ang1, The virus-deficient virus expressing mcherry, which expresses the normal angiogenic gene CompAng1 and can confirm the presence or absence of the virus, was prepared from the Comp-Ang1-mcherry vector.

The viruses were prepared as follows.

In order to delete the VGF (Vaccinia Growth Factor) in the virus, the DNA of the vector is introduced by introducing the vector pYSYAng1 mCherry having the foreign gene Ang1 or Comp-Ang1 and the mCherry therebetween with the homologous sequence with both (left and right) When the homologous portion of the sequence between the sequence and the vaccinia virus is exchanged, the foreign gene is inserted into the region of VGF together to obtain VCSYAng1mCherry and vYSYCompAng1mcherry, in which VGF is deleted and foreign genes Ang1 and mCherry are expressed. For homologous recombination, BSC-1 cells were used as host cells. Upon reaching 80% confluence, 0.01MOI wild type vaccinia virus (Wyeth strain, ATCC) was infected into the cells for 1 hour and then the medium was removed And transformed with the above vector using Lipofectamine 2000 (Life Technologies). The transformed mixture was prepared by adding Lipofectamine 2000 to a serum-free medium with 1-5 μg of the insertion vector so as to have a final volume of 150 μL, and then transfected into the virus-infected cells so that homologous recombination with the insertion vector Let it happen. The resulting new virus expresses mCherry, so that plaque expressing mCherry in more than three steps can be purified to select only a single new virus plaque.

As a result, as shown in Fig. 3C, vYSYAng1mCherry (indicated by a light blue dotted box, vYSYAng1); And as shown in FIG. 4C, vYSYCompAngmCherry (red arrow) was confirmed.

Example  3. vYSYTRAILAng1  Production of viruses

Using the viruses prepared in Examples 1 and 2, the present inventors found that expression of LacZ, in which the Tk and VGF regions disappeared, and the expression of TRAIL, which is a cancer suicide gene, Of vancomycin virus vYSYTRAILAng1 expressing mcherry was prepared.

The vYSYTRAILAng1 virus was prepared as follows.

In order to delete the Tk and VGF (Vaccinia Growth Factor) in the virus, the vYSYTRAILLACZ virus, which has already deleted Tk and expresses TRAIL and LacZ, is used, and has homologous sequences with both sides of the VGF gene (left and right) In the meantime, when the homologous portion between the DNA sequence of the vector and the sequence of Vaccinia virus is exchanged by introducing the vector pYSYAng1mCherry having the foreign gene Ang1 and mCherry, the foreign gene is inserted into the region of VGF together to delete VGF, And vaccinia virus vYSYTRAILAng1 expressing mCherry together can be obtained. For homologous recombination, BSC-1 cells were used as a host. Upon reaching 80% confluence, vYSYTRAILLACZ of 0.01 MOI was infected to the cells for 1 hour, then the medium was removed and the cells were treated with Lipofectamine 2000 (Life Technologies) Transformed with the above vector. The transformed mixture was prepared by adding Lipofectamine 2000 to a serum-free medium with 1-5 μg of the insertion vector so as to have a final volume of 150 μL, and transforming the virus-infected cells into homologous recombination with the insertion vector Let it happen. Since the new viruses express mCherry with LacZ, they can be obtained by screening only a single new virus plaque by purifying plaques that simultaneously express mCherry with LacZ in more than three steps.

As a result, as shown in Fig. 3C, vYSYTRAILAng1 (red dotted box) was confirmed.

Example  4: Do it  Cancer cell death by virus

The present inventors have confirmed the death of cancer cells by the avian influenza virus.

As a control group, wild-type vaccinia virus (ATCC VR1536 or less, WT) was used. As a treatment group 1, JX594 and 496GFP in which TK was deleted in wild-type vaccinia (JX594 deletion of TK region of wild type vaccinia virus and insertion of GM- Vaccine virus double-deleted (VVDD) virus in which TK of wild-type vaccinia and VGF are deleted together as treatment group 2, and 496GFP is a virus in which the TK region of the SLJ496 virus is deleted and GFP is inserted) And the cancer cells were killed at various concentrations.

