Antitumor activity of CTRP3 protein, recombinant oncolytic virus encoding it, preparation method and application

Abstract

This invention discloses the antitumor activity of the CTRP3 protein, a recombinant oncolytic virus encoding it, its preparation method, and its applications, relating to the field of gene therapy. The invention involves inserting the CTRP3 gene, which inhibits tumor growth, into the viral genome to induce viral replication and generate CTRP3. This invention effectively combines gene therapy and viral therapy for malignant tumors, preparing a recombinant oncolytic virus that efficiently expresses CTRP3, thus enhancing its tumor-killing ability compared to gene therapy or viral therapy alone.

Description

Technical Field

[0001] This invention relates to the field of gene therapy, and particularly to the antitumor effect of CTRP3 protein, the recombinant oncolytic virus encoding it, its preparation method, and its application. By inserting the CTRP3 gene, which inhibits tumor growth, into the viral genome to perform viral replication, CTRP3 is generated. Increased CTRP3 expression improves the anticancer and cancer-preventing efficiency of the recombinant oncolytic virus. Background Technology

[0002] C1q tumor necrosis factor (TNF)-associated protein 3 (CTRP3) is a newly discovered adipokines belonging to the C1q / TNF superfamily and showing high homology with adiponectin. CTRP3 exerts various effects on cardiovascular, metabolic, and inflammatory diseases via an endocrine pathway. However, its application in cancer is largely unreported. This study found that CTRP3 can effectively inhibit tumor growth. Therefore, using oncolytic viruses carrying CTRP3 to treat cancer holds promise as a novel possibility.

[0003] Oncolytic virus therapy, as a novel form of biological immunotherapy, exerts its anti-tumor effects through direct oncolysis and induction of the body's immune activity. Subsequently, with the development of the "cancer-targeting gene virotherapy (CTGVT)" strategy (i.e., inserting anti-cancer genes into oncolytic virus vectors, organically combining viral therapy with gene therapy to create a highly effective gene-virus therapy), oncolytic viruses loaded with targeting genes or cytokines have achieved great success in targeted tumor therapy. Oncolytic vaccinia virus (PVV) is a novel vector for viral therapy, with advantages including good viral stability, low pathogenicity, high gene transfection efficiency, and good safety. PVV can selectively infect tumor cells, replicate within the cells, and kill tumor cells, while exhibiting minimal toxicity to normal tissues and cells.

[0004] Therefore, studying the mechanism by which CTRP3 inhibits tumor growth and applying it to the treatment of tumors or cancer is a current research hotspot and challenge. Summary of the Invention

[0005] Existing methods for treating cancer are relatively ineffective. Therefore, the technical problem this invention aims to solve is to provide a novel method for treating cancer or tumors, specifically, the antitumor activity of the CTRP3 protein, a recombinant oncolytic virus encoding it, its preparation method, and its application. Specifically, this involves administering a recombinant oncolytic virus containing an inserted exogenous gene sequence encoding the CTRP3 gene to a subject. Furthermore, this invention provides the use of the above-mentioned recombinant oncolytic virus in the preparation of pharmaceutical compositions for treating cancer and / or tumors.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The CTRP3 protein or the gene encoding the CTRP3 protein is used in the prevention or treatment of tumors and / or cancer.

[0008] Since there are virtually no reports on the application of CTRP3 in tumors, this study is the first to discover that CTRP3 can effectively inhibit tumor growth.

[0009] This invention is achieved through the following technical solution:

[0010] The present invention also provides a recombinant oncolytic virus, wherein the recombinant oncolytic virus is operable to insert a foreign gene sequence, the foreign gene sequence encoding the CTRP3 protein.

