CN112725290A - Recombinant oncolytic virus and preparation method and application thereof - Google Patents

Abstract

The present invention relates to recombinant oncolytic viruses for the treatment and prevention of cancer, methods for their production and uses thereof. The micro RNA gene inhibiting the abnormal expression of the C-MYC gene or being inhibited by the abnormal expression of the C-MYC gene is inserted into a virus genome to carry out virus replication, tumor inhibiting factors such as miR-34a, let-7 and the like are generated by coding, the expression quantity of the let-7 is increased to inhibit the expression of C-MYC protein, the expression quantity of the C-MYC protein is reduced, the inhibition on the expression quantity of miR-34a is reduced, the expression quantity of miR-34a is increased, and the recombinant oncolytic virus also expresses miR-34a, so that the anticancer and cancer-preventing efficiency of the recombinant oncolytic virus is greatly improved.

Description

Recombinant oncolytic virus and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a recombinant oncolytic virus, a foreign gene expressing a specific anti-cancer factor of the recombinant oncolytic virus, an oncolytic virus, a kit and application of the kit.

Background

The protooncoprotein C-MYC plays an important role in cell proliferation and differentiation, cell cycle, metabolism and apoptosis. As a transcription factor protein, C-MYC directly combines with a CACGTG sequence on DNA by forming a heterodimer with MAX protein, thereby playing a wide transcription regulation role. Under normal physiological conditions, the expression of C-MYC is strictly regulated and increased when stimulated by extracellular stimuli such as growth factors. When chromosomal translocations or signal pathway gene mutations occur, C-MYC undergoes growth factor-independent stimulation of amplification, resulting in uncontrolled cell proliferation and tumor production. C-MYC is uncontrolled and aberrantly expressed in about 70% of human tumors. By inhibiting the over-expression of C-MYC, the processes of tumor generation, proliferation and the like can be inhibited. C-MYC has become a tumor therapy target of interest to academia (see International J.Oncology, vol.4, 4, 4.2017, 278 and 281).

Oncolytic virus therapy, a novel biological immunotherapy, can exert an anti-tumor effect through direct oncolytic action and by inducing immune activity in the body. Then, with the proposal of a "Cancer Targeting Gene Viral Therapy (CTGVT)" strategy (i.e., inserting an anti-cancer gene into an oncolytic virus vector, organically combining viral therapy with gene therapy to become a gene-virus therapy means with a strong killing effect), oncolytic viruses loaded with targeting genes or cytokines have been greatly successful in tumor targeting therapy. Oncolytic Vaccinia Virus (oncolytica Virus) is a novel vector for viral therapy with the advantages: good virus stability, low pathogenicity, high gene transfection efficiency and good safety. Oncolytic vaccinia virus selectively infects tumor cells, replicates in cells, kills tumor cells, and has little toxicity to normal tissues and cells.

Micro RNA (micro RNA, mi RNA) is a single-stranded non-coding RNA with the total length of about 20-22 nt, not only regulates gene expression at the level after transcription, but also plays an important role in the interaction of transcription level regulation and signal transduction pathways (cross-talk).

Therefore, the research on the abnormal expression of the proto-oncoprotein C-MYC and the action mechanism of micro RNA in the abnormal expression is a hotspot and difficulty of the current research on the application of the proto-oncoprotein C-MYC in the treatment of tumors or cancers.

Disclosure of Invention

The present inventors have found that the prior art methods of treating cancer are less effective. Therefore, the technical problem to be solved by the present invention is to provide a novel method for treating cancer or tumor. Specifically, the method comprises the step of administering to a subject a recombinant oncolytic virus inserted with an exogenous gene sequence encoding a factor inhibiting the abnormal expression product of the C-MYC gene or/and being inhibited by the expression product of the C-MYC gene. In another aspect, the present invention provides the use of the above recombinant oncolytic virus for the preparation of a pharmaceutical composition for the treatment of cancer or/and tumors.

More preferably, the exogenous gene is a microRNA-34a gene, a let7 gene or a gene formed by connecting microRNA-34a and let 7.

The invention is realized by the following technical scheme:

the recombinant oncolytic virus is operable to insert a foreign gene sequence encoding a factor that inhibits abnormal expression products of the C-MYC gene or/and is inhibited by expression products of the C-MYC gene.

The basic action mechanism of the C-MYC protooncoprotein is clear, and the inhibition of the expression of the C-MYC protooncoprotein can inhibit the pathogenesis of the corresponding cancer, so that the cancer or the tumor can be prevented and treated from a molecular and genetic level. The abnormal expression of the C-MYC proto-oncoprotein can inhibit the expression of some anti-cancer factors, and the pathogenesis of the corresponding cancer or tumor is indirectly pre-paid and inhibited from the molecular level and the gene level by improving the expression of the anti-cancer factors inhibited by the expression product of the C-MYC gene.

Preferably, the foreign gene sequence is inserted into the TK gene. The TK gene (TK) is a Thymidine kinase gene, and participates in DNA synthesis during cell proliferation, and when oncolytic virus replicates, a high-concentration nucleotide pool can be formed under the action of TK to ensure that progeny DNA replicates smoothly. In normal cells, the concentration of nucleotides is low, so TK is essential for normal cell proliferation; tumor cells have a higher concentration of nucleotides, and thus TK is not essential for tumor cell proliferation. From the above, it is known that oncolytic viruses are able to replicate preferentially in tumor cells, which are supplied with sufficient nucleotides, but not in normal cells, and that deletion of the TK gene may enhance the properties of oncolytic viruses to replicate within tumor cells. Exogenous gene segments are inserted into a TK gene sequence by using a gene recombination technology, so that an oncolytic virus vector is deleted for the TK sequence, and then an oncolytic vaccinia virus carrying microRNA is further constructed, so that the damage of the recombinant oncolytic virus to normal cells is reduced, the specific recognition of tumor cells is improved, the curative effect rate is improved, and the side effect is reduced.

Preferably, the factor inhibiting the abnormal expression product of the C-MYC gene is microRNA. Human microRNAs account for about 1-4% of total genes, regulate about one third of protein coding genes of human, each microRNA can regulate a plurality of target genes, and several microRNAs can also regulate the same gene, and the microRNAs have the characteristics of simple structure and fine regulation, thereby being more convenient for the precise treatment of cancers and tumors.

Preferably, the exogenous gene sequence comprises one or more of: miR-34a and let-7; the sequence of the miR-34a is shown as SEQ ID NO.1, and the sequence of the let-7 is shown as SEQ ID NO. 2. miR-34a is considered to be a tumor suppressor and can play an anti-tumor biological function by targeting bcl-2, notch1, sirt-1, foxp1 and the like. let-7 is a tumor suppressor family of tumor or cancer related microRNAs, and C-MYC is a target of let-7, and the expression of the C-MYC can be suppressed by let-7.

Preferably, the exogenous gene sequence comprises miR-34a and let-7, and the miR-34a and let-7 gene sequences are connected through an IRES sequence. The exogenous gene sequence simultaneously comprises miR-34a and let-7. The high expression of let-7 inhibits the abnormal expression of the C-MYC gene, thereby inhibiting and preventing tumors and cancers caused by the abnormal expression of the C-MYC gene. The expression of the C-MYC protein is reduced, the expression of miR-34a tumor inhibition factors is reduced, the miR-34a is expressed by the recombinant oncolytic virus, and the miR-34a quantity is increased to play a role in inhibiting tumors and cells.

IRES (internal ribosome entry site) sequences were found in the RNA genomes of Poliovirus (PV) and encephalomyocarditis virus (EMCV) in 1988 by the Nahum Sonenberg and Eckard Wimmer laboratories, respectively. IRES are typically located in the 5 'UTR of RNA viruses, allowing RNA translation in a 5' cap-independent manner. The IRES is used for connecting two genes, the IRES and the two genes are transcribed into the same mRNA under the control of an upstream promoter, the translation initiation of the IRES upstream gene follows the cap-independent mode of eukaryotic genes during translation, and the downstream gene recruits ribosome through the IRES to enter the translation initiation gene, so that the two genes connected by the IRES sequence share the same transcription unit for expression.