FIG. 5 shows that the virus (VVD) in which TK was deleted from wild-type vaccinia virus (WT) had a high killing ability in various cancer cells (colon cancer cells: HT29, DLD-1, SNU-5, HCT8, HCT116, Cell line: AGS, SNU638, Katoiii, liver cancer cell line: SNU354, SNU475). From this, it is shown that the deletion of TK increases cancer affinity. GFP is a fluorescent element inserted in the deleted region of TK in VVD.

FIG. 6 shows that the virus (VVDD +) in which the deletion of TK and the deletion of VGF occurred simultaneously or the virus (VVDD +) in which the functional gene is inserted into VVDD further enhances the assassination effect than VVD. Therefore, it is clear that the new virus proposed by the present inventors is superior in anticancer effect to the existing virus.

<110> Pusan National University Industry-University Cooperation Foundation <120> Novel Oncolytic Virus, Method for Preparing, and Uses thereof <130> PNUIUCF1-204p-1 <150> KR 10-2014-0135675 <151> 2014-10-08 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 8085 <212> DNA <213> Artificial Sequence <220> <223> pYSYTRAIL-LacZ <400> 1 ctgaacggtc tggttatagg tacattgagc aactgactga aatgcctcaa aatgttcttt 60 acgatgccat tgggatatat caacggtggt atatccagtg atttttttct ccattttagc 120 ttccttagct cctgaaaatc tcgataactc aaaaaatacg cccggtagtg atcttatttc 180 attatggtga aagttggaac ctcttacgtg ccgatcaacg tctcattttc gccaaaagtt 240 ggcccagggc ttcccggtat caacagggac accaggattt atttattctg cgaagtgatc 300 ttccgtcaca ggtatttatt cgaagacgaa agggcctcgt gatacgccta tttttatagg 360 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 420 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 480 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 540 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 600 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 660 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 720 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc 780 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 840 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 900 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 960 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 1020 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa 1080 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 1140 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 1200 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 1260 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 1320 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 1380 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 1440 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 1500 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 1560 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 1620 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 1680 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 1740 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 1800 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 1860 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 1920 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 1980 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 2040 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 2100 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 2160 cccgagtcag tctcatgttc tcaccggtat aaatacttaa taatctcatt tcagctgaat 2220 atgaaggagc aaaaggttgt aacattttat