CN111518833B - Construction method and application of an oncolytic adenovirus carrying AIM2 gene - Google Patents

Abstract

The invention relates to a method for constructing an oncolytic adenovirus carrying an AIM2 gene, belonging to the technical field of biomedicine. First, construct the CAIX promoter vector pZD55‑CAIX through PCR reaction and cloning technology, then construct the adenovirus shuttle plasmid pZD55‑CAIX‑AIM2, and finally co-transfect HEK293 with the identified plasmid pZD55‑CAIX‑AIM2 and the adenovirus backbone plasmid pBHGE3 cells, after virus plaques appeared on the cells, the target oncolytic adenovirus Ad-CAIX-AIM2 was obtained. The oncolytic adenovirus can specifically reach tumor cell sites, efficiently and specifically kill tumor cells without affecting normal cells, and has strong replication ability in tumor tissues.

Description

Construction method and application of an oncolytic adenovirus carrying AIM2 gene

technical field

The invention relates to a construction method of an oncolytic adenovirus, in particular to a construction method and application of a tumor-specific oncolytic adenovirus carrying an AIM2 therapeutic gene, and belongs to the technical field of biomedicine.

Background technique

Renal cell carcinoma (RCC), referred to as kidney cancer, accounts for 2%-3% of adult malignant tumors and is the most lethal tumor of the urinary system. Its incidence rate is increasing at a rate of about 2.5% per year. Renal cancer is highly malignant and difficult to detect early. 30% of patients have metastasized at the time of diagnosis, and 20% to 30% of patients still develop metastases after radical operation. The 2-year mortality rate of metastatic patients is as high as 95%. Radiotherapy, chemotherapy and cytokine therapy are not ideal. At present, sorafenib (Sorafenib) is the representative tyrosine kinase inhibitor in the clinical treatment of metastatic patients, but its drug resistance is high, and the disease progression can be controlled for only 6 months. . Therefore, there is an urgent need for new treatments for advanced RCC.

Oncolytic adenovirus (OAd) refers to a type of adenovirus that has been genetically engineered to replicate and kill tumor cells, releasing progeny viruses to continue to function. In 1996, Onyx Company reported in Science that the adenovirus Onyx-015, which knocked out the E1B 55KD protein coding gene, only replicated in tumor cells and killed them without affecting normal cells, thereby turning on the oncolytic virus (OV ) the door to the treatment of tumors. OV has always been a hot spot in the research of tumor therapy. Currently, adenovirus, herpes simplex virus (HSV), coxsackie virus, poxvirus, poliovirus, measles virus, reovirus and many other OVs have entered clinical trials. Since 2000, 18 phase I-III clinical trials of Onyx-015 in the treatment of tumors have been completed. In 2006, the domestically developed similar virus H101 was launched on the market, becoming the world's first OAd drug. In 2015, the FDA approved Amgen's genetically modified HSV-1 product T-Vec for the treatment of advanced melanoma, which became a milestone in the treatment of tumors with OV. At present, T-Vec has been widely used in the United States, Europe and Australia. In May 2017, Jiangsu Hengrui Medicine spent US$102 million to purchase OBP-301, an OAd drug regulated by the hTERT promoter from Oncolys BioPharma in Japan. OAV is once again attracting great attention. Although clinical trials have proved that Onyx-015 has high treatment safety, its anti-tumor effect is weak, and the curative effect of single use is only 15%. In order to improve the anti-tumor effect of Onyx-015, the present inventors used the vector pZD55 modified by Onyx-015 to carry anti-cancer genes, which improved the effect of Onyx-015 in treating renal cancer to a certain extent. However, the E1B 55KD protein coding gene was deleted during Onyx-015 transformation, which reduced its replication ability and affected the therapeutic effect.