[0011] Preferably, the exogenous gene sequence is inserted into the TK gene. The TK gene (Thymidine kinase gene, TK) is involved in DNA synthesis during cell proliferation. During oncolytic virus replication, a high concentration of nucleotides is formed under the action of TK to ensure the smooth replication of progeny DNA. In normal cells, the concentration of nucleotides is low, therefore TK is essential for normal cell proliferation; in tumor cells, the concentration of nucleotides is high, therefore TK is not essential for tumor cell proliferation. As can be seen from the above, oncolytic viruses can preferentially replicate in tumor cells that provide sufficient nucleotides, but cannot replicate in normal cells. Deleting the TK gene can enhance the replication characteristics of oncolytic viruses in tumor cells. We use gene recombination technology to insert an exogenous gene fragment into the TK gene sequence, so that the oncolytic virus vector lacks the TK sequence. On this basis, we further construct an oncolytic vaccinia virus carrying the exogenous gene, which reduces the damage of recombinant oncolytic virus to normal cells, improves the specific recognition of tumor cells, thereby improving treatment efficiency and reducing side effects.

[0012] Preferably, the exogenous gene sequence CTRP3, as shown in SEQ ID NO.1, can exert its anti-tumor biological function, or it can be a sequence with added, reduced, or replaced sequences based on the sequence in SEQ ID NO.1, but with the same or similar function as SEQ ID NO.1.

[0013] Those skilled in the art will understand that, for sequenced genes, inventors generally cannot isolate and purify enough similar substances to detect their performance patterns. Therefore, based on the exogenous gene sequence CTRP3 (SEQ ID NO.1) of this application, any sequence that has a certain degree of homology (i.e., sequence structural similarity) with SEQ ID NO.1, and whose sequence is added, removed, or replaced on the basis of the specific sequence, and whose function is the same as or similar to SEQ ID NO.1, should be considered to be within the scope of protection of this application.

[0014] Preferably, the exogenous gene sequence is composed of a nucleotide sequence that is at least 60% identical to the sequence in SEQ ID NO. 1.

[0015] Preferably, the recombinant oncolytic virus includes, but is not limited to, selected replicating recombinant oncolytic viruses and / or non-replicating oncolytic viruses.

[0016] Preferably, the selectively replicating oncolytic virus is derived from, but is not limited to, adenoviruses, poxviruses, herpes simplex viruses, measles viruses, Semliki Forest virus, vesicular stomatitis virus, polioviruses, retroviruses, reoviruses, Seneca Valley virus, Ek enteroviruses, Coxsackie virus, Newcastle disease virus, and Malaba virus.

[0017] Preferably, the non-replicating recombinant oncolytic virus is derived from, but is not limited to, adenoviruses, poxviruses, herpes simplex viruses, measles viruses, Semliki Forest virus, vesicular stomatitis virus, polioviruses, retroviruses, reoviruses, Seneca Valley virus, Ek enteroviruses, Coxsackie virus, Newcastle disease virus, and Malaba virus.

[0018] Specifically, the recombinant oncolytic virus is a recombinant oncolytic poxvirus.

[0019] The present invention also provides the application of the above-mentioned recombinant oncolytic viruses, that is, all of the above-mentioned recombinant oncolytic viruses can be made into therapeutic agents containing therapeutically effective amounts.

[0020] In preferred embodiments, all of the above-mentioned recombinant oncolytic viruses are formulated for administration via adjacent normal injection, intratumoral injection, intraperitoneal administration, subarachnoid intracavitary administration, or intravenous administration.

[0021] Preferably, the therapeutic agents described above further comprise 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 injectable solutions and aqueous and non-aqueous sterile suspensions. The sterile injectable solutions may optionally contain antioxidants, buffers, bacteriostatic agents, and solutes that enable the formulation to be isobaric with the blood of the intended recipient. The sterile suspensions may include suspending agents and thickeners. The formulations may be present in single-dose or multi-dose containers, such as sealed ampoules, and may be stored under freeze-dried conditions, requiring only the addition of a sterile liquid carrier, such as water for injection, before immediate use.

[0022] The active ingredient of this invention can optionally be combined with a solid excipient, and, if necessary, the mixture of particles can be processed after the addition of suitable adjuvants to obtain the desired dosage form. Suitable excipients are particularly fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose or starch preparations; gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If necessary, disintegrants such as croscarmellose, agar, or alginate or its salts, such as sodium alginate, can be added.

