CN115634281A - Anti-tumor effect of CTRP3 protein, recombinant oncolytic virus encoding same, preparation method and application - Google Patents

Abstract

The invention discloses an anti-tumor effect of a CTRP3 protein, a recombinant oncolytic virus encoding the same, a preparation method and application thereof, and relates to the field of gene therapy. The invention effectively combines gene therapy and virus therapy of malignant tumors to prepare the recombinant oncolytic virus capable of efficiently expressing CTRP3, and enhances the killing capacity on tumors compared with simple gene therapy or virus therapy.

Description

Anti-tumor effect of CTRP3 protein, recombinant oncolytic virus encoding same, preparation method and application

Technical Field

The invention relates to the field of gene therapy, in particular to an anti-tumor effect of a CTRP3 protein, a recombinant oncolytic virus encoding the same, a preparation method and application thereof, wherein the CTRP3 gene for inhibiting tumor growth is inserted into a virus genome to carry out virus replication, the CTRP3 is generated by encoding, and the expression quantity of the CTRP3 is increased to improve the anti-cancer and cancer-prevention efficiency of the recombinant oncolytic virus.

Background

C1q Tumor Necrosis Factor (TNF) -related protein 3 (CTRP 3) is a newly discovered adipokine belonging to the C1q/TNF superfamily, which has high homology to adiponectin. The CTRP3 can play a plurality of roles in cardiovascular, metabolic, inflammatory and other related diseases in an internal secretion mode. However, CTRP3 has not been reported in terms of tumors, and it was found in this study that CTRP3 can effectively inhibit tumor growth. Therefore, the use of oncolytic virus carrying CTRP3 for the treatment of tumors is expected to be a new possibility.

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 Targeted Gene Virotherapy (CTGVT)" strategy (i.e., inserting an anti-cancer gene into an oncolytic virus vector, organically combining the viral therapy with the gene therapy to become a gene viral therapy means with a strong killing effect), the oncolytic virus loaded with the targeted gene or the cytokine has been greatly successful in the tumor targeted therapy. Oncolytic Vaccinia Virus (oncolytica Virus) is a novel vector for viral therapy with the advantages: the virus has good stability, low pathogenicity, high gene transfection efficiency and good safety. The oncolytic vaccinia virus can selectively infect tumor cells, replicate in the cells and kill the tumor cells, and has low toxicity to normal tissues and cells.

Therefore, the research on the action mechanism of the CTRP3 for inhibiting the tumor growth and the application thereof in the treatment of tumors or cancers is a hot spot and difficulty of the current research.

Disclosure of Invention

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, namely, the anti-tumor effect of CTRP3 protein, the recombinant oncolytic virus encoding the same, and the preparation method and the application thereof. In particular, a recombinant oncolytic virus comprising an insertion of a foreign gene sequence encoding the CTRP3 gene is administered to a subject. 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 tumor.

In order to achieve the above object, the present invention provides the following technical solutions:

the CTRP3 protein or the gene for coding the CTRP3 protein is applied to preventing or treating tumors and/or cancers.

Because the current research of the CTRP3 on the tumor is not reported basically, the research discovers that the CTRP3 can effectively inhibit the growth of the tumor for the first time.

The invention is realized by the following technical scheme:

the invention also provides a recombinant oncolytic virus, wherein the recombinant oncolytic virus can be operably inserted into an exogenous gene sequence, and the exogenous gene sequence codes and generates the CTRP3 protein.

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 can enhance the properties of oncolytic viruses to replicate within tumor cells. Foreign gene segments are inserted into a TK gene sequence by using a gene recombination technology, so that an oncolytic virus vector is subjected to TK sequence deletion, and an oncolytic vaccinia virus carrying a foreign gene is further constructed on the basis, so that the damage of the recombinant oncolytic virus to normal cells is reduced, the specific recognition of tumor cells is improved, the treatment efficiency is improved, and the side effect is reduced.

Preferably, the sequence of the exogenous gene sequence CTRP3 is shown as SEQ ID NO.1, and can play the role of the biological function of resisting tumors, or is a sequence which is added, reduced or replaced on the basis of the sequence of the SEQ ID NO.1 and has the same or similar function with the sequence of the SEQ ID NO. 1.

As the skilled person knows, for the sequenced gene, the inventor can not isolate and purify enough similar substances to detect the performance rules, therefore, based on the exogenous gene sequence CTRP3 sequence (SEQ ID NO. 1) of the present application, the addition, reduction or replacement of the sequence on the basis of the specific sequence and the same or similar function as SEQ ID NO.1 should be considered as being within the protection scope of the present application, if the sequence has a certain degree of homology (i.e. similar degree of sequence structure).