Carbonic anhydrase 9 (Carbonic anhydrase IX, CAIX), also known as G250, is one of the isomers of the carbonic anhydrase family. It is a transmembrane glycoprotein composed of acidic amino acids and plays an important role in regulating cell proliferation and transformation. CAIX is highly expressed in renal cancer, liver cancer, gastric cancer, bladder cancer, etc., but rarely expressed and expressed in normal tissues, such as stomach, bile duct, pancreas, small intestine and vas deferens epithelium. Therefore, CAIX serves as a relevant specific antigen for these solid tumors. Studies have shown that the OAd of the CAIX promoter can specifically target and kill renal cancer cells.

Melanoma deficiency factor 2 (Absent in melanoma, AIM2) is a member of the interferon-induced protein family, which can recognize double-stranded DNA as a cytoplasmic DNA sensor, thereby recruiting the cytoplasmic adapter protein ASC, and inducing the assembly of the inflammatory complex, which in turn causes Activation of Caspase-1 and production of IL-1β, IL-18. AIM2 is also involved in tumor development as a tumor suppressor gene. The inactivation, deletion or mutation of AIM2 gene has been found in some malignant tumors such as endometrial cancer, breast cancer, liver cancer, prostate cancer, squamous cell carcinoma, gastric cancer and colon cancer. During melanomagenesis, overexpression of AIM2 reversed the tumor phenotype of melanoma and delayed the proliferation of fibroblasts in mice. Loss of AIM2 expression in tumor cells of colorectal cancer patients is closely related to poor prognosis. AIM2 is low-expressed in renal cancer solid tumors, and overexpression of AIM2 inhibits the growth, metastasis and invasion of tumor cells. These studies suggest that changes in AIM2 gene expression play an important role in tumor progression.

Contents of the invention

The purpose of the present invention is to solve the shortcomings of existing drugs for the treatment of kidney cancer and the weak tumor killing effect and low curative effect of the oncolytic virus Onyx-015, and provide a method for constructing an oncolytic adenovirus carrying the AIM2 gene.

Technical solutions

The present inventors found that AIM2, as an immune regulation and tumor suppressor gene, is beneficial to enhance the effect of CAIX promoter OAd in treating solid tumors. Therefore, a tumor-specific conditional OAd was constructed to regulate the transcriptional activity of the E1A gene in the hypoxic environment of the tumor through the CAIX promoter, and improve the replication ability of the virus in the tumor tissue; in addition, the virus carries the AIM2 gene, which can pass the therapeutic gene AIM2. The expression enhances the immune killing and growth inhibition of CAIX promoter OAd on tumor cells, so as to achieve efficient and specific killing of tumor cells. The specific plan is as follows:

A method for constructing an oncolytic adenovirus carrying an AIM2 gene, comprising the steps of:

(1) Construction of CAIX promoter vector pZD55-CAIX

Through PCR reaction and cloning technology, the 218bp CAIX core promoter fragment was obtained from the plasmid pGL3-CAIX, and then inserted into the adenovirus plasmid pZD55 to obtain pZD55-CAIX;

(2) Construction of adenovirus shuttle plasmid pZD55-CAIX-AIM2

Using pEF-BOS-AIM2 as a template, the 1029bp AIM2 sequence without terminator was obtained by PCR amplification, which was cloned into the HindIII and XbaI sites of the proliferation-deficient adenovirus vector pCA13 to obtain the pCA13-AIM2 plasmid; from pCA13-AIM2 Obtain the CMV+AIM2+PolyA expression cassette from the plasmid and insert it into pZD55-CAIX to obtain the recombinant adenovirus shuttle plasmid pZD55-CAIX-AIM2;

(3) Preparation of oncolytic adenovirus carrying AIM2 gene

The identified plasmid pZD55-CAIX-AIM2 and the adenovirus backbone plasmid pBHGE3 were co-transfected into HEK293 cells, and the target oncolytic adenovirus Ad-CAIX-AIM2 was obtained after virus plaques appeared in the cells.