[0023] Preferably, the recombinant oncolytic virus or therapeutic agent described above can be used for the prevention or treatment of tumors and / or cancer.

[0024] The effective amount of the active ingredient of the present invention can be any amount for treating tumors or cancer, and the determination of the effective amount is within the capabilities of those skilled in the art, particularly based on the guidance provided herein.

[0025] According to the present invention, the pharmaceutical products (medications, preparations) or pharmaceutical compositions of the present invention can be administered to the subject at any effective number of doses. Preferably, the pharmaceutical products (medications, preparations) or pharmaceutical compositions of the present invention can be administered in multiple doses, for example from about 2 to about 15 doses, more preferably from about 4 to 10 doses, and most preferably about 6 doses. In a particularly preferred embodiment, during administration, the pharmaceutical products (medications, preparations) or pharmaceutical compositions of the present invention are administered to the subject at a frequency of about once every three weeks, for example by injection, infusion, or oral administration. In a particularly preferred embodiment, administration is by injection to the tumor-bearing site.

[0026] It should be understood that the pharmaceutical products (drugs, preparations) or pharmaceutical compositions of the present invention can be formulated in any suitable manner for administration via any suitable route.

[0027] The dosage units of the pharmaceutical products (medications, pharmaceutical preparations) or pharmaceutical compositions of the present invention are based on subjects who are routinely administered the medication. For example, the dosage units may be administered more than once daily, once weekly, once monthly, etc. The dosage units may be administered on a twice-weekly basis, i.e., twice a week, for example, once every three days.

[0028] Preferably, the tumors and / or cancers include, but are not limited to: 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, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, bile duct cancer, bladder cancer, ureteral cancer, glioma, neuroblastoma, meningioma, spinal cord tumors, osteochondroma, chondrosarcoma, Ewing's sarcoma, carcinoma of unknown primary site, carcinoid tumor, fibrosarcoma, Paget's disease, cervical cancer, gallbladder cancer, ocular cancer, Kaposi's sarcoma, prostate cancer, testicular cancer, squamous cell carcinoma of the skin, mesothelioma. Multiple myeloma, ovarian cancer, pancreatic endocrine tumor, glucagonoma, pancreatic cancer, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, gastric cancer, thymic carcinoma, trophoblastic carcinoma, hydatidiform mole, endometrial cancer, vaginal cancer, vulvar cancer, mycosis fungoides, insulinoma, cardiac cancer, meningeal cancer, hematologic malignancies, peritoneal cancer, and pleural cancer. More preferably, the tumor and / or cancer is liver cancer.

[0029] Preferably, the kit comprising the aforementioned recombinant oncolytic virus or therapeutic agent includes a container, and the container may be a single set. One set of containers contains the recombinant oncolytic virus or therapeutic agent containing CTRP3 for convenient administration.

[0030] The package insert for the pharmaceutical product may include the following: indications (e.g., liver cancer), dosage (e.g., as exemplified above), and possible side effects, etc.

[0031] The design principle of this invention: The inventors discovered that CTRP3 can effectively inhibit tumor growth, and its anti-tumor biological function can be exerted by oncolytic viruses carrying CTRP3.

[0032] Preferably, the preparation of the required recombinant oncolytic virus includes the following steps:

[0033] 1) Gene synthesis methods for synthesizing exogenous gene sequences;

[0034] 2) Synthesize the pCB shuttle plasmid based on the spectrum and sequence;

[0035] 3) The exogenous gene sequence and pCB shuttle plasmid were double-digested with BgLII and EcoRI restriction enzymes respectively to obtain the linearized plasmids BgLII-exogenous gene sequence-EcoRI and TKL-pCB-TKR.

[0036] 4) Use DNA ligase to ligate the linearized plasmids BgLII-exogenous gene sequence-EcoRI and TKL-pCB-TKR to obtain the pCB-exogenous gene sequence plasmid.