Preferably, the sequence of the exogenous gene is formed by a nucleotide sequence which has at least 60 percent of identity with the sequence of SEQ ID NO. 1.

Preferably, the recombinant oncolytic virus includes, without limitation, a selectively replicating recombinant oncolytic virus and/or a non-replicating oncolytic virus.

Preferably, the selectively replicating recombinant oncolytic virus is derived from a group including, but not limited to, adenoviruses, poxviruses, herpes simplex viruses, measles viruses, semliki forest viruses, vesicular stomatitis viruses, polioviruses, retroviruses, reoviruses, seneca valley viruses, enteroviruses of the ekg type, coxsackieviruses, newcastle disease viruses, and malaba viruses that have an oncolytic effect.

Preferably, the non-replicating recombinant oncolytic virus is derived from a group including, but not limited to, adenovirus, poxvirus, herpes simplex virus, measles virus, semliki forest virus, vesicular stomatitis virus, poliovirus, retrovirus, reovirus, seneca valley virus, enterovirus of the type ekg, coxsackie virus, newcastle disease virus and malaba virus having oncolytic effect.

In particular, the recombinant oncolytic virus is a recombinant oncolytic poxvirus.

The invention also provides the use of the recombinant oncolytic viruses described above, all of which can be formulated to contain a therapeutically effective amount of a therapeutic agent.

Methods of use preferably, all of the recombinant oncolytic viruses described above are formulated for administration by paracancerous injection, intratumoral injection, intraperitoneal administration, subarachnoid space 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 polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

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 the 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 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, 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 based on conventional administration to a subject. 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 includes, but is 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 tumor, osteochondrosis, 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, skin squamous cell carcinoma, mesothelioma, multi-cuspid tumor, ovarian cancer, pancreatic endocrine tumor, glucagon tumor, pancreatic cancer, penile cancer, pituitary cancer, chondrosarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, trophoblastic cell carcinoma, fetal cancer, endometrial cancer, uterine cancer, myeloma, vaginal cancer, meningeal cancer, mycosis, meningioma, breast cancer, meningeal cancer, mycosis, and meningeal cancer. More preferably, the tumor and/or cancer is liver cancer.

Preferably, the kit comprising the recombinant oncolytic virus or therapeutic agent described above comprises a container, which may comprise 1 set. 1 set of containers contains the CTRP3 containing recombinant oncolytic virus or therapeutic agent for convenient administration.

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

The design principle of the invention is as follows: the inventor researches and finds that the CTRP3 can effectively inhibit the growth of tumors, and can exert the anti-tumor biological function of the CTRP3 carried by oncolytic virus.

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) Synthesizing a pCB shuttle plasmid according to a map and a sequence;

3) Carrying out double digestion on the foreign gene sequence and the pCB shuttle plasmid by BgLII and EcoRI endonucleases respectively to obtain digestion products BgLII-foreign gene sequence-EcoRI and TKL-pCB-TKR linearized plasmids;

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

5) Screening and cloning of pCB-foreign Gene sequence plasmids: transforming E.coli strain DH5 alpha competence with pCB-exogenous gene sequence plasmid, and screening colibacillus resistance gene to obtain 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.

Wherein the foreign gene sequence codes and generates a CTRP3 protein.

Compared with the prior art, the invention has the advantages that:

a) The invention effectively combines gene therapy and virus therapy of malignant tumor to prepare the recombinant oncolytic virus which can efficiently express and has tumor inhibition effect, and enhances the killing ability to tumor compared with simple gene therapy or virus therapy.

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

c) The invention adopts a mode of virus replication related gene deletion, ensures the intratumoral specific replication of the virus and greatly enhances the safety of the oncolytic vaccinia virus vector.

d) CTRP3 is a cytokine that is released into the tumor microenvironment after its expression, and inhibits tumor growth in a wide range, and has one-to-many therapeutic effects compared to non-secretory genes.

e) The invention effectively combines gene therapy and virus therapy of malignant tumors to prepare the recombinant oncolytic virus capable of efficiently expressing CTRP3, and enhances the killing capacity on tumors compared with simple gene therapy or virus therapy.

f) According to the invention, the recombinant oncolytic vaccinia virus carrying CTRP3 is constructed, so that a good anti-solid tumor effect is exerted, and 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 microenvironment.