Further, in step (1), the specific method for obtaining the CAIX core promoter fragment of 218bp from the plasmid pGL3-CAIX is as follows: design primer 1, and introduce the Xho I restriction site in the upstream primer of primer 1, and in primer 1 The SnaBI restriction site was introduced into the downstream primers, wherein the upstream primer sequence was: 5'-TTGTCTCGAGACCTGCCCCTCACTC-3', and the downstream primer sequence was: 5'-TTGCTACGTACATGCGGCTGACTG-3'; using the designed primer 1 and plasmid pGL3-CAIX, PCR The CAIX promoter fragment was amplified to obtain a PCR product, and the PCR product was double-digested with XhoI and SnaBI restriction endonucleases. After incubation, 1.5% agarose gel electrophoresis was performed, and the gel was recovered to obtain a 218bp CAIX core promoter fragment.

Further, in step (2), the upstream primer sequence used for PCR amplification is 5'-GATAAGCTTATGGAGAGTAAATACAAGGAGATACTCTT-3', and the downstream primer sequence is 5'-TGCTCTAGACTATGTTTTTTTTTTGGCCTTAATAA-3'; the PCR cycle condition parameters are: 94°C for 3min, 94°C 1min, 55°C 1min, 72°C 1min 30s, 32 cycles, 72°C 10min.

Use of the above-mentioned oncolytic adenovirus carrying the AIM2 gene in the preparation of drugs for treating kidney cancer.

Beneficial effects of the present invention: the oncolytic adenovirus carrying the AIM2 gene constructed in the present invention can specifically reach tumor cell sites, efficiently and specifically kill tumor cells without affecting normal cells, and has strong replication ability in tumor tissues. Among them, on the one hand, AIM2 acts as a tumor suppressor, which can induce the apoptosis of tumor cells through the inflammasome pathway or autophagy pathway; at the same time, as a tumor immune regulation gene, AIM2 induces cytokines such as IL-1β through the inflammasome signaling pathway. And the production of IL-18, thereby promoting the apoptosis of tumor cells.

Description of drawings

Fig. 1 is the agarose gel electrophoresis pattern of the CAIX promoter fragment amplified by PCR after double enzyme digestion; Swimming lane 1.DNAMarker (DL2000); The CAIX promoter after double enzyme digestion in swimming lane 2.XhoI and SnaBI restriction;

Figure 2 is the agarose electrophoresis image of the pZD55 vector after double digestion; lane 1. DNAMarker (22kb plus); lane 2. pZD55 vector after double digestion with XhoI and SnaBI restriction enzymes;

Figure 3 is the PCR identification result of oncolytic adenovirus Ad-CAIX-AIM2;

Fig. 4 is the test result of E1A and AIM2 protein expression in the OSRC-2 cell of different virus infection;

Figure 5 is the test result of the replication ability of different viruses in 786-O and OSRC-2 cells;

Figure 6 is the test result of the virulence effect of different viruses on 786-O and OSRC-2 cells;

Figure 7 is the effect of different viruses on the proliferation of 786-O and OSRC-2 cells;

Fig. 8 is the HE staining photograph of mouse organ after different virus treatment;

Fig. 9 is the ELISA detection result of the level of IL-6 cytokine in the serum of mice after different virus treatments;

Fig. 10 is the test result of the tumor volume of mice after different virus treatments;

Figure 11 is the test results of tumor inhibition rate in mice treated with different viruses.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

In the following examples, the plasmids pZD55 and pGL3-CAIX were all from the Tumor Biotherapy Laboratory of Xuzhou Medical University; XhoI and SnaBI restriction enzymes, T4 ligase were purchased from Beijing NEB Company; the gel recovery kit was purchased from AxyGen Company ; Agarose was purchased from Amerasco; Effectene transfection reagent was purchased from Qiagen; DMEM medium was purchased from Invitrogen.