[0037] 5) Screening and cloning of pCB-exogenous gene sequence plasmid: Transform the pCB-exogenous gene sequence plasmid into E. coli strain DH5α competent cells, and obtain pCB-exogenous gene sequence positive clones by screening for E. coli resistance genes;

[0038] 6) Infect human cells with oncolytic virus, then transfect pCB-exogenous gene sequence plasmid, and obtain recombinant oncolytic virus with inserted exogenous gene sequence by screening the resistance gene carried by recombinant oncolytic virus.

[0039] The exogenous gene sequence encodes the CTRP3 protein.

[0040] Compared with the prior art, the advantages of this invention are:

[0041] a) This invention effectively combines gene therapy and viral therapy for malignant tumors, and prepares a recombinant oncolytic virus that can be efficiently expressed and has a tumor-suppressing effect, which enhances the tumor-killing ability compared to gene therapy or viral therapy alone.

[0042] (b) This invention employs a tumor-targeted therapy strategy, which can effectively target tumor cells and specifically proliferate within them. This significantly enhances the safety of the oncolytic vaccinia virus vector.

[0043] c) This invention employs a method of deleting virus replication-related genes to ensure specific intratumoral replication of the virus, greatly enhancing the safety of the oncolytic vaccinia virus vector.

[0044] d) CTRP3 is a cytokine that, after expression, is released into the tumor microenvironment to inhibit tumor growth on a large scale, providing a one-to-many therapeutic effect compared to non-secretory genes.

[0045] e) This invention effectively combines gene therapy and viral therapy for malignant tumors, and prepares a recombinant oncolytic virus that can efficiently express CTRP3, which enhances the tumor-killing ability compared to gene therapy or viral therapy alone.

[0046] f) This invention demonstrates a good anti-solid tumor effect by constructing a recombinant oncolytic vaccinia virus carrying CTRP3. The oncolytic vaccinia virus VV-CTRP3 can effectively inhibit tumor growth; the CTRP3 expressed by the oncolytic vaccinia virus VV-CTRP3 can inhibit tumor growth through the microenvironment. Attached Figure Description

[0047] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0048] Figure 1 Schematic diagram of the exogenous gene insertion structure of oncolytic vaccinia virus;

[0049] Figure 2 PCR / agarose gel electrophoresis was used to identify whether the recombinant oncolytic vaccinia virus contained wild-type virus contamination.

[0050] Figure 3 PCR / agarose gel electrophoresis was used to identify whether the recombinant oncolytic vaccinia virus carried the exogenous gene CTRP3.

[0051] Figure 4 RT-PCR was used to detect the expression level of CTRP3 in SMMC-7721 and Hep 3B cells after infection with recombinant oncolytic vaccinia virus carrying CTRP3.

[0052] Figure 5 and Figure 6 MTT assay was used to detect the killing effect of recombinant oncolytic vaccinia virus OVV-CTRP3 on SMMC-7721 and Hep 3B cells;

[0053] Figure 7 PCB vectors containing the TK gene sequence and the gpt gene. Detailed Implementation

[0054] The following discloses some embodiments of the present invention. Those skilled in the art can appropriately modify the process parameters to achieve the desired results based on the content of this document. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of the present invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0055] Example 1: The preparation method of recombinant oncolytic vaccinia virus is as follows:

[0056] Prepare pCB shuttle plasmids by referring to the following literature: Fang Yourong, Li Hongyu, Chen Keda, Zhou Wenshuo, Yan Hui. Construction of recombinant vaccinia virus vectors with Zeocin and GFP double-selection markers, International Journal of Epidemiology and Infectious Diseases, June 2012, Vol. 39, No. 3; Panicali D, Paoletti E. Construction of Poxviruses ascloning vectors; insertion of the thymidine kinase gene from herpes simplexvirus info the DNA of infectious vaccinia virus. Proc Nail Acad sci USA, 1982, 79(16); 4927-4931;

[0057] The vTK-L, vTK-R, and gpt gene sequences were obtained from NCBI (https: / / www.ncbi.nlm.nih.gov / ); Nanjing GenScript Biotech Co., Ltd. was commissioned to synthesize the vTK-L, vTK-R, and gpt gene sequences, and these genes were inserted into blank pCB plasmids respectively.