taccgtgtgg gatataaaag tccttgatcc 2280 attgatctgg aaacgggcat ctccatttaa gactagacgc cacggggttt aaaatactaa 2340 tcatgacatt ttgtagagcg taattactta gtaaatccgc cgtactaggt tcatttcctc 2400 ctcgtttgga tctcacatca gaaattaaaa taatcttaga aggatgcagt tgttttttga 2460 tggatcgtag atattcctca tcaacgaacc gagtcactag agtcacatca cgcaatccat 2520 ttaaaatagg atcatgatgg cggccgtcaa ttagcatcca tttgatgatc actcctaaat 2580 tatagaaatg atctctcaaa taacgatatat gtgtaccggg agcagatcct atatacacta 2640 cggtggcacc atctaatata ccgtgtcgct gtaacttact aagaaaaaat aattctccta 2700 gtaatagttt taactgtcct tgatacggca gtttttttgc gacctcattt gcactttctg 2760 gttcgtaatc taactcatta tcaatttcct caaaatacat aaacggttta tctaacgaca 2820 caacatccat ttttaagtat tatattaaaa tttaatcaat gtttattttt agttttttag 2880 ataaaaaata taatattatg agtcgatgta acactttcta cacaccgatt gatacatatc 2940 attacctcct attatttcta tctcggtttc ctcacccaat cgtttagaaa aggaagcctc 3000 cttaaagcat ttcatacaca cagcagttag ttttaccacc atttcagata atggaataag 3060 attcaaaata ttattaaacg gtttacgttg aaatgtccca tcgagtgcgg ctactataac 3120 tatttttcct tcgtttgcca tacgctcaca gaattggatc ccccctgccc ggttattatt 3180 atttttgaca ccagaccaac tggtaatggt agcgaccggc gctcagctgg aattccgccg 3240 atactgacgg gctccaggag tcgtcgccac caatccccat atggaaaccg tcgatattca 3300 gccatgtgcc ttcttccgcg tgcagcagat ggcgatggct ggtttccatc agttgctgtt 3360 gactgtagcg gctgatgttg aactggaagt cgccgcgcca ctggtgtggg ccataattca 3420 attcgcgcgt cccgcagcgc agaccgtttt cgctcgggaa gacgtacggg gtatacatgt 3480 ctgacaatgg cagatcccag cggtcaaaac aggcggcagt aaggcggtcg ggatagtttt 3540 cttgcggccc taatccgagc cagtttaccc gctctgctac ctgcgccagc tggcagttca 3600 ggccaatccg cgccggatgc ggtgtatcgc tcgccacttc aacatcaacg gtaatcgcca 3660 tttgaccact accatcaatc cggtaggttt tccggctgat aaataaggtt ttcccctgat 3720 gctgccacgc gtgagcggtc gtaatcagca ccgcatcagc aagtgtatct gccgtgcact 3780 gcaacaacgc tgcttcggcc tggtaatggc ccgccgcctt ccagcgttcg acccaggcgt 3840 tagggtcaat gcgggtcgct tcacttacgc caatgtcgtt atccagcggt gcacgggtga 3900 actgatcgcg cagcggcgtc agcagttgtt ttttatcgcc aatccacatc tgtgaaagaa 3960 agcctgactg gcggttaaat tgccaacgct tattacccag ctcgatgcaa aaatccattt 4020 cgctggtggt cagatgcggg atggcgtggg acgcggcggg gagcgtcaca ctgaggtttt 4080 ccgccagacg ccactgctgc caggcgctga tgtgcccggc ttctgaccat gcggtcgcgt 4140 tcggttgcac tacgcgtact gtgagccaga gttgcccggc gctctccggc tgcggtagtt 4200 caggcagttc aatcaactgt ttaccttgtg gagcgacatc cagaggcact tcaccgcttg 4260 ccagcggctt accatccagc gccaccatcc agtgcaggag ctcgttatcg ctatgacgga 4320 acaggtattc gctggtcact tcgatggttt gcccggataa acggaactgg aaaaactgct 4380 gctggtgttt tgcttccgtc agcgctggat gcggcgtgcg gtcggcaaag accagaccgt 4440 tcatacagaa ctggcgatcg ttcggcgtat cgccaaaatc accgccgtaa gccgaccacg 4500 ggttgccgtt ttcatcatat ttaatcagcg actgatccac ccagtcccag acgaagccgc 4560 cctgtaaacg gggatactga cgaaacgcct gccagtattt agcgaaaccg ccaagactgt 4620 tacccatcgc gtgggcgtat tcgcaaagga tcagcgggcg cgtctctcca ggtagcgaaa 4680 gccatttttt gatggaccat ttcggcacag ccgggaaggg ctggtcttca tccacgcgcg 4740 cgtacatcgg gcaaataata tcggtggccg tggtgtcggc tccgccgcct tcatactgca 4800 ccgggcggga aggatcgaca gatttgatcc agcgatacag cgcgtcgtga ttagcgccgt 4860 ggcctgattc attccccagc gaccagatga tcacactcgg gtgattacga tcgcgctgca 4920 ccattcgcgt tacgcgttcg ctcatcgccg gtagccagcg cggatcatcg gtcagacgat 4980 tcattggcac catgccgtgg gtttcaatat tggcttcatc caccacatac aggccgtagc 5040 ggtcgcacag cgtgtaccac agcggatggt tcggataatg cgaacagcgc acggcgttaa 5100 agttgttctg cttcatcagc aggatatcct gcaccatcgt