Drawings

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

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

FIG. 2 PCR/agarose gel electrophoresis to identify whether recombinant oncolytic vaccinia virus contains wild-type viral contamination;

FIG. 3 PCR/agarose gel electrophoresis to identify whether recombinant oncolytic vaccinia virus carries an exogenous gene CTRP3;

FIG. 4RT-PCR assay of the expression level of CTRP3 in recombinant oncolytic vaccinia virus carrying CTRP3 after infection of SMMC-7721 and Hep3B cells;

FIGS. 5 and 6 show that the MTT method detects the killing effect of recombinant oncolytic vaccinia virus OVV-CTRP3 on SMMC-7721 and Hep3B cells;

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

Detailed Description

Some embodiments of the invention are disclosed below, and those skilled in the art can appropriately modify the process parameters to achieve the invention according to the disclosure herein. It is expressly intended that all such alterations and modifications which are obvious to those skilled in the art are deemed to be incorporated herein by reference. While the method and application of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the method and application described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

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

the pCB shuttle plasmid was prepared, and prepared with reference to the following references: construction of a recombinant vaccinia virus vector with double screening markers of Zeocin and GFP, international epidemiology infectious disease journal, no. 39, no. 3 in 6 months of 2012; panicali D, paolettie. Construction of Poxviruses as cloningvectors; 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 to 4931;

obtaining vTK-L, vTK-R and gpt gene sequences by inquiring in NCBI (https:// www.ncbi.nlm.nih.gov /); the genes of Nanjing King Musrey Biotechnology Limited company are entrusted to synthesize vTK-L, vTK-R and gpt gene sequences, and the genes are respectively inserted into blank pCB plasmids.

The pCB (as SEQ ID NO. 2) plasmid carrying the Vkl-L, vTK-R and gpt genes as described in FIG. 7, pCB sequence was constructed by the above procedure.

The CTRP3 gene oncolytic vaccinia virus specifically comprises the following construction steps:

the cDNA sequence of CTRP3 obtained by the Gene Synthesis method is as follows

Obtaining the mRNA sequence of the CTRP3 by searching in NCBI (https:// www.ncbi.nlm.nih.gov /), entrusting Nanjing Kingsley Biotech limited to synthesize a DNA (such as SEQ ID NO. 1) sequence according to the mRNA sequence gene, and inserting a T vector for sequencing;

it should be noted that other pCBs, such as shuttle plasmids pCBs without selectable markers, may also be used in the present invention.

After double digestion of the correctly sequenced DNA with BgLII and EcoRI, the target gene fragment is recovered according to the instruction of the DNA gel recovery kit, and the digestion product BgLII-CTRP3-EcoRI is obtained. Meanwhile, the pCB original plasmid is also subjected to BgLII and EcoRI double enzyme digestion to form a TKL-pCB-TKR linearized plasmid, and the reaction system is as follows: CTRP3/pCB 20ul, bgLII 1ul, ecoRI,10xTango Buffer 6ul, ddH2O 2ul; carrying out enzyme digestion at 37 ℃ for 2-3h, carrying out 1% agarose gel electrophoresis after the enzyme digestion is finished, observing by a gel imager, and recovering a digestion product through a DNA gel recovery kit.

BgLII-CTRP3-EcoRI was ligated to TKL-pCB-TKR to form the pCB-CTRP3 shuttle plasmid, the ligation was as follows: TKL-pCB-TKR 2ul, bgLII-CTRP3-EcoRI 8ul, and ligation High ver.210ul. Water bath connection at 16 ℃ is carried out overnight, the connection product is transformed into E.coli strain DH5 alpha competence, and after agar plate coating, the E.coli strain is cultured in a biochemical incubator at 37 ℃ for 12h. Selecting a growing bacterial monoclonal, carrying out shake amplification for 10 hours at 37 ℃ in an LB solution containing 1 per mill ampicillin, and carrying out enzyme digestion identification and screening of pCB-CTRP3 positive clones.