[0058] The above steps are used to construct, as follows: Figure 7 The pCB plasmid (e.g., SEQ ID NO.2) carrying the Vkl-L, vTK-R, and gpt genes described in the pCB sequence.

[0059] The specific steps for constructing the CTRP3 gene-based oncolytic vaccinia virus are as follows:

[0060] The cDNA sequence of CTRP3 was obtained through gene synthesis as follows:

[0061] The mRNA sequence of CTRP3 was obtained from NCBI (https: / / www.ncbi.nlm.nih.gov / ). Nanjing Genscript Biotech Co., Ltd. was commissioned to synthesize a DNA sequence (such as SEQ ID NO.1) based on its mRNA sequence and insert it into a T vector for sequencing.

[0062] It should be noted that other pCBs can also be used in this invention, such as shuttle plasmids pCBs without selective labeling.

[0063] After double digestion of the sequenced DNA with BgLII and EcoRI, the target gene fragment was recovered according to the DNA gel recovery kit instructions to obtain the digestion product BgLII-CTRP3-EcoRI. Simultaneously, the original pCB plasmid was also double-digested with BgLII and EcoRI to form the linearized TKL-pCB-TKR plasmid. The reaction system was as follows: CTRP3 / pCB 20 μL, BgLII 1 μL, EcoRI, 10x Tango Buffer 6 μL, ddH2O 2 μL; digestion at 37℃ for 2-3 h. After digestion, 1% agarose gel electrophoresis was performed, and the digestion products were observed using a gel imaging system and recovered using a DNA gel recovery kit.

[0064] BgLII-CTRP3-EcoRI was ligated with TKL-pCB-TKR to form the pCB-CTRP3 shuttle plasmid. The ligation system was as follows: TKL-pCB-TKR 2 μL, BgLII-CTRP3-EcoRI 8 μL, and ligation High ver. 210 μL. The ligation was incubated overnight at 16°C. The ligation product was transformed into E. coli strain DH5α competent cells, plated on agar plates, and cultured in a biochemical incubator at 37°C for 12 h. Single colonies of the grown bacteria were picked and amplified in LB solution containing 1‰ ampicillin at 37°C in a shaker for 10 h. Restriction enzyme digestion was performed to screen for pCB-CTRP3 positive clones.

[0065] The dosage is 1×10 4 VP wild-type vaccinia virus (ATCC, USA) was used to infect HEK293 cells (human embryonic kidney cell line, purchased from the Chinese Academy of Sciences Cell Bank) for 2 hours. Then, the HEK293 cells infected with vaccinia virus were transfected with 5 μg of pCB-CTRP3 plasmid. After 48 hours, a mixture of recombinant oncolytic vaccinia virus OVV-CTRP3 and wild-type virus was obtained.