The two genes connected by the IRES sequence are micro RNA genes, and experimental data of subsequent embodiments prove that the two micro RNAs are expressed, so that a foundation is laid for connecting a plurality of micro RNAs in an oncolytic virus expression vector. On the basis, the recombinant oncolytic virus constructed by inserting the microRNAs with multiple tumor inhibition effects provides an active research direction for targeted therapy of cancers or tumors.

Preferably, the IRES sequence is shown as SEQ ID NO. 3.

Preferably, the exogenous gene encoding miR-34a can be a polynucleotide sequence which hybridizes with the nucleotide sequence of miR-34a under stringent conditions and encodes miR-34a, a polynucleotide sequence with 60% and more identity of the polynucleotide sequence, or a complementary sequence of the polynucleotide sequence;

the exogenous gene encoding let-7 can be a polynucleotide sequence which hybridizes with a nucleotide sequence of let-7 under stringent conditions and encodes let-7, a polynucleotide sequence which is 60% or more identical to the polynucleotide sequence of let-7, or a complementary sequence thereof.

The "stringent conditions" as used herein may be any of low stringency conditions, medium stringency conditions or high stringency conditions, and preferably high stringency conditions. Illustratively, "low stringency conditions" can be conditions of 30 ℃, 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 52% formamide; "Medium stringency conditions" can be 40 ℃, 5 XSSC, 5 XDenhardt solution, 0.5% SDS, 52% formamide; the "high stringency conditions" can be 50 ℃ in 5 XSSC, 5 XDenhardt's solution, 0.5% SDS, 52% formamide. It will be appreciated by those skilled in the art that higher temperatures will result in polynucleotides with high homology. In addition, one skilled in the art can select the temperature, probe concentration, probe length, ionic strength, time, salt concentration, etc., which affect the stringency of hybridization to form a composite result to achieve the corresponding stringency.

The polynucleotides that can hybridize to each other may be those that have a sequence of about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or the like, as calculated by homology search software such as FASTA, BLAST, and the like using default parameters set by the system, 99% or more, 99.1 or more, 99.2 or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more, identical. The identity of nucleotide sequences can be determined using the algorithm rules BLAST of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-. The programs BLASTN, BLASTX based on the rules of the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215:403,1990). When BLASTN is used to analyze a nucleotide sequence, the parameters are set to score 100 and wordlength 12, for example.

Preferably, the recombinant oncolytic virus comprises a selectively replicating recombinant oncolytic virus.

Preferably, the selectively replicating recombinant oncolytic virus is derived from an adenovirus, a poxvirus, a herpes simplex virus, a measles virus, a semliki forest virus, a vesicular stomatitis virus, a poliovirus, a retrovirus, a reovirus, a seneca valley virus, an enterovirus of the eke type, a coxsackievirus, a newcastle disease virus and a malaba virus having an oncolytic effect.

Preferably, all of the recombinant oncolytic viruses described above can be formulated to contain a therapeutically effective amount of a therapeutic agent.

Preferably, all of the above recombinant oncolytic viruses are formulated for administration by intratumoral injection, intraperitoneal administration, subarachnoid intracavity administration or intravenous administration.

Preferably, the above-mentioned therapeutic agent further comprises a pharmaceutically acceptable carrier, and other active ingredients for treating cancer. For example, formulations suitable for injection or infusion include aqueous and non-aqueous sterile injection solutions, which may optionally contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

The active ingredients of the invention may optionally be combined with solid excipients and, if desired, after addition of suitable auxiliaries, the mixture of granules is processed to give the desired dosage form. Suitable excipients are in particular fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose or starch preparations, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate.

Preferably, the recombinant oncolytic virus of the invention further comprises other active ingredients for treating gastric cancer, colon cancer and pancreatic cancer, such as paclitaxel.

Preferably, the recombinant oncolytic virus or the therapeutic agent comprising the recombinant oncolytic virus can be used for preventing or treating tumors or/and cancers.

An effective amount of an active ingredient of the invention can be any amount that treats a tumor or cancer, and determination of an effective amount is within the ability of those skilled in the art, particularly in light of the disclosure provided herein.

According to the present invention, the pharmaceutical product (drug, medicament) or pharmaceutical composition of the present invention may be administered to a subject in any effective number of doses. Preferably, the pharmaceutical product (drug, medicament) or pharmaceutical composition of the invention may be administered in multiple doses, for example from about 2 to about 15 doses, more preferably from about 4-10 doses, most preferably about 6 doses. In a particularly preferred embodiment, the pharmaceutical product (drug, medicament) or pharmaceutical composition of the invention is administered to the subject during the course of administration, e.g. injection, infusion or oral administration, at a frequency of about once every three weeks. In a particularly preferred embodiment, the administration is by injection to the tumor-bearing site.

It will be appreciated that the pharmaceutical product (drug, medicament) or pharmaceutical composition of the invention may be formulated in any suitable manner for administration by any suitable route.

Dosage units of the pharmaceutical products (drugs, medicaments) or pharmaceutical compositions of the invention are administered to a subject on a regular basis. For example, a dosage unit may be administered more than once daily, once weekly, once monthly, etc. Dosage units may be administered on a twice/week basis, i.e., twice weekly, e.g., once every three days.

Preferably, wherein the tumor and/or cancer comprises: breast cancer, head and neck tumor, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactin tumor, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, paget's disease, cervical cancer, gall bladder cancer, eye cancer, kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multitip myeloma, ovarian cancer, pancreatic endocrine tumor, glucagon tumor, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, hematological cancer, peritoneal cancer and pleural cancer.

Preferably, wherein the tumor and/or cancer comprises: lymphoma.

More particularly diffuse large B-cell lymphoma.

Lymphomas are a group of hematological malignancies originating from lymphocytes, and are mainly classified into two types, non-Hodgkin lymphoma (NHL, 90%) and Hodgkin lymphoma (HL, 10%). Among them, diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma (NHL). First-line treatment regimens (R-CHOP) are currently used to treat patients <60 years of age with a recurrence-free survival rate of 74.3% in 6 years; patients aged >60 years have poor long-term survival, with a progression-free survival rate of only 36.5% over 10 years. MYC gene abnormal expression (MYC gene amplification/rearrangement/high expression) accounts for about 30% of Diffuse Large B Cell Lymphoma (DLBCL), and the abnormal high expression of the gene is a main reason for treatment difficulty and relapse of DLBCL, so the MYC signaling pathway is an important target for treating DLBCL.

Expression of mir-34a and let7 is down-regulated in DLBCL, interacting with the MYC pathway, and emphasizing aberrant expression of MYC, leading to poor prognosis. Therefore, the method for improving the expression of the two microRNAs in the DLBCL cell so as to inhibit the abnormal high expression of the MYC gene is a novel method for treating DLCBL.

Preferably, the kit comprising the recombinant oncolytic virus or therapeutic agent described above comprises a container, and the contained container can be 1 or more sets. The 1-set container can contain a recombinant oncolytic virus or therapeutic agent comprising mir-34a, or can contain a recombinant oncolytic virus or therapeutic agent comprising let7, or can contain a recombinant oncolytic virus or therapeutic agent comprising mir-34a and let7 linked together. Combinations of any 1-6 of the above 6 viruses or therapeutic agents may also be incorporated to facilitate administration. The virus or therapeutic agent in each container of the plurality of sets may be single or mixed to facilitate administration.

The instructions contained in relation to the pharmaceutical product may contain the following: indications (e.g., lymphoma), dosages administered (e.g., as exemplified above), and possible side effects, among others.