With a dosage of 1X 10 ⁴ vp wild vaccinia virus (ATCC company in USA) is used for infecting HEK293 cells (human embryonic kidney cell line, purchased from cell bank of Chinese academy of sciences) for 2 hours, then 5 mu g of pCB-CTRP3 plasmid is transfected to the HEK293 cells infected with vaccinia virus, and a recombinant oncolytic vaccinia virus OVV-CTRP3 and wild virus mixed solution is obtained after 48 hours;

screening and plaque selection of the recombinant oncolytic vaccinia virus: considering that the genome of the recombinant oncolytic vaccinia virus carries a gene for resisting xanthine guanine phosphotransferase gene gpt, mycophenolic acid, xanthine and hypoxanthine are used for screening, and virus monoclonal plaques are picked, so that the OVV-CTRP3 of the vaccinia virus without the pollution of the wild type vaccinia virus is obtained. (screening principle: mycophenolic acid as an inhibitor of hypoxanthine dehydrogenase inhibits Guanine (GMP) synthesis, thereby rendering the cell incapable of synthesizing DNA and dead, and inhibits replication of vaccinia virus in the cell, whereas the xanthine guanine phosphotransferase gene (gpt gene) salvages synthetic GMP via xanthine phosphate intermediates in a medium containing xanthine, thereby allowing nucleic acid synthesis to proceed normally, and viral replication to proceed normally. 5 'TGTGAAGACGATAATTAATGATC-3', R: 5 'GTTTGCCATACGCTCAAG-3', wherein the primer can amplify 816bp in the middle part sequence of the TK gene, and identify the wild type virus contamination of the oncolytic virus OVV-CTRP3, the result is shown in FIG. 2, lane 2 does not show the PCR amplification product of 816bp, lane 3 shows the PCR amplification product of 816bp, as a positive control, the result shows that the recombinant oncolytic virus corresponding to lane 2 has no PCR band product amplified by the TK gene primer, which indicates that the virus has no wild type virus contamination, as shown in FIG. 3, lane 2 shows the PCR amplification product of 741bp, indicating that oncolytic virus OVV-CTRP3 carries the CTRP3 gene.

Example 2:

and (3) detecting the expression level of the CTRP3 when the recombinant oncolytic vaccinia virus carrying the CTRP3 infects SMMC-7721 and Hep3B cells by RT-PCR.

Recombinant oncolytic vaccinia virus carrying CTRP3 (OVV-CTRP 3) infected SMMC-7721 and Hep3B cells at a dose of 1MOI,

the control groups were:

1) Control group: an equal amount of PBS;

2) Recombinant Oncolytic Vaccinia Virus (OVV) alone;

37℃，5％CO ₂ incubated 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: 95 ℃ for 1min,95 ℃ for 15s,60 ℃ for 1min,40 cycles. The results were analyzed by applied biosystems 7300real-time PCR instrument software. The results are shown in FIG. 4, after the OVV-CTRP3 treats SMMC-7721 and Hep3B cells, the expression level of CTRP3 is obviously increased on mRNA level, which indicates that OVV-CTRP3 can stably and highly express CTRP3 in hepatoma cells.

Example 3: recombination with CTRP3

Effect of oncolytic vaccinia virus on the proliferative activity of hepatoma cells.

Human hepatoma cells SMMC-7721 and Hep3B were cultured in DMEM (purchased from Gibco) containing 10% Fetal Bovine Serum (FBS) (37 ℃, 5% CO2, saturation humidity), and the fourth generation cells were inoculated at a concentration of 2X 104/ml into 100. Mu.l of 96-well plates. A control group, a recombinant oncolytic vaccinia virus group (OVV), and a recombinant oncolytic vaccinia virus group carrying CTRP3 (OVV-CTRP 3) were set, and each group was plated with 3 replicate wells. OVV (0.5 MOI, 1MOI, 2MOI, 4MOI, 8 MOI), OVV-CTRP3 (0.5 MOI, 1MOI, 2MOI, 4MOI, 8 MOI) were added, the culture was continued for 72 hours, 20. Mu.l of the MTT reaction solution was added, the culture was terminated after the culture was continued for 4 hours, the supernatant culture solution was discarded, 150l of DMSO was added for dissolution, the absorbance (A value) of each well was measured at a wavelength of 450nm on a Thermo Varioskan Flash full-automatic enzyme calibrator, 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: cell inhibition (%) = (negative control a value-drug added a value)/negative control a value × 100%.

The results are shown in figure 5, with the increase of the dosage, the inhibition effect of OVV and OVV-CTRP3 on the proliferation of the hepatoma cells is more obvious, and the inhibition effect of OVV-CTRP3 on the proliferation of human hepatoma cells SMMC-7721 and Hep3B is obviously stronger than that of OVV. As shown in figure 6, with the increase of treatment time, the inhibition effect of OVV and OVV-CTRP3 on the proliferation of hepatoma carcinoma cells is more obvious, and the inhibition effect of OVV-CTRP3 on the proliferation of human hepatoma carcinoma cells SMMC-7721 and Hep3B is obviously stronger than that of OVV.