Example 1

A method for constructing an oncolytic adenovirus carrying the AIM2 gene, comprising the steps of:

(1) Construction of vector pZD55-CAIX

1) Design primer 1, and introduce the Xho I restriction site in the upstream primer of primer 1, and introduce the SnaBI restriction site in the downstream primer, wherein the sequence of the upstream primer is: 5'-TTGTCTCGAGACCTGCCCCTCACTC-3' (SEQ IDNo. 1), the downstream primer sequence is: 5'-TTGCTACGTACATGCGGCTGACTG-3' (SEQ ID No.2). Synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.;

2) Utilize designed primer 1 and plasmid pGL3-CAIX (the CAIX promoter sequence in the plasmid pGL3-CAIX sequence is shown in SEQ ID No.3), PCR amplifies the CAIX promoter fragment, obtains PCR product, utilizes XhoI and SnaBI restriction The PCR product was double-digested with endonuclease, incubated at 37°C for 4 hours, and then subjected to 1.5% agarose gel electrophoresis (Figure 1), and the gel was recovered to obtain a 218bp CAIX core promoter fragment;

3) Digest the adenoviral plasmid pZD55 with XhoI and SnaBI restriction enzymes, incubate at 37° C. for 4 hours, perform 1% agarose gel electrophoresis ( FIG. 2 ), and recover the linear pZD55 plasmid fragment;

4) Ligate the pZD55 fragment with the CAIX core promoter fragment overnight at 16°C;

5) Transformation, bacterial picking, plasmid extraction, and sequencing verification are correct, indicating that the vector pZD55-CAIX was successfully constructed.

(2) Construction of adenovirus shuttle plasmid pZD55-CAIX-AIM2

Primers were designed according to the AIM2 sequence (NM-004833), the sequence of the upstream primer was 5'-GATAAGCTTATGGAGAGTAAATACAAGGAGATACTCTT-3' (SEQ ID No.4), and the sequence of the downstream primer was 5'-TGCTCTAGACTATGTTTTTTTTTTGGCCTTAATAA-3' (SEQ ID No.5) , synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., using pEF-BOS-AIM2 as a template, PCR amplified to obtain the AIM2 sequence excluding the terminator 1029bp, and cloned it into the HindIII and XbaI of the proliferation-deficient adenovirus vector pCA13 site to obtain the pCA13-AIM2 plasmid; obtain the CMV+AIM2+PolyA expression cassette from the pCA13-AIM2 plasmid by BglII digestion, insert it into pZD55-CAIX, and obtain the recombinant adenovirus shuttle plasmid pZD55-CAIX-AIM2, pZD55-CAIX- The nucleotide sequence of the AIM2 plasmid is shown in SEQ ID No.6, and sequencing confirmed that the plasmid was constructed successfully;

(3) Preparation of oncolytic adenovirus carrying AIM2 gene

Co-transfect HEK293 cells with plasmid pZD55-CAIX-AIM2 and adenovirus backbone plasmid pBHGE3, the specific method is as follows:

1) One day before transfection, 293 cells were plated in a 6-well plate, with 5×10 ⁵ cells per well;

2) Add 2 μg of the constructed shuttle plasmid pZD55-CAIX-AIM2 and 2 μg of the backbone plasmid pBHGE3 to 200 μl of serum-free medium, shake for 1 second, let stand at room temperature for 5 minutes, add 25 μl of Effectene transfection reagent, invert and mix five times, and store at room temperature Incubate for 10 minutes;

3) Replace the medium in the 6-well plate with fresh medium of 5% fetal bovine serum (FBS), and add 1ml of fresh culture medium based on the above plasmid mixture, mix and add to the cell culture dish, 37°C, 5% After carbon dioxide culture for 6-12 hours, suck out the medium, add 2ml of fresh growth medium, and cultivate overnight;

4) Aspirate the medium, add 6ml of low-melting glue (5% FBS, 1.25% Agarose) for culture, and cover with nutrient solution for 1-2 times in the middle, usually virus plaques appear in 9-14 days.

After the virus plaques appeared on the cells, carry out PCR identification. After the successful preparation of Ad-CAIX-AIM2 was identified, the virus was amplified in the same way, HERK293 cells were spread on a cell culture dish with a diameter of 10 cm or 15 cm, and an appropriate volume of virus was added, and the virus was added at 37 Continue culturing in a 5% CO ₂ incubator at ℃, until 2-4 days after the cells are completely pathological (CPE), collect the culture medium in a 50ml centrifuge tube, centrifuge at 1,500rpm for 5min, remove the supernatant, and resuspend with the corresponding amount of buffer Cells were frozen and thawed three times at -80°C/37°C, centrifuged at 12,000 rpm and 4°C for 15 minutes, and the supernatant was collected, which was the oncolytic adenovirus Ad-CAIX-AIM2 carrying the AIM2 gene. After the virus was aliquoted, -80 ℃ refrigerator.