ctgctcatcc atgacctgac 5160 catgcagagg atgatgctcg tgacggttaa cgcctcgaat cagcaacggc ttgccgttca 5220 gcagcagcag accattttca atccgcacct cgcggaaacc gacatcgcag gcttctgctt 5280 caatcagcgt gccgtcggcg gtgtgcagtt caaccaccgc acgatagaga ttcgggattt 5340 cggcgctcca cagtttcggg ttttcgacgt tcagacgtag tgtgacgcga tcggcataac 5400 cccacgctc atcgataatt tcaccgccga aaggcgcggt gccgctggcg acctgcgttt 5460 caccctgcca taaagaaact gttacccgta ggtagtcacg caactcgccg cacatctgaa 5520 cttcagcctc cagtacagcg cggctgaaat catcattaaa gcgagtggca acatggaaat 5580 cgctgatttg tgtagtcggt ttatgcagca acgagacgtc acggaaaatg ccgctcatcc 5640 gccacatatc ctgatcttcc agataactgc cgtcactcca gcgcagcacc atcaccgcga 5700 ggcggttttc tccggcgcgt aaaaatgcgc tcaggtcaaa ttcagacggc aaacgactgt 5760 cctggccgta accgacccag cgcccgttgc accacagatg aaacgccgag ttaacgccat 5820 caaaaataat tcgcgtctgg ccttcctgta gccagctttc atcaacatta aatgtgagcg 5880 agtaacaacc cgtcggattc tccgtgggaa caaacggcgg attgaccgta atgggatagg 5940 tyacgttggt gtagatgggc gcatcgtaac cgtgcatctg ccagtttgag gggacgacga 6000 cagtatcggc ctcaggaaga tcgcactcca gccagctttc cggcaccgct tctggtgccg 6060 gaaaccaggc aaagcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg 6120 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa 6180 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggga tctcccatgc 6240 tcgagttatg atctacttcc ttaccgtgca ataaattaga atatattttc tacttttacg 6300 agaaattaat tattgtattt attatttatg ggtgaaaaac ttactataaa aagcgggtgg 6360 gtttggaatt agtgaaagct taaaaattga aattttattt tttttttttg gaatataaat 6420 aagctcgaag tcgacatggc tatgatggag gtccaggggg gacccagcct gggacagacc 6480 tgcgtgctga tcgtgatctt cacagtgctc ctgcagtctc tctgtgtggc tgtaacttac 6540 gtgtacttta ccaacgagct gaagcagatg caggacaagt actccaaaag tggcattgct 6600 tgtttcttaa aagaagatga cagttattgg gaccccaatg acgaagagag tatgaacagc 6660 ccctgctggc aagtcaagtg gcaactccgt cagctcgtta gaaagatgat tttgagaacc 6720 tctgaggaaa ccatttctac agttcaagaa aagcaacaaa atatttctcc cctagtgaga 6780 gaaagaggtc ctcagagagt agcagctcac ataactggga ccagaggaag aagcaacaca 6840 ttgtcttctc caaactccaa gaatgaaaag gctctgggcc gcaaaataaa ctcctgggaa 6900 tcatcaagga gtgggcattc attcctgagc aacttgcact tgaggaatgg tgaactggtc 6960 atccatgaaa aagggtttta ctacatctat tcccaaacat actttcgatt tcaggaggaa 7020 ataaaagaaa acacaaagaa cgacaaacaa atggtccaat atatttacaa atacacaagt 7080 tatcctgacc ctatattgtt gatgaaaagt gctagaaata gttgttggtc taaagatgca 7140 gaatatggac tctattccat ctatcaaggg ggaatatttg agcttaagga aaatgacaga 7200 atttttgttt ctgtaacaaa tgagcacttg atagacatgg accatgaagc cagttttttc 7260 ggggcctttt tagttggcta acccgggtaa ttaattaatt tttatcgatc taattcaaca 7320 atgtctggaa agaactgtcc ttcatcgata cctatcacgg agaaatctgt aattgattcc 7380 aagacatcac atagtttagt tgcttccaat gcttcaaaat tattcttatc atgcgtccat 7440 agtcccgttc cgtatctatt atcgttagaa tattttatag tcacgcattt atattgagct 7500 atttgataac gtctaactcg tctaattaat tctgtacttt tacctgaaaa catggggccg 7560 attatcaact gaatatgtcc gccgttcatg atgacaataa agaattaatt attgttcact 7620 ttattcgact ttaatatatc catcacgtta gaaaatgcga tattgcgacg aggatctatg 7680 tatctaacag gatctattgc ggtggtagct agagaggatt cttttttgaa tcgcatcaaa 7740 ctaatcacaa agtcgaacaa atatccttta ttaagtttga cccttccatc tgtaacaata 7800 gggaccttgt taaacagttt tttaaaatct tgaaagtctg tgaattttgt caattgtctg 7860 tattcctctg aaagagattc ataacaatga cccacggctt ctaatttatt ttttgattgg 7920 atcaacagaaa gtctagatat tgagtgattt gcaatatatc agataatgaa 7980 gatttcatca tcttgactag ccaaatactt aaaaaatgaa tcatcatctg cgaagaacat 8040 