In conclusion, the improvement of the expression level of the CTRP3 can realize good anticancer and cancer-suppressing effects, especially the prevention and treatment of liver cancer.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> tip of Chinese zodiac

<120> antitumor effect of CTRP3 protein, recombinant oncolytic virus encoding same, 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 caaaccggag gactaccccc agactgcagt 120

aagtgttgtc atggagacta cagctttcga ggctaccaag gcccccctgg gccaccgggc 180

cctcctggca ttccaggaaa ccatggaaac aatggcaaca atggagccac tggtcatgaa 240

ggagccaaag gtgagaaggg cgacaaaggt gacctggggc ctcgagggga gcgggggcag 300

catggcccca aaggagagaa gggctacccg gggattccac cagaacttca gattgcattc 360

atggcttctc tggcaaccca cttcagcaat cagaacagtg ggattatctt cagcagtgtt 420

gagaccaaca ttggaaactt ctttgatgtc atgactggta gatttggggc cccagtatca 480

ggtgtgtatt tcttcacctt cagcatgatg aagcatgagg atgttgagga agtgtatgtg 540

taccttatgc acaatggcaa cacagtcttc agcatgtaca gctatgaaat gaagggcaaa 600

tcagatacat ccagcaatca tgctgtgctg aagctagcca aaggggatga ggtttggctg 660

cgaatgggca atggcgctct ccatggggac caccaacgct tctccacctt tgcaggattc 720

ctgctctttg aaactaagta a 741

<210> 2

<211> 4867

<212> DNA

<213> pCB Sequence carrying Vkl-L, vTK-R and gpt genes (Artificial Sequence)

<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

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> 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 (10)

1. Use of CTRP3 protein or a gene encoding said CTRP3 protein in the prevention or treatment of a tumour and/or cancer.
2. 2. A recombinant oncolytic virus operable to insert a foreign gene sequence encoding the CTRP3 protein of claim 1 into a host cell.
3. 3. The recombinant oncolytic virus of claim 2, wherein the exogenous gene sequence is inserted into a TK gene.
4. 4. The recombinant oncolytic virus of claim 2, wherein the sequence of the exogenous gene is shown as SEQ ID No.1 or is a sequence which is added, reduced or replaced on the basis of the sequence of the SEQ ID No.1 and has the same or similar function as the sequence of the SEQ ID No. 1.
5. 5. The recombinant oncolytic virus of claim 2, wherein the recombinant oncolytic virus comprises a selectively replicating recombinant oncolytic virus and/or a non-replicating recombinant oncolytic virus.
6. 6. The recombinant oncolytic virus of claim 5, wherein the selectively replicative recombinant oncolytic virus is derived from a group consisting of, but not limited to, 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 oncolytic effects;

   the non-replicating recombinant oncolytic virus is derived from a group including, but not limited to, adenoviruses, poxviruses, herpes simplex viruses, measles viruses, semliki forest viruses, vesicular stomatitis viruses, polioviruses, retroviruses, reoviruses, seneca valley viruses, enteroviruses of the eke type, coxsackieviruses, newcastle disease viruses, and malaba viruses that have an oncolytic effect.
7. 7. Use of the recombinant oncolytic virus of claims 2-6, wherein the recombinant oncolytic virus is for the prevention or treatment of a tumor and/or cancer.
8. 8. The use of the recombinant oncolytic virus of claim 7, wherein the recombinant oncolytic virus is formulated into a formulation comprising, but not limited to, by paraneoplastic injection, intratumoral injection administration, intraperitoneal administration, subarachnoid intracavitary administration, or intravenous administration; the recombinant oncolytic virus is formulated to contain a therapeutically effective amount of a therapeutic agent by the addition of pharmaceutically acceptable carriers, adjuvants and/or other active ingredients for the treatment of cancer.
9. 9. Use of the recombinant oncolytic virus of claim 7, wherein the tumor and/or cancer comprises, but is 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 tumor, osteochondrosis, 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, trophoblastic carcinoma, placental cancer, endometrial cancer, vaginal cancer, meningioma, mycosis, meningioma, pleural cancer, and pleural cancer.
10. 10. A method of producing a recombinant oncolytic virus according to claims 1-5 and/or claims 7-9, comprising the steps of:

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

    2) Synthesizing pCB shuttle plasmid according to map and sequence;

    3) 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;

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

    5) Screening and cloning of pCB-exogenous gene sequence plasmid: transforming E.coli strain DH5 alpha competence with pCB-exogenous gene sequence plasmid, and screening colibacillus resistance gene to obtain pCB-exogenous gene sequence positive clone;

    6) 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.

Images