[0066] Screening and plaque selection of recombinant oncolytic vaccinia virus: Given that the recombinant oncolytic vaccinia virus genome carries the xanthine-guanine phosphotransferase gene gpt resistance gene, mycophenolic acid, xanthine, and hypoxanthine were used for screening, and single-clonal plaques of the virus were selected to obtain vaccinia virus OVV-CTRP3 that was not contaminated with wild-type vaccinia virus. (Screening Principle: Mycophenolic acid, as an inhibitor of hypoxanthine dehydrogenase, inhibits guanine (GMP) synthesis, thereby causing cells to lose their ability to synthesize DNA and die, thus inhibiting the replication of vaccinia virus in cells. The xanthine-guanine phosphotransferase gene (gpt gene) synthesizes GMP via a xanthine phosphate intermediate in a xanthine-containing medium, allowing nucleic acid synthesis to proceed normally and viral replication to occur. Recombinant oncolytic vaccinia virus carries the gpt gene and is therefore unaffected by mycophenolic acid inhibition, enabling normal replication. Wild-type vaccinia virus, lacking the gpt gene, is blocked from guanine synthesis by mycophenolic acid, preventing viral replication. Therefore, after the addition of mycophenolic acid, positive clones are not inhibited and continue to survive, while negative clones are inhibited and die, thus achieving the screening objective. The structure of oncolytic vaccinia virus OVV-CTRP3 is shown below.) Figure 1 Next, we identified the constructed oncolytic virus. Using the intermediate sequence of the oncolytic virus TK gene that does not contain the Vkl-L and vTK-R sequences, we designed suitable PCR primers. The primer sequences were F: 5'-TGTGAAGACGATAAATTAATGATC-3', R: 5'-GTTTGCCATACGCTCACAG-3'. This primer amplified the full-length 816bp intermediate sequence of the TK gene. We then identified the wild-type viral contamination of the oncolytic virus OVV-CTRP3. The results are as follows: Figure 2 As shown, lane 2 did not display an 816bp PCR amplification product, while lane 3 displayed an 816bp PCR amplification product, serving as a positive control. The results indicate that the recombinant oncolytic viruses corresponding to lane 2 did not produce PCR bands amplified by the TK gene primers, suggesting that the viruses were not contaminated with wild-type viruses. Figure 3 As shown, lane 2 displays a 741bp PCR amplification product, indicating that the oncolytic virus OVV-CTRP3 carries the CTRP3 gene.

[0067] Example 2:

[0068] RT-PCR was used to detect the expression level of CTRP3 in SMMC-7721 and Hep3B cells infected with recombinant oncolytic vaccinia virus carrying CTRP3.

[0069] SMMC-7721 and Hep3B cells were infected with a recombinant oncolytic vaccinia virus carrying CTRP3 (OVV-CTRP3) at a dose of 1 MOI.

[0070] The control groups were:

[0071] 1) Control group: Equal volume of PBS;

[0072] 2) Recombinant oncolytic vaccinia virus (OVV) only;

[0073] The cells were cultured at 37℃ and 5% CO2 for 24 hours. Total RNA was extracted from the cells using the TRIZOL method, and cDNA was reverse transcribed using the extracted total RNA as a template. Primers were designed using Primer 5.0 software. The cDNA was amplified by real-time quantitative PCR using SYBR qPCR Mix in a 20 μL volume.

[0074] The amplification parameters were: 95℃ for 1 min, 95℃ for 15 s, 60℃ for 1 min, for 40 cycles. Results were analyzed using AppliedBiosystems 7300 real-time PCR software. See below for results. Figure 4 After treatment of SMMC-7721 and Hep 3B cells with OVV-CTRP3, the expression level of CTRP3 was significantly increased at the mRNA level, indicating that OVV-CTRP3 can stably and highly express CTRP3 in liver cancer cells.

[0075] Example 3: Recombination carrying CTRP3

[0076] The effect of oncolytic vaccinia virus on the proliferative activity of liver cancer cells.

[0077] Human hepatocellular carcinoma cells SMMC-7721 and Hep3B were cultured in DMEM (purchased from Gibco) containing 10% fetal bovine serum (FBS) (37℃, 5% CO2, saturated humidity). Fourth-generation cells were seeded at a concentration of 2 × 10⁴ cells / ml, with 100 μl per well in 96-well plates. Control, recombinant oncolytic vaccinia virus (OVV), and recombinant oncolytic vaccinia virus carrying CTRP3 (OVV-CTRP3) groups were established, with each group prepared in triplicate. OVV (0.5 MOI, 1 MOI, 2 MOI, 4 MOI, 8 MOI) and OVV-CTRP3 (0.5 MOI, 1 MOI, 2 MOI, 4 MOI, 8 MOI) were added respectively, and cultured for 72 hours. Then, 20 μl of MTT reaction solution was added, and cultured for another 4 hours. The culture was then terminated, the supernatant was discarded, and 150 μl of DMSO was added to dissolve the culture. The absorbance (A value) of each well was measured at 450 nm using a Thermo Varioskan Flash automated microplate reader. The experiment was repeated three times. The cell inhibition rate was calculated based on the A value using the formula: Cell inhibition rate (%) = (A value of negative control group - A value of drug-treated group) / A value of negative control group × 100%.