Preferably, the preparation of the desired recombinant oncolytic virus comprises the following steps:

1) synthesizing an exogenous gene sequence by a gene synthesis method;

2) carrying out double enzyme digestion on the exogenous gene sequence and the PCB original plasmid by BgLII and EcoRI endonucleases respectively to obtain enzyme digestion products BgLII-exogenous gene sequence-EcoRI and TKL-pCB-TKR linearized plasmids;

3) DNA ligase is used for connecting the plasmids linearized by the BgLII-exogenous gene sequence-EcoRI and TKL-pCB-TKR to obtain a PCB-exogenous gene sequence plasmid;

4) screening and cloning of the PCB-foreign Gene sequence plasmid: transforming E.coli strain DH5 alpha competence with the PCB-exogenous gene sequence plasmid, and screening escherichia coli resistance genes to obtain a PCB-exogenous gene sequence positive clone;

5) infecting human body cell with oncolytic virus, transfecting PCB-exogenous gene sequence plasmid, and screening resistance gene carried by recombinant oncolytic virus to obtain recombinant oncolytic virus inserted with exogenous gene sequence.

Definition of

The terms "tumor," "cancer," "tumor cell," "cancer cell" encompass meanings commonly recognized in the art.

The term "oncolytic virus" refers to a virus that is capable of selectively replicating in and lysing tumor cells.

The term "therapeutically effective amount" refers to an amount of a functional agent or pharmaceutical composition that is capable of exhibiting a detectable therapeutic or inhibitory effect, or that exerts an anti-tumor effect. The effect can be detected by any assay known in the art.

The term "administering" or "administering" refers to providing a compound, complex, or composition (including viruses and cells) to a subject.

The term "patient" refers to a human or non-human organism. Thus, the recombinant oncolytic viruses and therapeutics described herein are useful for human and veterinary disease. In some embodiments, the patient has a tumor. In some instances, the patient is concurrently suffering from one or more types of cancer.

The term "vector" refers to a polynucleotide construct designed to transduce/transfect one or more cell types. The vector may be, for example, a "cloning vector" designed for the isolation, propagation and replication of inserted nucleotides, an "expression vector" designed for the expression of nucleotide sequences in a host cell, or a "viral vector" designed to result in the production of recombinant viruses or virus-like particles, or a "shuttle vector" comprising the attributes of more than one type of vector.

The term "operably inserted" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.

The term "recombinant" refers to a virus that has been altered by genetic engineering, modification or manipulation of the genetic material of the virus so that it is different from a naturally occurring virus or naturally occurring variant of a virus.

The design principle of the invention is as follows: miR-34a in microRNA is considered to be a tumor suppressor and can play the role of resisting tumors by targeting bcl-2, notch1, sirt-1, foxp1 and the like, whereas miR-34a is inhibited by C-MYC with high expression in cancer or tumor cells. let-7 is a tumor suppressor family of tumor or cancer related microRNAs, and C-MYC is a target of let-7, and the expression of the C-MYC can be suppressed by let-7. The expression pathway of C-MYC is shown in detail in FIG. 6.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention effectively combines gene therapy and virus therapy of malignant tumor, prepares the recombinant oncolytic virus which can efficiently express and is related to C-MYC proto-oncoprotein and has tumor inhibition effect, and enhances the killing capability to tumor compared with simple gene therapy or virus therapy.

2. The invention adopts a tumor targeted therapy strategy, can effectively target tumor cells and specifically proliferate in the tumor cells. Thereby greatly enhancing the safety of oncolytic vaccinia virus vectors.

3. The invention adopts a virus replication related gene deletion mode to ensure the intratumoral specific replication of the virus and greatly enhance the safety of the oncolytic vaccinia virus vector.

4. The IRES sequence is used as a connecting sequence of different micro RNA genes, so that different micro RNAs are expressed together in the same recombinant oncolytic virus, and the application range of the recombinant oncolytic virus is enlarged.

5. The invention effectively combines gene therapy and virus therapy of malignant tumors to prepare the recombinant oncolytic virus capable of efficiently expressing miRNA-34a and let7, and the killing capability to tumors is enhanced compared with simple gene therapy or virus therapy.

6. The invention develops a better effect of resisting diffuse large B cell lymphoma by constructing the recombinant oncolytic vaccinia virus carrying the microRNA-34a and the let 7.

As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional unrecited elements or method steps. The term "comprising" in any of the expressions herein, particularly in describing the method, use or product of the invention, is to be understood as including those products, methods and uses which consist essentially of and consist of the recited components or elements or steps. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

For a more clear illustration of the invention, reference is now made in detail to the following examples, which are intended to be purely exemplary of the invention and are not to be interpreted as limiting the application.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1a is a schematic diagram showing the insertion structure of an exogenous gene of an oncolytic vaccinia virus;

FIG. 1b PCR/agarose gel electrophoresis to identify whether recombinant oncolytic vaccinia virus contains wild-type viral contamination;

FIG. 2 level of expression of the infectious capacity of lymphoma cell lines by oncolytic vaccinia virus carrying a green fluorescent protein reporter (OVV-GFP);

FIG. 3 RT-PCR detects the expression level of the recombinant oncolytic vaccinia virus carrying microRNA-34a and let7 infected lymphoma OCI-LY3 cell and the microRNA-34a and let 7;

FIG. 4 cck8 method to detect the killing effect of recombinant oncolytic vaccinia virus OVV-mir34a-IRES-let7 on lymphoma cell strain;

FIG. 5A is a flow staining graph of OVV-GFP, OVV-mir34a, OVV-let7, OVV-mir34a-IRES-let7 for inducing apoptosis in lymphoma cells OCI-LY3, pfeiffer and Farage, respectively, stained with the Annexin V/7-AAD apoptosis detection kit, with the abscissa representing the fluorescence intensity of the cell Annexin V-PE and the ordinate representing the fluorescence intensity of the cell 7-AAD. The graph is divided into four quadrants (Q3-1 represents Annexin V-/7-AAD +; Q3-2 represents Annexin V +/7-AAD +; Q3-3 represents Annexin V-/7-AAD-; Q3-4 represents Annexin V +/7-AAD-; and the sum of the cell rates of Q3-2 plus Q3-4 represents the total apoptotic cell rate;

FIG. 5B is a histogram of the quantitative statistics of the apoptotic cell rates of each of the three replicates of lymphoma cells OCI-LY3, pfeiffer and Farage induced apoptosis by OVV-GFP, OVV-mir34a, OVV-let7, OVV-mir34a-IRES-let7, respectively;

FIG. 6 shows the mechanism of action of tumor suppressor in the expression pathway of C-MYC proto-oncoprotein in large B-cell lymphoma (DLBCL);

FIG. 7 PCB vector containing TK gene sequence and gpt gene.

Detailed Description

Example 1, a method for preparing recombinant oncolytic vaccinia virus, as follows:

PCB shuttle plasmids were prepared, with reference to the following literature: construction of a recombinant vaccinia virus vector with double screening markers of Zeocin and GFP, International J.Snowski infectious disease, volume 39, No.3 in 2012; panicali D, Paolettie. Construction of Poxviruses as cloning vectors; insertion of the enzyme gene from microorganisms complex virus info the DNA of infection of vaccinia virus. Proc Nail acid sci USA,1982,79 (16); 4927-;

the vTK-L, vTK-R and gpt gene sequences were obtained by searching in NCBI (https:// www.ncbi.nlm.nih.gov /); the vTK-L, vTK-R and gpt gene sequences were synthesized by entrusting Nanjing Kingsrei Biotech Co., Ltd and the genes were inserted into blank PCB plasmids, respectively.

Through the above steps, a PCB plasmid carrying Vkl-L, vTK-R and gpt genes, as shown in FIG. 7, and having a sequence shown in SEQ ID NO.5, was constructed.