cgttaagaga tactggttgt gatccattta ttgatcgcaa aagct 8085 <210> 2 <211> 6018 <212> DNA <213> Artificial Sequence <220> <223> pYSY-Ang1-mCherry <400> 2 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600 acttacttct gacaaggatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900 ggccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1380 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctta acggggggtt 1560 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2220 accatgatta cgccaagctt gggctgcagg tcgatcttgt tatcgattga tagtggtgtc 2280 ctttttcatc cttgctatca aagttacgca tgccgtggtg taacaatatc tttaatacag 2340 atggattaaa tcgtgtattc atcgtatagc aatgtaatgg agagttacct cgtttattca 2400 gatcgcagtg tttaataact agcttaaaca gatgagacga tgtatccaca tcaaagaacg 2460 tgaaatacat atgacagaca ttgttgacag aaacgtgacc ttcattctta ccgtcgtcca 2520 taaatacgtt aggtatgtac cacatactgt cgcgaacgat gcgtacaatc tcgtccatct 2580 cataatgatt tactttttca taattaaaga tgtgaaagaa aaacagaaca atatattttt 2640 ttagtaatgt ttatgcgaga catataaaat aaactccgtg tttatgatca tttttaacag 2700 caacacattc aatattgtat tgttattttt atattattta cacaattaac aatatattat 2760 tagtttatat tactgaatta ataatataaa attcccaatc ttgtcataaa cacacactga 2820 gaaacagcat aaacacaaaa tccatcaaaa atgtcgatga aatatctgat gttgttgttc 2880 gctgctatgc agatctgcag atctgttgga catgtttgcg gatccgcaaa catgtccaac 2940 tagaagcgat gctacgctag tcacaatcac cactttcata tttagaatat atgtatgtaa 3000 aaatatagta gaatttcatt ttgttttttt ctatgctata agaattcctc gagatgcttg 3060 tcaatctttg cactaaagaa ggtgttttac taaagggagg aaaaagagag gaagagaaac 3120 catttagaga ctgtgcagat gtatatcaag ctggttttaa taaaagtgga atctacacta 3180 tttatattaa taatatgcca gaacccaaaa aggtgttttg caatatggat gtcaatgggg 3240 gaggttggac tgtaatacaa catcgtgaag atggaagtct agatttccaa agaggctgga 3300 aggaatataa aatgggtttt ggaaatccct ccggtgaata ttggctgggg aatgagttta 3360 tttttgccat taccagtcag aggcagtaca tgctaagaat tgagttaatg gactgggaag 3420 ggaaccgagc ctattcacag tatgacagat tccacatagg aaatgaaaag caaaactata 3480 ggttgtattt aaaaggtcac actgggacag caggaaaaca gagcagcctg atcttacacg 3540 gtgctgattt cagcactaaa gatgctgata atgacaactg tatgtgcaaa tgtgccctca 3600 tgttaacagg aggatggtgg tttgatgctt gtggcccctc caatctaaac ggaatgttct 3660 atactgcggg gcaaaaccat ggaaaactga atgggataaa gtggcactac ttcaaagggc 3720 ccagttactc cttacgttcc acaactatga tgattcgacc tttagatttt tgagcggccg 3780 cggatcccgc ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata 3840 aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt 3900 gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct 3960 cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc 4020 ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga 4080 caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc 4140 ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctc cctcaagcgt 4200 attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg 4260 gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaacgtct aggccccccg 4320 aaccacgggg acgtggtttt cctttgaaaa acacgatgat aatatggtga gcaagggcga 4380 ggaggataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc 4440 cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac 4500 ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct 4560 gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga 4620 ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga 4680 cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa 4740 ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat 4800 gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat 4860 caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta 4920 caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat 4980 cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca 5040 ctccaccggc ggcatggacg agctgtacaa gtgaataata ataaccgggc aggggggatc 5100 ccccagatct gtaaagtgta tgtcttatgt atatttataa aaatgctaag tatgcgatgt 5160 atctatgtta tttgtattta tctaaacaat acctctacct ctagatatta tacaaaaatt 5220 ttttatttcg gcatattaaa gtaaaatcta gttaccttga aaatgaatac agtgggtggt 5280 tccgtatcac cagtaagaac ataatagtcg aatacagtat ccgattgaga ttttgcatac 5340 aatactagtc tagaaagaaa tttgtaatca tcttctgtga cgggagtcca tatatctgta 5400 tcatcgtcta gtttatcagt gtcccatgct atattcctgt tatcatcatt agttaatgaa 5460 aataactctc gtgattcaga aaagtcaaat attgtatcca tacatacatc tccaaaacta 5520 tcgcttatac gtttatcttt aacgatacct atacctagat ggttatttac taacagacat 5580 tttccagatc gactctagag gatccccggg cgagctcgaa ttcactggcc gtcgttttac 5640 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc 5700 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc 5760 gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta 5820 tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc 5880 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 5940 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 6000 catcaccgaa acgcgcga 6018 <210> 3 <211> 6237 <212> DNA <213> Artificial Sequence <220> <223> pYSY-Comp-Ang1-mCherry <400> 3 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600 acttacttct gacaaggatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900 ggccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1380 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctta acggggggtt 1560 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2220 accatgatta cgccaagctt gggctgcagg tcgatcttgt tatcgattga tagtggtgtc 2280 ctttttcatc cttgctatca aagttacgca tgccgtggtg taacaatatc tttaatacag 2340 atggattaaa tcgtgtattc atcgtatagc aatgtaatgg agagttacct cgtttattca 2400 gatcgcagtg tttaataact agcttaaaca gatgagacga tgtatccaca tcaaagaacg 2460 tgaaatacat atgacagaca ttgttgacag aaacgtgacc ttcattctta ccgtcgtcca 2520 taaatacgtt aggtatgtac cacatactgt cgcgaacgat gcgtacaatc tcgtccatct 2580 cataatgatt tactttttca taattaaaga tgtgaaagaa aaacagaaca atatattttt 2640 ttagtaatgt ttatgcgaga catataaaat aaactccgtg tttatgatca tttttaacag 2700 caacacattc aatattgtat tgttattttt atattattta cacaattaac aatatattat 2760 tagtttatat tactgaatta ataatataaa attcccaatc ttgtcataaa cacacactga 2820 gaaacagcat aaacacaaaa tccatcaaaa atgtcgatga aatatctgat gttgttgttc 2880 gctgctatgc agatctgcag atctgttgga catgtttgcg gatccgcaaa catgtccaac 2940 tagaagcgat gctacgctag tcacaatcac cactttcata tttagaatat atgtatgtaa 3000 aaatatagta gaatttcatt ttgttttttt ctatgctata agaattcctc gagatgaaga 3060 cgatcatcgc cctgagctac atcttctgcc tggtattcgc cgactacaag gacgatgatg 3120 acaaggggat cttagacctg ggcccgcaga tgcttcggga actgcaggaa accaacgcgg 3180 cgctgcagga cgtgcgggac ttgctgcggc agcaggtcag ggagatcacg ttcctgaaaa 3240 acacggtgat ggagtgtgac gcgtgcgggg gatcccttgt caatctttgc actaaagaag 3300 gtgttttact aaagggagga aaaagagagg aagagaaacc atttagagac tgtgcagatg 3360 tatatcaagc tggttttaat aaaagtggaa tctacactat ttatattaat aatatgccag 3420 aacccaaaaa ggtgttttgc aatatggatg tcaatggggg aggttggact gtaatacaac 3480 atcgtgaaga tggaagtcta gatttccaaa gaggctggaa ggaatataaa atgggttttg 3540 gaaatccctc cggtgaatat tggctgggga atgagtttat ttttgccatt accagtcaga 3600 ggcagtacat