1. PCR identification of oncolytic adenovirus Ad-CAIX-AIM2

After a small amount of viral plaques were picked and amplified, the viral genomic DNA was extracted, and the extracted viral genomic DNA was used as a template, and the sequence of AIM2 upstream primer 3 was added to be 5'-GATAAGCTTATGGAGAGTAAATACAAGGAGATACTCTT-3'(SEQ ID No.4) , The sequence of the downstream primer 4 is 5'-TGCTCTAGACTATGTTTTTTTTTTGGCCTTAATAA-3' (SEQ ID No.5), 2×Taq enzyme mixture, nuclease-free water. Perform PCR amplification, the program is: 94°C for 3min, 32 cycles (94°C for 1mins, 55°C for 1min, 72°C for 1min for 30s), 72°C for 10min. The PCR amplification product was subjected to 1% agarose gel electrophoresis, and the electrophoresis diagram is shown in FIG. 3 , wherein, lane 1 is DNAMarker (DL2000bp); lane 2 is the AIM2 target gene amplified by PCR.

It can be seen from Figure 3 that using the viral genome as a template, the AIM2-specific amplification primers can be used to amplify a PCR product at a position of about 1 kb, which is verified to be the full-length sequence of AIM2 by sequencing. Therefore, the PCR method can identify the viral plaques as the oncolytic adenovirus Ad-CAIX-AIM2.

2. Titer determination of oncolytic adenovirus Ad-CAIX-AIM2

1) Prepare about 20ml of 293 cell suspension containing ¹⁰⁵ cells/ml, inoculate into two 96-well plates, 100μl per well;

2) Prepare virus dilution:

A. In duplicate, prepare 8 serial dilutions in sterile centrifuge tubes; add 0.9ml of DMEM culture solution to the first test tube, and add 1.8ml to the other tubes.

B. Add 0.1 ml of virus stock solution to the first test tube. Pipette up and down 5 times, mix well, this is 10 ^-1 dilution series.

C. Transfer 0.2ml of 10 ^-1 dilution to the second test tube, dilute to the 8th highest dilution sequentially, and complete the second serial dilution with the same virus stock solution.

3) After adding the diluted virus, culture it in a _CO2 incubator at 37°C for 10 days, observe each well with an inverted microscope, and count the CPE observed in each row. Determine the percentage of positive wells for each row.

4) Calculate the measured titer tissue culture median infectious dose (TCID50) value.

The TCID50 of the oncolytic adenovirus Ad-CAIX-AIM2 was measured to be 4×10 ⁹ pfu/ml.

3. In vitro tumor killing effect test of oncolytic adenovirus Ad-CAIX-AIM2

Three kinds of kidney cancer 786-O, OSRC-2 and normal HK-2 cells were cultured in vitro, and Ad-Ctrl (control empty vector virus), Ad-CAIX, Ad-AIM2, and Ad-CAIX-AIM2 were added to observe the The effect of viruses on the growth of experimental cells.

(1) Targeted detection

Ad-Ctrl, Ad-CAIX, Ad-AIM2, and Ad-CAIX-AIM2 various viruses infect OSRC-2 cells with multiplicity of infection (MOI=10), and after 48 hours, flow cytometry detects virus component E1A, AIM2 gene in Expression changes in renal carcinoma cells. The test results of E1A and AIM2 protein expression levels in OSRC-2 cells infected with different viruses are shown in FIG. 4 .

In Figure 4, Figure 4A is the test result of E1A protein expression in OSRC-2 cells infected with different viruses, and Figure 4B is the test result of AIM2 protein expression in OSRC-2 cells infected with different viruses, P<0.05,** P<0.01, ***P<0.001 was considered significant difference. It can be seen that the oncolytic adenovirus Ad-CAIX-AIM2 constructed in the present invention can effectively express virus core element E1A and AIM2 proteins in renal cancer cell OSRC-2 cells.