[0078] See results Figure 5 With increasing dosage, OVV and OVV-CTRP3 showed increasingly significant inhibitory effects on the proliferation of liver cancer cells, and OVV-CTRP3 showed significantly stronger inhibitory effects on the proliferation of human liver cancer cells SMMC-7721 and Hep3B than OVV. Figure 6 As shown, with increasing treatment time, the inhibitory effects of OVV and OVV-CTRP3 on the proliferation of liver cancer cells became more significant, and the inhibitory effect of OVV-CTRP3 on the proliferation of human liver cancer cells SMMC-7721 and Hep 3B was significantly stronger than that of OVV.

[0079] In conclusion, increasing the expression level of CTRP3 can achieve good anti-cancer and cancer-suppressing effects, especially for the prevention and treatment of liver cancer.

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

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[0085] sequence list <110> Xiao Boduan <120> Antitumor activity of CTRP3 protein, recombinant oncolytic virus encoding it, preparation method and application <140> 2021108221642 <141> 2021-07-20 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 741 <212> DNA <213> CTRP3 sequence (Artificial Sequence) <400> 1 atgctttgga ggcagctcat ctattggcaa ctgctggctt tgtttttcct ccctttttgc 60 ctgtgtcaag atgaatacat ggagtctcca caaccggag gactaccccc agactgcagt 120 aagtgttgtc atggagacta cagctttcga ggctaccaag gcccccctgg gccaccgggc 180 cctcctggca ttccaggaaa ccatgggaaac aatggcaaca atggagccac tggtcatgaa 240 ggagccaaag gtgagaaggg cgacaaaggt gacctggggc ctcgaggggga gcgggggcag 300 catggcccca your area gggctacccg gggattccac cagaacttca gattgcattc 360 atggcttctc tggcaaccca cttcagcaat cagaacagtg ggattatctt cagcagtgtt 420 gagaccaaa ttggaaactt ctttgatgtc atgactggta gatttggggc cccagtatca 480 ggtgtgtatt tcttcacctt cagcatgatg aagcatgagg atgttgagga agtgtatgtg 540 taccttatgc aaatggcaa cacagtcttc agcatgtaca gctatgaaat gaagggcaaa 600 tcagatacat ccagcaatca tgctgtgctg aagctagcca aaggggatga ggtttggctg 660 cgaatgggca atggcgctct ccatggggac caccaacgct tctccacctt tgcaggattc 720 ctgctctttg aaactaagtga a 741 <210> 2 <211> 4867 <212> DNA <213> pCB sequence carrying Vkl-L, vTK-R, and gpt genes (Artificial SequenceArtificialSequence) <400> 2 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 atacccgca agatacctgg attgaacagc cgtgggatat gggcgtcgta ttcgtcccgc 840 caatctccgg tcgctaatct tttcaacgcc tggcactgcc gggcgttgtt cttttaact 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 taaaataaa cattgattaa atttaatat aatacttaaa aatggatgtt gtgtcgttag 1320 aaaccgtt tatgtatttt gaggaaatttg ataatgagtt agattacgaa ccagaaagtg 1380 caaatgaggt cgcaaaaaaa ctgccgtac aaggacagtt aaactatta ctaggagaat 1440 tattttttct tagtaagtta cagcgacacg gtatattaga tggtgccacc gtagtgtata 1500 taggatctgc tcccggtaca catatacgtt atttgagaga tcattctat aatttaggag 1560 tgatcatcaa atggatgcta attgacggcc gccatcatga tcctatttta atggattgc 1620 gtgatgtgac tctagtgact cggttcgttg atgaggaata tctacgatcc atcaaaaac 1680 aactgcatcc ttctaagatt attttaattt ctgatgtgag atccaacga ggaggaatg 1740 aacctagtac ggcggattta ctaagtaatt acgctctaca aaatgtcatg attagtattt 1800 taaaccccgt ggcgtctagt cttaatgga gatgcccgtt tccagatca tggatcagg 1860 acttttatat cccacggt aaaaatgt tacaactt tgctccttca tattcagctg 1920 aaatgagatt attaagtatt tataccggtg agaacatgag actgactcgg gccgcgttgc 1980 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaatcga cgctcaagctc 2040 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 2100 tcgtgcgctc tcctgttccg acctgccgc 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 gcggtggtt 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 acctactca gcgatctgtc tattcgttc atccatagtt gcctgactcc 2820 ccgtcgtgta gatactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 2880 taccgcgaga cccacgctca ccggctccag attackcagc ataaaccag cccaccggaa 2940 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct atttattgtt 3000 gccgggaagc taagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 3060 ctgcaggcat cgtgtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 3120 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaagcggtt agctccttcg 3180 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 3240 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 3300 actcaaccaa gtcattctga gatagtgta tgcggcgacc gagttgctt tgcccggcgt 3360 siacacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaac 3420 gttcttcggg gcgaaaactc tcaggatct taccgctgtt gagatccagt tcgatgtac 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 tttttaagt atttggctag tcagatgat gatcttcat tattctgat attgcaaatc 4260 actcaatg tagtagact ttctgttatt attattgatc caatcaaaa attaattaga 4320 agccgtgggt cattgttatg aatctctttc agaggaatac agacaattga caaattcac 4380 agactttca gattttaaa aactgtttaa caggtccct attgacagat ggaagggtca 4440 aacttaataa aggatatttg ttcgactttg tgattagttt gatgcgattc aaaaaagaat 4500 cctctctagc taccaccgca atagatccctg ttagatacat agatcctcgt cgcaatcg 4560 cattttctaa cgtgatggat atattaagt cgaataagt gacaataat taattcttta 4620 ttgtcatcat gaacggcgga catattcagt tgataatcgg cccatgttt tcaggtaaaa 4680 gtacagaatt aattagacga gttagacgtt atcaatagc tcaataaa tgcgtgacta 4740 taaaatattc taacgataat agatacggaa cgggactatg gacccatgat agaatatt 4800 ttgaagcatt ggaagcact aaactatgtg atctcttgga atcattaca gatttctccg 4860 tag 4867 <210> 3 <211> 24 <212> DNA <213> Primer sequence (Artificial Sequence) <400> 3 tgtgaagacg ataaattaat gatc 24 <210> 4 <211> 19 <212> DNA <213> Primer sequence (Artificial Sequence) <400> 4 gtttgccata cgctcacag 19