gctaagaatt gagttaatgg actgggaagg gaaccgagcc tattcacagt 3660 atgacagatt ccacatagga aatgaaaagc aaaactatag gttgtattta aaaggtcaca 3720 ctgggacagc aggaaaacag agcagcctga tcttacacgg tgctgatttc agcactaaag 3780 atgctgataa tgacaactgt atgtgcaaat gtgccctcat gttaacagga ggatggtggt 3840 ttgatgcttg tggcccctcc aatctaaacg gaatgttcta tactgcgggg caaaaccatg 3900 gaaaactgaa tgggataaag tggcactact tcaaagggcc cagttactcc ttacgttcca 3960 caactatgat gattcgacct ttagattttt gagcggccgc ggatcccgcc cctctccctc 4020 ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta 4080 tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc 4140 tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct 4200 gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt 4260 agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa 4320 gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg 4380 gatagttgtg gaaagagtca aatggctcac ctcaagcgta ttcaacaagg ggctgaagga 4440 tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac 4500 atgtgtttag tcgaggttaa aaaacgtcta ggccccccga accacgggga cgtggttttc 4560 ctttgaaaaa cacgatgata atatggtgag caagggcgag gaggataaca tggccatcat 4620 caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga 4680 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt 4740 gccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg 4800 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc 4860 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac 4920 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa 4980 cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga 5040 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa 5100 ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca 5160 gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta 5220 caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga 5280 gctgtacaag tgaataataa taaccgggca ggggggatcc cccagatctg taaagtgtat 5340 gtcttatgta tatttataaa aatgctaagt atgcgatgta tctatgttat ttgtatttat 5400 ctaaacaata cctctacctc tagatattat acaaaaattt tttatttcgg catattaaag 5460 taaaatctag ttaccttgaa aatgaataca gtgggtggtt ccgtatcacc agtaagaaca 5520 taatagtcga atacagtatc cgattgagat tttgcataca atactagtct agaaagaaat 5580 ttgtaatcat cttctgtgac gggagtccat atatctgtat catcgtctag tttatcagtg 5640 tcccatgcta tattcctgtt atcatcatta gttaatgaaa ataactctcg tgattcagaa 5700 aagtcaaata ttgtatccat acatacatct ccaaaactat cgcttatacg tttatcttta 5760 acgataccta tacctagatg gttatttact aacagacatt ttccagatcg actctagagg 5820 atccccgggc gagctcgaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc 5880 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 5940 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc 6000 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca 6060 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac 6120 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 6180 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcga 6237

Claims (10)

As oncolytic viruses,
The cancer-
(TK) gene and VGF (Vaccinia Growth Factor) gene are deleted, and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) gene and Ang1 (Angiopoietin 1) is introduced. delete delete delete delete delete delete The virus according to claim 1, wherein the cancer-solving virus is vaccinia virus. A pharmaceutical composition for preventing and treating cancer comprising the cancer-solving virus of claim 1 as an active ingredient. delete

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