Taking miR-34a and let-7 double-gene oncolytic vaccinia virus as an example, the specific construction steps are as follows:

obtaining a DNA sequence of miR-34a-IRES-let7 by a gene synthesis method, which comprises the following specific steps:

inquiring in NCBI (https:// www.ncbi.nlm.nih.gov /) to obtain a miR-34a precursor sequence, a let-7 precursor sequence and an IRES linker sequence, entrusting Nanjing Kingsry Biotechnology Co., Ltd to synthesize miR-34a-IRES-let7 with a sequence shown as SEQ ID NO.4, and inserting a T vector for sequencing;

it is to be understood that other PCBs, such as shuttle plasmid PCBs without selectable markers, may be used in the present invention.

After double enzyme digestion is carried out on the miRNA with correct sequencing by BgLII and EcoRI, the target gene fragment is recovered according to the instruction of a DNA gel recovery kit, and the enzyme digestion product BgLII-miR-34a-IRES-let7-EcoRI is obtained. Meanwhile, the pCB original plasmid is subjected to BgLII and EcoRI double digestion to form a TKL-pCB-TKR linearized plasmid, and the reaction system is as follows: miR-34a-IRES-let7/pCB 20ul, BgLII 1ul, EcoRI,10xTango Buffer 6ul, ddH2O 2 ul; carrying out enzyme digestion for 2-3h at 37 ℃, running 1% agarose gel electrophoresis after the enzyme digestion is finished, observing by a gel imager, and recovering the enzyme digestion product by a DNA gel recovery kit.

And (2) connecting the BgLII-miR-34a-IRES-let7-EcoR and the TKL-pCB-TKR to form a pCB-miR-34a-IRES-let7 shuttle plasmid, wherein the connecting system is as follows: TKL-pCB-TKR 2ul, BgLII-miR-34a-IRES-let7-EcoR 8ul, ligation High ver.210ul. The ligation was performed overnight in a water bath at 16 ℃ and the ligation product was transformed into E.coli strain DH 5. alpha. competent, which was plated on agar plates and then cultured in a biochemical incubator at 37 ℃ for 12 hours. Selecting a growing bacterial monoclonal, carrying out shake amplification for 10h in an LB solution containing 1 per mill ampicillin at 37 ℃, and carrying out enzyme digestion identification to screen a positive clone of pCB-miR-34a-IRES-let7 (with the sequence of SEQ ID NO. 6).

HEK293 cells (human embryonic kidney cell line purchased from Chinese academy of sciences) were infected with wild-type vaccinia virus (ATCC company, USA) at a dose of 1X104vp for 2 hours, and then the HEK293 cells infected with vaccinia virus were transfected with pCB-miR34a-IRES-let7 plasmid 5 μ g, and after 48 hours, a mixture of recombinant oncolytic vaccinia virus OVV-miR-34a-IRES-let7 and wild-type virus was obtained;

screening and plaque selection of the recombinant oncolytic vaccinia virus: considering that the recombinant oncolytic vaccinia virus genome carries a xanthine guanine phosphotransferase gene gpt resistance gene, mycophenolic acid, xanthine and hypoxanthine are used for screening, and virus monoclonal plaques are picked, so that vaccinia virus OVV-mir34a-IRES-let7 without wild-type vaccinia virus pollution is obtained. (the screening principle: mycophenolic acid as hypoxanthine dehydrogenase inhibitor can inhibit the synthesis of Guanine (GMP), so that the cell loses the ability of synthesizing DNA and dies, and inhibits the replication of vaccinia virus in the cell. the xanthine guanine phosphate transferase gene (gpt gene) is used for remedying and synthesizing GMP in a culture medium containing xanthine through a xanthine phosphate intermediate, so that the nucleic acid synthesis is normally carried out, the virus normally replicates We identify the constructed oncolytic virus, select an appropriate sequence from the intermediate sequence not containing Vkl-L, vTK-R sequence in the TK gene of the oncolytic virus to design a PCR primer, the sequence of the primer is shown in brackets (F: 5'-TGTGAAGACGATAAATTAATGATC-3', R: 5'-GTTTGCCATACGCTCACAG-3'), the primer can amplify the full length of 816bp of the intermediate sequence of the TK gene, and identify the wild type virus pollution condition of each oncolytic virus, the result is shown in figure 1b, the 3 rd lane shows PCR amplification products of 816bp, and the rest 2 blank controls and 45678 lanes correspond to recombinant oncolytic viruses which do not have PCR strip products amplified by the TK gene primer, which indicates that each virus does not have wild type virus pollution.

The miR-34a monogene oncolytic vaccinia virus and the let-7 monogene oncolytic vaccinia virus are independently constructed, and the steps are the same except that the artificially synthesized monogene sequences are miR-34a and let-7 respectively.

An oncolytic vaccinia virus with a GFP gene was constructed separately, and the steps were the same except that the target gene sequence was the GFP gene.

Example 2: detecting the infection capacity of the recombinant oncolytic vaccinia virus carrying the green fluorescent protein reporter gene on a lymphoma cell line by using a flow cytometer.

Inoculation in six well plates 5X 10⁵OCI-LY3, OCI-LY8, pfeiffer, Farage, Raji, Jurkat cells (human lymphoma cell line, purchased from cell bank of Chinese academy of sciences) per mL, each group of cells was added with recombinant oncolytic vaccinia virus carrying GFP at 1MOI, 5MOI, 10MOI, respectively, the control group was added with an equal amount of PBS, 5% CO at 37 deg.C₂And (5) culturing. After 24h and 48h, fluorescence was observed under a fluorescence microscope, and after the observation, the cells were collected and the GFP expression ratio of each group of samples was analyzed by a flow cytometer (BD Accuri C6). The results are shown in FIG. 2, where the expression level of GFP increased with increasing doses of OVV-GFP, indicating that the recombinant oncolytic vaccinia virus is effective in attacking lymphoma cells.

Example 3: and (3) detecting the expression levels of the microRNA-34a and the let7 when the recombinant oncolytic vaccinia virus carrying the microRNA-34a and the let7 infects OCI-LY3 cells by RT-PCR.

Recombinant oncolytic vaccinia virus carrying microRNA-34a, let7 (OVV-mir34a-IRES-let7) was infected with OCI-LY3 at a dose of 1MOI,

the control groups were: 1) control group: an equal amount of PBS;

2) recombinant oncolytic vaccinia virus carrying only GFP (OVV-GFP);

3) recombinant oncolytic vaccinia virus carrying only let7 (OVV-let 7);

4) carrying only microRNA-34a recombinant oncolytic vaccinia virus (OVV-mir34 a);

37℃，5％CO₂incubated under conditions for 24 hours. Extracting total RNA of cells according to a TRIZOL method, performing reverse transcription to form cDNA by using the extracted total RNA as a template, and designing a primer by using Primer5.0 software. The cDNA was subjected to fluorescent quantitative PCR by SYBR qPCR Mix in an amplification system of 20. mu.L.

The amplification parameters were: 1min at 95 ℃, 15s at 95 ℃, 1min at 60 ℃ and 40 cycles. The results were analyzed by Applied Biosystems 7300real-time PCR instrument software. The results are shown in FIG. 3, and expression levels of microRNA-34a and let7 are remarkably increased on mRNA level after the OCI-LY3 cells are treated by OVV-mir34a-IRES-let7, which indicates that OVV-mir34a-IRES-let7 can stably and highly express microRNA34a and let7 in lymphoma cells.

Example 4: effect of recombinant oncolytic vaccinia virus carrying microRNA-34a, let7 on proliferation activity of diffuse large B-cell lymphoma cells.