(2) Detection of virus replication ability

Ad-Ctrl, Ad-CAIX, Ad-AIM2, and Ad-CAIX-AIM2 viruses infected 786-O and OSRC-2 cells at a certain multiplicity of infection (MOI=10). After 48 hours, the cell supernatant and cells were collected, frozen and thawed three times at -80°C to 37°C to release the virus, centrifuged to take the supernatant, and stored at -80°C for later use. The replication ability of each virus was determined according to the TCID ₅₀ method. The results are shown in Figure 5.

Figure 5 is the test result of the replication ability of different viruses in 786-O and OSRC-2 cells, wherein, Figure 5A is the test result of the replication ability of different viruses in 786-O cells, and Figure 5B is the test result of the replication ability of different viruses in OSRC-2 cells 2 The test results of the replication ability in cells, P<0.05, **P<0.01, ***P<0.001 are considered to have significant differences. It can be seen that the oncolytic adenovirus Ad-CAIX-AIM2 constructed in the present invention can replicate efficiently in renal cancer cells.

(3) Cytotoxicity detection

Add 2ml of culture solution containing 3× ¹⁰⁵ cells (DMEM culture solution containing 10% fetal bovine serum) to each well of a 6-well cell culture plate, and cultivate in a carbon dioxide incubator with 5% CO2 at 37°C for 2 to 3 hours. Cell wall. Cells were infected according to MOIs: 0, 0.1, 1, 10 and 100, with 3 replicate wells for each dilution. After culturing for 5 days, carefully wash once with PBS, add 0.1ml of 10% methanol solution to each well to fix the cells for 30s. Aspirate the methanol solution, add 0.1ml crystal violet staining solution to each well, and place at room temperature for 20 minutes. Gently shake off the staining solution, wash each well with distilled water, place the culture plate upside down on absorbent paper to absorb the water, and let it dry. Under an inverted microscope, the toxicity of the virus to 786-O and OSRC-2 cells was observed.

Figure 6 is the test result of the virulence effect of different viruses on 786-O and OSRC-2 cells, wherein Figure 6A is the test result of the virulence effect of different viruses on 786-O cells, and Figure 6B is the test result of different viruses on OSRC-2 cells Toxicity effect test results, P<0.05, **P<0.01, ***P<0.001 were considered to be significantly different; ns, were considered to have no significant difference. It can be seen that when MOI=10, the oncolytic adenovirus Ad-CAIX-AIM2 constructed by the present invention has a great virulence effect on 786-O and OSRC-2 cells, which shows that Ad-CAIX-AIM2 has a great effect at MOI=10 Can effectively kill kidney cancer cells.

(4) Detection of cell proliferation ability

The cells in the logarithmic growth phase (786-O and OSRC-2) were digested, spread on a 96-well plate, inoculated ³ ×10 cells per well, and cultivated in a 37°C, 5% CO ₂ incubator for 24 hours; the virus (Ad-Ctrl , Ad-CAIX, Ad-AIM2 and Ad- _CAIX -AIM2) were added to each well of cells according to the multiplicity of infection of MOI=10, 3 duplicate wells, 100 μl/well; 72h; after reaching each time point, replace the 96-well plate with 100μl serum-free medium, add 10μl CCK-8 solution, and mix well; put it in the incubator and continue to incubate for 2h, use a microplate reader to detect each The optical density value of the well.

Figure 7 is the effect of different viruses on the proliferation of 786-O and OSRC-2 cells, wherein Figure 7A is the effect of different viruses on the proliferation of 786-O cells, and Figure 7B is the effect of different viruses on the proliferation of OSRC-2 cells, P< 0.05, **P<0.01, ***P<0.001 was considered significant difference; ns, no significant difference was considered. It can be seen that as the time of Ad-CAIX-AIM2 virus infection of renal cancer cells increases, the proliferation of cells is significantly inhibited.