Claims

1. The application of recombinant oncolytic virus in the preparation of drugs for treating liver cancer, characterized in that, The recombinant oncolytic virus is operable to insert a foreign gene sequence, the foreign gene sequence encoding the CTRP3 protein, and the sequence of the gene encoding the CTRP3 protein is shown in SEQ ID NO.1; The drug is a drug that highly expresses the CTRP3 protein.

2. The application according to claim 1, characterized in that, The recombinant oncolytic virus is formulated into a preparation for administration via adjacent normal injection, intratumoral injection, intraperitoneal administration, subarachnoid intracavitary administration, or intravenous administration; the recombinant oncolytic virus is further formulated into a therapeutic agent containing a therapeutically effective amount by adding a pharmaceutically acceptable carrier, adjuvants, and / or active ingredients for treating cancer.

3. The application according to claim 1, characterized in that, The exogenous gene sequence was inserted into the thymidine kinase gene.

4. The application according to claim 1, characterized in that, The recombinant oncolytic virus includes selectively replicating recombinant oncolytic viruses and / or non-replicating recombinant oncolytic viruses.

5. The application according to claim 4, characterized in that, The recombinant oncolytic viruses include adenoviruses, poxviruses, herpes simplex viruses, measles viruses, Semliki Forest virus, vesicular stomatitis virus, polioviruses, retroviruses, reoviruses, Seneca Valley virus, Ek enteroviruses, Coxsackie virus, Newcastle disease virus, and Malaba virus.

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