Human diffuse large B-cell lymphoma cells OCI-LY3, pfeiffer and Farage were cultured in RPMI (purchased from Gibco) containing 10% Fetal Bovine Serum (FBS) (37 ℃, 5% CO2, saturation humidity), and the fourth generation of cells was replaced at 2X 10⁴Mu.l of the suspension was inoculated into a 96-well plate at a concentration of 100 ml. A control group, a recombinant oncolytic vaccinia virus group (OVV-GFP) and a recombinant oncolytic vaccinia virus group (OVV-mir34a-IRES-let7) carrying microRNA-34a and let7 are set, and each group is provided with 3 more wells. OVV-GFP (0.01MOI, 0.1MOI, 1MOI, 10MOI), OVV-mir34a (0.01MOI, 0.1MOI, 1MOI, 10MOI), OVV-let7(0.01MOI, 0.1MOI, 1MOI, 10MOI) and OVV-mir34a-IRES-let7(0.01MOI, 0.1MOI, 1MOI, 10MOI) were added, 10. mu.l of CCK8 reaction solution was added after 48 hours of incubation, the incubation was terminated after 2 hours of incubation, the absorbance (A value) of each well was measured at a wavelength of 450nm on a Thermo Varioskan Flash full-automatic enzyme standard apparatus, and the above experiment was repeated 3 times. And calculating the cell inhibition rate according to the value A, wherein the calculation formula is as follows: the cell inhibition ratio (%) was (negative control group a value-addition group a value)/negative control group a value × 100%.

The results are shown in FIG. 4, with increasing doses, OVV-GFP, OVV-mir34a, OVV-let7, OVV-mir34a-IRES-let7 had an increasingly significant effect on inhibition of proliferation of diffuse large B-cell lymphoma, and OVV-mir34a-IRES-let7 had a significantly greater effect on inhibition of proliferation of lymphoma cells OCI-LY3, pfeiffer and Farage than OVV-GFP, OVV-mir34a, OVV-let 7.

Example 5: the recombinant oncolytic vaccinia virus carrying microRNA-mir34a and let7 has the effect of promoting apoptosis of lymphoma cells.

Inoculation in six well plates 5X 10⁵one/mL OCI-LY3, pfeiffer and Farage cells were added with 10MOI of OVV-mir34a, OVV-let7 and OVV-mir34a-IRES-let7 and empty-load oncolytic vaccinia virus (OVV-GFP), respectively, and the control group was added with the same amount of PBS, 5% CO at 37 deg.C₂And (5) culturing. And (3) after 48h, referring to an Annexin V/PI apoptosis detection kit operation instruction: cells were harvested and washed with pre-chilled PBSThe cells were resuspended in 100. mu.L of 1 XBinding Buffer, 5. mu.L of Annexin-V and 5. mu.L of 7-AAD were added, and after 15min of RT photophobia, 400. mu.L of 1 XBinding Buffer was added and analyzed by flow cytometry.

The results are shown in FIG. 5, and OVV-GFP, OVV-mir34a-IRES-let7, OVV-mir34a and OVV-let7 all induced apoptosis, with OVV-mir34a-IRES-let7 treatment group having the most significant pro-apoptotic effect.

The expression pathway of C-MYC and the relationship between mir-34 and let7 in FIG. 6 are as follows:

three pathways leading to diffuse large B-cell lymphoma (DLBCL) are shown: the BCR signal pathway expression quantity is increased, the apoptosis is weakened, or the cell differentiation and division are enhanced. The p53 cancer suppressor gene DNA gene damage causes the reduction of p53 expression quantity, drives the reduction of mir-34a expression quantity, and increases the expression quantity of corresponding targets (SIRT1 and the like) regulated and controlled by mir-34a, thereby influencing the abnormal BCR expression pathway and generating Diffuse Large B Cell Lymphoma (DLBCL). The abnormal BCR expression pathway promotes the increase of the expression of the proto-oncogene c-MYC, thereby promoting the increase of cell differentiation and division to generate Diffuse Large B Cell Lymphoma (DLBCL).

The reduction of mir-34a expression level can also lead to the increase of the expression level of proto-oncogene Bcl-2, the Bcl-2 product plays a role in inhibiting apoptosis, and the reduction of apoptosis leads to the generation of Diffuse Large B Cell Lymphoma (DLBCL).

The let-7 gene product inhibits the abnormal expression of the c-MYC gene.

The abnormal expression of the c-MYC gene can also inhibit the expression of mir-34 a.

In conclusion of the mechanism, the c-MYC gene expression pathway can be regulated and controlled at a molecular level and a gene level by improving the expression quantity of the let-7 and the mir-34a, so that good anticancer and cancer-suppressing effects are achieved, and particularly the prevention and treatment of Diffuse Large B Cell Lymphoma (DLBCL) are realized.

Although the present invention has been described in the above-mentioned embodiments, it is to be understood that the present invention may be further modified and changed without departing from the spirit of the invention, and that such modifications and changes are within the scope of the present invention.

Sequence listing

<110> Zhejiang province people hospital

<120> recombinant oncolytic virus and preparation method and application thereof

<141> 2020-06-26

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 509

<212> DNA

<213> Artificial Sequence

<400> 1

tggctaattt ttaaaatttt tttgtagaga tggagtcttg ctagttgcct gggctggtct 60

tgaactcctg gcctgaagcg atcctcccac ctcggcctcc tgcatccttt ctttcctccc 120

cacatttcct tcttatcaac aggtgctggg gagaggcagg acaggcctgt cccccgagtc 180

ccctccggat gccgtggacc ggccagctgt gagtgtttct ttggcagtgt cttagctggt 240

tgttgtgagc aatagtaagg aagcaatcag caagtatact gccctagaag tgctgcacgt 300

tgtggggccc aagagggaag atgaagcgag agatgcccag accagtggga gacgccagga 360

cttcggaagc tcttctgcgc cacggtgggt ggtgagggcg gctgggaaag tgagctccag 420

ggccccagga gcagcctgct cgtgggtgcg gaaggaaaaa ggcacagggg cttggtgtgg 480

gcggcttttg gctgggagaa gtttgcacg 509

<210> 2

<211> 473

<212> DNA

<213> Artificial Sequence

<400> 2

atgtccccac tcccagggtt tcggcgtccc agccccctgc gcccaccgcg cctgcccgcc 60

agaatccctg tgcccttggt gcgtgtggcc tgccgagcct cgagcccctg ttctcctcag 120

ccctctttcc tcccgcgtcc ccaggaggtg cctctggaag ccacggagtc ccatcggcac 180

caagaccgac tgccctttgg ggtgaggtag taggttgtat agtttggggc tctgccctgc 240

tatgggataa ctatacaatc tactgtcttt cctgaagtgg ctgtaatatc tgcggtggac 300

agagcgtctg gaaccctggc tgggagcggg cagggccagg tttgggggca gccttggcag 360

cagtcggggg caggggccgc ctacactgag aagtctgaca ggcctaggtg ccacttgctg 420

tgtgaccttg gacaggcccc tgatctctct gggtctcagt ttcctcctct gta 473

<210> 3

<211> 608

<212> DNA

<213> Artificial Sequence

<400> 3

ggaaggtcgt ctccttgtgg gttgtggcaa gcttatcatc gtgtttttca aaggaaaacc 60

acgtccccgt ggttcggggg gcctagacgt ttttttaacc tcgactaaac acatgtaaag 120

catgtgcacc gaggccccag atcagatccc atacaatggg gtaccttctg ggcatccttc 180

agccccttgt tgaatacgct tgaggagagc catttgactc tttccacaac tatccaactc 240

acaacgtggc actggggttg tgccgccttt gcaggtgtat cttatacacg tggcttttgg 300

ccgcagaggc acctgtcgcc aggtgggggg ttccgctgcc tgcaaagggt cgctacagac 360

gttgtttgtc ttcaagaagc ttccagagga actgcttcct tcacgacatt caacagacct 420

tgcattcctt tggcgagagg ggaaagaccc ctaggaatgc tcgtcaagaa gacagggcca 480

ggtttccggg ccctcacatt gccaaaagac ggcaatatgg tggaaaataa catatagaca 540

aacgcacacc ggccttattc caagcggctt cggccagtaa cgttaggggg gggggaggga 600

gaggggcg 608

<210> 4

<211> 1590

<212> DNA

<213> Artificial Sequence

<400> 4

tggctaattt ttaaaatttt tttgtagaga tggagtcttg ctagttgcct gggctggtct 60

tgaactcctg gcctgaagcg atcctcccac ctcggcctcc tgcatccttt ctttcctccc 120

cacatttcct tcttatcaac aggtgctggg gagaggcagg acaggcctgt cccccgagtc 180

ccctccggat gccgtggacc ggccagctgt gagtgtttct ttggcagtgt cttagctggt 240

tgttgtgagc aatagtaagg aagcaatcag caagtatact gccctagaag tgctgcacgt 300

tgtggggccc aagagggaag atgaagcgag agatgcccag accagtggga gacgccagga 360

cttcggaagc tcttctgcgc cacggtgggt ggtgagggcg gctgggaaag tgagctccag 420

ggccccagga gcagcctgct cgtgggtgcg gaaggaaaaa ggcacagggg cttggtgtgg 480

gcggcttttg gctgggagaa gtttgcacgg gaaggtcgtc tccttgtggg ttgtggcaag 540

cttatcatcg tgtttttcaa aggaaaacca cgtccccgtg gttcgggggg cctagacgtt 600

tttttaacct cgactaaaca catgtaaagc atgtgcaccg aggccccaga tcagatccca 660

tacaatgggg taccttctgg gcatccttca gccccttgtt gaatacgctt gaggagagcc 720

atttgactct ttccacaact atccaactca caacgtggca ctggggttgt gccgcctttg 780

caggtgtatc ttatacacgt ggcttttggc cgcagaggca cctgtcgcca ggtggggggt 840

tccgctgcct gcaaagggtc gctacagacg ttgtttgtct tcaagaagct tccagaggaa 900

ctgcttcctt cacgacattc aacagacctt gcattccttt ggcgagaggg gaaagacccc 960

taggaatgct cgtcaagaag acagggccag gtttccgggc cctcacattg ccaaaagacg 1020

gcaatatggt ggaaaataac atatagacaa acgcacaccg gccttattcc aagcggcttc 1080

ggccagtaac gttagggggg ggggagggag aggggcgatg tccccactcc cagggtttcg 1140

gcgtcccagc cccctgcgcc caccgcgcct gcccgccaga atccctgtgc ccttggtgcg 1200

tgtggcctgc cgagcctcga gcccctgttc tcctcagccc tctttcctcc cgcgtcccca 1260

ggaggtgcct ctggaagcca cggagtccca tcggcaccaa gaccgactgc cctttggggt 1320

gaggtagtag gttgtatagt ttggggctct gccctgctat gggataacta tacaatctac 1380

tgtctttcct gaagtggctg taatatctgc ggtggacaga gcgtctggaa ccctggctgg 1440

gagcgggcag ggccaggttt gggggcagcc ttggcagcag tcgggggcag gggccgccta 1500

cactgagaag tctgacaggc ctaggtgcca cttgctgtgt gaccttggac aggcccctga 1560

tctctctggg tctcagtttc ctcctctgta 1590

<210> 5

<211> 4867

<212> DNA

<213> Artificial Sequence

<400> 5

cgcgcgtaat acgactcact atagggcgaa ttggagctct ttttatctgc gcggttaacc 60

gcctttttat ccatcaggtg atctgttttt attgtggagt ctagagaatt cgatatcagg 120

cctagatctg tcgacttcga gcttatttat attccaaaaa aaaaaaataa aatttcaatt 180

tttaagcttt cactaattcc aaacccaccc gctttttata gtaagttttt cacccataaa 240

taataaatac aataattaat ttctcgtaaa agtagaaaat atattctaat ttattgcacg 300

gtaaggaagt agatcataac gatctctata atctcgcgca acctattttc ccctcgaaca 360

ctttttaagc cgtagataaa caggctggga cacttcacat gagcgaaaaa tacatcgtca 420

cctgggacat gttgcagatc catgcacgta aactcgcaag ccgactgatg ccttctgaac 480

aatggaaagg cattattgcc gtaagccgtg gcggtctggt accgggtgcg ttactggcgc 540

gtgaactggg tattcgtcat gtcgataccg tttgtatttc cagctacgat cacgacaacc 600

agcgcgagct taaagtgctg aaacgcgcag aaggcgatgg cgaaggcttc atcgttattg 660

atgacctggt ggataccggt ggtactgcgg ttgcgattcg tgaaatgtat ccaaaagcgc 720

actttgtcac catcttcgca aaaccggctg gtcgtccgct ggttgatgac tatgttgttg 780

atatcccgca agatacctgg attgaacagc cgtgggatat gggcgtcgta ttcgtcccgc 840

caatctccgg tcgctaatct tttcaacgcc tggcactgcc gggcgttgtt ctttttaact 900

tcaggcgggt tacaatagtt tccagtaagt attctggagg ctgcatccat gacacaggca 960

aacctgcgga tcccagcttt tgttcccttt agtgagggtt aattgcgcgc agttatagta 1020

gccgcactcg atgggacatt tcaacgtaaa ccgtttaata atattttgaa tcttattcca 1080

ttatctgaaa tggtggtaaa actaactgct gtgtgtatga aatgctttaa ggaggcttcc 1140

ttttctaaac gattgggtga ggaaaccgag atagaaataa taggaggtaa tgatatgtat 1200

caatcggtgt gtagaaagtg ttacatcgac tcataatatt atatttttta tctaaaaaac 1260

taaaaataaa cattgattaa attttaatat aatacttaaa aatggatgtt gtgtcgttag 1320

ataaaccgtt tatgtatttt gaggaaattg ataatgagtt agattacgaa ccagaaagtg 1380

caaatgaggt cgcaaaaaaa ctgccgtatc aaggacagtt aaaactatta ctaggagaat 1440

tattttttct tagtaagtta cagcgacacg gtatattaga tggtgccacc gtagtgtata 1500

taggatctgc tcccggtaca catatacgtt atttgagaga tcatttctat aatttaggag 1560

tgatcatcaa atggatgcta attgacggcc gccatcatga tcctatttta aatggattgc 1620

gtgatgtgac tctagtgact cggttcgttg atgaggaata tctacgatcc atcaaaaaac 1680

aactgcatcc ttctaagatt attttaattt ctgatgtgag atccaaacga ggaggaaatg 1740

aacctagtac ggcggattta ctaagtaatt acgctctaca aaatgtcatg attagtattt 1800

taaaccccgt ggcgtctagt cttaaatgga gatgcccgtt tccagatcaa tggatcaagg 1860

acttttatat cccacacggt aataaaatgt tacaaccttt tgctccttca tattcagctg 1920

aaatgagatt attaagtatt tataccggtg agaacatgag actgactcgg gccgcgttgc 1980

tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 2040

agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 2100

tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 2160

cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg 2220

ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 2280

ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 2340

ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 2400

ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 2460

cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 2520

gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 2580

atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 2640

ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 2700

gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 2760

tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 2820

ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 2880

taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 2940

gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 3000

gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 3060

ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 3120

aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 3180

gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 3240

cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 3300

actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 3360

caacacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 3420

gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 3480

ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 3540

caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 3600

tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 3660

gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 3720

cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa 3780

ataggcgtat cacgaggccc tttcgtcttc gaataaatac ctgtgacgga agatcacttc 3840

gcagaataaa taaatcctgg tgtccctgtt gataccggga agccctgggc caacttttgg 3900

cgaaaatgag acgttgatcg gcacgtaaga ggttccaact ttcaccataa tgaaataaga 3960

tcactaccgg gcgtattttt tgagttatcg agattttcag gagctaagga agctaaaatg 4020

gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat 4080

tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca gagcttttgg 4140

gatcaataaa tggatcacaa ccagtatctc ttaacgatgt tcttcgcaga tgatgattca 4200

ttttttaagt atttggctag tcaagatgat gaatcttcat tatctgatat attgcaaatc 4260

actcaatatg tagctagact ttctgttatt attattgatc caatcaaaaa ataaattaga 4320

agccgtgggt cattgttatg aatctctttc agaggaatac agacaattga caaaattcac 4380

agactttcaa gattttaaaa aactgtttaa caaggtccct attgacagat ggaagggtca 4440

aacttaataa aggatatttg ttcgactttg tgattagttt gatgcgattc aaaaaagaat 4500

cctctctagc taccaccgca atagatcctg ttagatacat agatcctcgt cgcaatatcg 4560

cattttctaa cgtgatggat atattaaagt cgaataaagt gaacaataat taattcttta 4620

ttgtcatcat gaacggcgga catattcagt tgataatcgg ccccatgttt tcaggtaaaa 4680

gtacagaatt aattagacga gttagacgtt atcaaatagc tcaatataaa tgcgtgacta 4740

taaaatattc taacgataat agatacggaa cgggactatg gacgcatgat aagaataatt 4800

ttgaagcatt ggaagcaact aaactatgtg atctcttgga atcaattaca gatttctccg 4860

tgatagg 4867

<210> 6

<211> 6445

<212> DNA

<213> Artificial Sequence

<400> 6

cgcgcgtaat acgactcact atagggcgaa ttggagctct ttttatctgc gcggttaacc 60

gcctttttat ccatcaggtg atctgttttt attgtggagt ctagagaatt catgtctcct 120

cctttgactc tgggtctctc tagtccccgg acaggttcca gtgtgtcgtt caccgtggat 180

ccggacagtc tgaagagtca catccgccgg ggacgggggc tgacgacggt tccgacgggg 240

gtttggaccg ggacgggcga gggtcggtcc caaggtctgc gagacaggtg gcgtctataa 300

tgtcggtgaa gtcctttctg tcatctaaca tatcaatagg gtatcgtccc gtctcggggt 360

ttgatatgtt ggatgatgga gtggggtttc ccgtcagcca gaaccacggc taccctgagg 420

caccgaaggt ctccgtggag gacccctgcg ccctcctttc tcccgactcc tcttgtcccc 480

gagctccgag ccgtccggtg tgcgtggttc ccgtgtccct aagaccgccc gtccgcgcca 540

cccgcgtccc ccgaccctgc ggctttggga ccctcacccc tgtagcgggg agagggaggg 600

gggggggatt gcaatgaccg gcttcggcga accttattcc ggccacacgc aaacagatat 660

acaataaaag gtggtataac ggcagaaaac cgttacactc ccgggccttt ggaccgggac 720

agaagaactg ctcgtaagga tccccagaaa ggggagagcg gtttccttac gttccagaca 780

acttacagca cttccttcgt caaggagacc ttcgaagaac ttctgtttgt tgcagacatc 840

gctgggaaac gtccgtcgcc ttggggggtg gaccgctgtc cacggagacg ccggttttcg 900

gtgcacatat tctatgtgga cgtttccgcc gtgttggggt cacggtgcaa cactcaacct 960

atcaacacct ttctcagttt accgagagga gttcgcataa gttgttcccc gacttcctac 1020

gggtcttcca tggggtaaca taccctagac tagaccccgg agccacgtgt acgaaatgta 1080

cacaaatcag ctccaatttt tttgcagatc cggggggctt ggtgcccctg caccaaaagg 1140

aaactttttg tgctactatt cgaacggtgt tgggtgttcc tctgctggaa gggcacgttt 1200

gaagagggtc ggttttcggc gggtgtggtt cggggacacg gaaaaaggaa ggcgtgggtg 1260

ctcgtccgac gaggaccccg ggacctcgag tgaaagggtc ggcgggagtg gtgggtggca 1320

ccgcgtcttc tcgaaggctt caggaccgca gagggtgacc agacccgtag agagcgaagt 1380

agaagggaga acccggggtg ttgcacgtcg tgaagatccc gtcatatgaa cgactaacga 1440

aggaatgata acgagtgttg ttggtcgatt ctgtgacggt ttctttgtga gtgtcgaccg 1500

gccaggtgcc gtaggcctcc cctgagcccc ctgtccggac aggacggaga ggggtcgtgg 1560

acaactattc ttcctttaca cccctccttt ctttcctacg tcctccggct ccaccctcct 1620

agcgaagtcc ggtcctcaag ttctggtcgg gtccgttgat cgttctgagg tagagatgtt 1680

tttttaaaat ttttaatcgg tagatctgtc gacttcgagc ttatttatat tccaaaaaaa 1740

aaaaataaaa tttcaatttt taagctttca ctaattccaa acccacccgc tttttatagt 1800

aagtttttca cccataaata ataaatacaa taattaattt ctcgtaaaag tagaaaatat 1860

attctaattt attgcacggt aaggaagtag atcataacga tctctataat ctcgcgcaac 1920

ctattttccc ctcgaacact ttttaagccg tagataaaca ggctgggaca cttcacatga 1980

gcgaaaaata catcgtcacc tgggacatgt tgcagatcca tgcacgtaaa ctcgcaagcc 2040

gactgatgcc ttctgaacaa tggaaaggca ttattgccgt aagccgtggc ggtctggtac 2100

cgggtgcgtt actggcgcgt gaactgggta ttcgtcatgt cgataccgtt tgtatttcca 2160

gctacgatca cgacaaccag cgcgagctta aagtgctgaa acgcgcagaa ggcgatggcg 2220

aaggcttcat cgttattgat gacctggtgg ataccggtgg tactgcggtt gcgattcgtg 2280

aaatgtatcc aaaagcgcac tttgtcacca tcttcgcaaa accggctggt cgtccgctgg 2340

ttgatgacta tgttgttgat atcccgcaag atacctggat tgaacagccg tgggatatgg 2400

gcgtcgtatt cgtcccgcca atctccggtc gctaatcttt tcaacgcctg gcactgccgg 2460

gcgttgttct ttttaacttc aggcgggtta caatagtttc cagtaagtat tctggaggct 2520

gcatccatga cacaggcaaa cctgcggatc ccagcttttg ttccctttag tgagggttaa 2580

ttgcgcgcag ttatagtagc cgcactcgat gggacatttc aacgtaaacc gtttaataat 2640

attttgaatc ttattccatt atctgaaatg gtggtaaaac taactgctgt gtgtatgaaa 2700

tgctttaagg aggcttcctt ttctaaacga ttgggtgagg aaaccgagat agaaataata 2760

ggaggtaatg atatgtatca atcggtgtgt agaaagtgtt acatcgactc ataatattat 2820

attttttatc taaaaaacta aaaataaaca ttgattaaat tttaatataa tacttaaaaa 2880

tggatgttgt gtcgttagat aaaccgttta tgtattttga ggaaattgat aatgagttag 2940

attacgaacc agaaagtgca aatgaggtcg caaaaaaact gccgtatcaa ggacagttaa 3000

aactattact aggagaatta ttttttctta gtaagttaca gcgacacggt atattagatg 3060

gtgccaccgt agtgtatata ggatctgctc ccggtacaca tatacgttat ttgagagatc 3120

atttctataa tttaggagtg atcatcaaat ggatgctaat tgacggccgc catcatgatc 3180

ctattttaaa tggattgcgt gatgtgactc tagtgactcg gttcgttgat gaggaatatc 3240

tacgatccat caaaaaacaa ctgcatcctt ctaagattat tttaatttct gatgtgagat 3300

ccaaacgagg aggaaatgaa cctagtacgg cggatttact aagtaattac gctctacaaa 3360

atgtcatgat tagtatttta aaccccgtgg cgtctagtct taaatggaga tgcccgtttc 3420

cagatcaatg gatcaaggac ttttatatcc cacacggtaa taaaatgtta caaccttttg 3480

ctccttcata ttcagctgaa atgagattat taagtattta taccggtgag aacatgagac 3540

tgactcgggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 3600

aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 3660

ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 3720

tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc 3780

tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 3840

ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 3900

tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 3960

ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 4020

tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 4080

aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 4140

aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 4200

aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 4260

ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 4320

acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 4380

ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 4440

gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 4500

taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 4560

tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 4620

gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 4680

cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 4740

aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 4800

cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 4860

tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 4920

gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag 4980

tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 5040

gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 5100

ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 5160

cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 5220

agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 5280

gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca 5340

tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcga ataaatacct 5400

gtgacggaag atcacttcgc agaataaata aatcctggtg tccctgttga taccgggaag 5460

ccctgggcca acttttggcg aaaatgagac gttgatcggc acgtaagagg ttccaacttt 5520

caccataatg aaataagatc actaccgggc gtattttttg agttatcgag attttcagga 5580

gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa 5640

tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag 5700

accgttcaga gcttttggga tcaataaatg gatcacaacc agtatctctt aacgatgttc 5760

ttcgcagatg atgattcatt ttttaagtat ttggctagtc aagatgatga atcttcatta 5820

tctgatatat tgcaaatcac tcaatatgta gctagacttt ctgttattat tattgatcca 5880

atcaaaaaat aaattagaag ccgtgggtca ttgttatgaa tctctttcag aggaatacag 5940

acaattgaca aaattcacag actttcaaga ttttaaaaaa ctgtttaaca aggtccctat 6000

tgacagatgg aagggtcaaa cttaataaag gatatttgtt cgactttgtg attagtttga 6060

tgcgattcaa aaaagaatcc tctctagcta ccaccgcaat agatcctgtt agatacatag 6120

atcctcgtcg caatatcgca ttttctaacg tgatggatat attaaagtcg aataaagtga 6180

acaataatta attctttatt gtcatcatga acggcggaca tattcagttg ataatcggcc 6240

ccatgttttc aggtaaaagt acagaattaa ttagacgagt tagacgttat caaatagctc 6300

aatataaatg cgtgactata aaatattcta acgataatag atacggaacg ggactatgga 6360

cgcatgataa gaataatttt gaagcattgg aagcaactaa actatgtgat ctcttggaat 6420

caattacaga tttctccgtg atagg 6445

Claims (22)

1. A recombinant oncolytic virus operable to insert a foreign gene encoding an anti-cancer factor that inhibits aberrant expression of a C-MYC gene or/and is inhibited by expression of a C-MYC gene.

2. The recombinant oncolytic virus of claim 1, wherein the exogenous gene is inserted into the TK gene.

3. The recombinant oncolytic virus of claim 2, wherein the agent that inhibits aberrant expression of a C-MYC gene is a microrna and the agent that is inhibited by expression of a C-MYC gene is a microrna.

4. The recombinant oncolytic virus of claim 3, wherein the exogenous gene comprises one or more of: miR-34a and let-7; the sequence of the miR-34a is shown as SEQ ID NO.1, and the sequence of the let-7 is shown as SEQ ID NO. 2.

5. The recombinant oncolytic virus of claim 4, wherein the exogenous genes comprise miR-34a and let-7, and the miR-34a and let-7 gene sequences are linked by an IRES sequence.

6. The recombinant oncolytic virus of claim 5, wherein the IRES sequence is as set forth in SEQ ID No. 3.

7. The recombinant oncolytic virus of any one of claims 4 or 6,

the exogenous gene encoding miR-34a can be a polynucleotide sequence which hybridizes with a nucleotide sequence of miR-34a under a strict condition and encodes miR-34a, a polynucleotide sequence with 60 percent of identity and above or a complementary sequence thereof;

the exogenous gene encoding let-7 can be a polynucleotide sequence which hybridizes with a nucleotide sequence of let-7 under stringent conditions and encodes let-7, a polynucleotide sequence with 60% or more of identity of the polynucleotide sequence, or a complementary sequence of the polynucleotide sequence.

8. The recombinant oncolytic virus of any one of claims 1-6, wherein the recombinant oncolytic virus comprises a selectively replicating recombinant oncolytic virus.

9. The recombinant oncolytic virus of claim 8, wherein the selectively replicating recombinant oncolytic virus is derived from an adenovirus with oncolytic effects, a poxvirus, a herpes simplex virus, a measles virus, a semliki forest virus, a vesicular stomatitis virus, a poliovirus, a retrovirus, a reovirus, a seneca valley virus, an enterovirus of the eke type, a coxsackievirus, a newcastle disease virus, and a malaba virus.

10. A therapeutic agent comprising a therapeutically effective amount of the recombinant oncolytic virus of any one of claims 1-9.

11. The therapeutic agent according to claim 10, wherein the recombinant oncolytic virus is formulated for administration by intratumoral injection, intraperitoneal administration, subarachnoid intracavity administration or intravenous administration.

12. The therapeutic agent of claim 10, further comprising a pharmaceutically acceptable carrier, an additional active ingredient for the treatment of cancer.

13. The therapeutic agent of claim 12, wherein the pharmaceutically acceptable carrier comprises excipients and adjuvants that facilitate processing of the recombinant oncolytic vaccinia virus into a formulation.

14. The therapeutic agent of claim 12 wherein said additional active ingredient is paclitaxel.

15. Use of a recombinant oncolytic virus of any one of claims 1-9 in the manufacture of a medicament for the prevention or treatment of a tumor or/and cancer.

16. Use of a therapeutic agent according to any one of claims 10 to 14 in the manufacture of a medicament for the prevention or treatment of a tumour or/and cancer.

17. The use of claim 15 or 16, wherein the tumor and/or cancer comprises: breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactin tumor, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, paget's disease, cervical cancer, gall bladder cancer, eye cancer, kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma, multi-tip myeloma, ovarian cancer, pancreatic endocrine tumor, glucagon tumor, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, heart cancer, meningeal cancer, hematological cancer, peritoneal cancer and pleural cancer.

18. The use of claim 15 or 16, wherein the cancer is lymphoma.

19. The use of claim 18, wherein the cancer is diffuse large B-cell lymphoma.

20. A kit of synergistic combinations for the treatment of tumors and/or cancers comprising: a container comprising the recombinant oncolytic virus of any one of claims 1-9; instructions for timing and mode of administration are specified.

21. A kit of synergistic combinations for the treatment of tumors and/or cancers comprising: a container containing a therapeutic agent according to any one of claims 10-14; instructions for timing and mode of administration are specified.

22. The method of producing the recombinant oncolytic virus of claim 4 or 6: the method is characterized by comprising the following steps:

1) construction of a PCB plasmid containing a TK Gene sequence and a gpt resistance Gene

2) Synthesizing an exogenous gene sequence by a gene synthesis method;

3) carrying out double enzyme digestion on the exogenous gene sequence and the PCB plasmid containing the TK gene sequence and the gpt resistance gene by BgLII and EcoRI endonucleases to obtain enzyme digestion products BgLII-exogenous gene sequence-EcoRI and TKL-pCB-TKR linearized plasmids;

4) DNA ligase is used for connecting the plasmids linearized by the BgLII-exogenous gene sequence-EcoRI and TKL-pCB-TKR to obtain a PCB-exogenous gene sequence plasmid;

5) screening and cloning of the PCB-foreign Gene sequence plasmid: transforming E.coli strain DH5 alpha competence with the PCB-exogenous gene sequence plasmid, and screening escherichia coli resistance genes to obtain a PCB-exogenous gene sequence positive clone;

6) infecting human body cells with oncolytic virus, transfecting PCB-exogenous gene sequence plasmid, and screening resistance gene carried by recombinant oncolytic virus to obtain recombinant oncolytic virus inserted with exogenous gene sequence.

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