4. Tumor killing effect of oncolytic adenovirus Ad-CAIX-AIM2 on tumor-bearing mice

To establish a nude mouse kidney cancer OSRC-2 cell subcutaneous xenograft tumor model, the method: randomly select 4-6 week-old female BALB/c nude mice, collect OSRC-2 cells cultured to the logarithmic growth phase, wash with PBS, each 100 μl of cell suspension with a density of 2×10 ⁶ cells/ml was subcutaneously inoculated on the abdomen and back of mice.

When the tumor volume ^was 100-120 mm, they were randomly divided into 4 groups, 10 rats in each group, and 100 μl of virus (Ad-Ctrl, Ad-AIM2, Ad-CAIX, Ad-CAIX-AIM2 virus, all 2 ×10 ⁸ PFU), divided into 5 injections, once every other day. 24 hours after the last injection, 3 mice in each group were selected, and the hearts, kidneys, and lungs were removed, and HE staining was used to observe the inflammatory damage of each organ, and ELISA was used to detect the level of IL-6 cytokines in the serum of mice given virus treatment. levels to monitor the safety of viral treatments. The remainder were monitored for tumor growth weekly until the end of the experiment after 5 weeks. After the experiment is completed, the tumor volume is measured, the tumor tissue is removed, the tumor weight is weighed, and the tumor inhibition rate is calculated.

Fig. 8 is the HE staining photograph of the mouse organ after different virus treatment, and Fig. 9 is the ELISA detection result of the level of IL-6 cytokine in the serum of the mouse body after different virus treatment, P<0.05, **P< 0.01, ***P<0.001 was considered significant difference; ns, no significant difference was considered. It can be seen from Figure 8 and Figure 9 that the oncolytic adenovirus Ad-CAIX-AIM2 constructed by the present invention will have a transient inflammatory response at the initial stage of treatment, and the inflammation will return to a normal state after one week. The organs of the virus-treated mice also showed no inflammatory damage. These results indicate that the constructed oncolytic adenovirus has low toxicity and good safety during treatment.

Figure 10 is the test result of the tumor volume of mice after different virus treatment, and Figure 11 is the test result of the tumor inhibition rate of mice after different virus treatment, P<0.05, **P<0.01, ***P< A significant difference was considered to exist at 0.001. It can be seen that compared with the control group, the Ad-CAIX-AIM2 virus constructed in the present invention can significantly inhibit the growth of mouse kidney cancer tumors, reflecting an effective therapeutic effect.

sequence listing

<110> Xuzhou Medical University

<120> Construction method and application of an oncolytic adenovirus carrying AIM2 gene

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 25

<212>DNA

<213> Oncolytic adenovirus

<400> 1

ttgtctcgag acctgcccct cactc 25

<210> 2

<211> 24

<212>DNA

<213> Oncolytic adenovirus

<400> 2

ttgctacgta catgcggctg actg 24

<210> 3

<211> 221

<212>DNA

<213> Oncolytic adenovirus

<400> 3

acctgcccct cactccaccc ccatcctagc tttggtatgg gggagaggggc acagggccag 60

acaaacctgt gagactttgg ctccatctct gcaaaagggc gctctgtgag tcagcctgct 120

cccctccagg cttgctcctc ccccacccag ctctcgtttc caatgcacgt acagcccgta 180

cacaccgtgt gctgggacac cccacagtca gccgcatgta c 221

<210> 4

<211> 38

<212>DNA

<213> Oncolytic adenovirus

<400> 4

gataagctta tggagagtaa atacaaggag atactctt 38

<210> 5

<211> 35

<212>DNA

<213> Oncolytic adenovirus

<400> 5

tgctctagac tatgttttttttttggcctt aataa 35

<210> 6

<211> 11526

<212>DNA

<213> Oncolytic adenovirus

<400> 6

cccttccagc tctctgcccc ttttggattg aagccaatat gataatgagg gggtggagtt 60

tgtgacgtgg cgcggggcgt gggaacgggg cgggtgacgt agtagtgtgg cggaagtgtg 120

atgttgcaag tgtggcggaa cacatgtaag cgacggatgt ggcaaaagtg acgtttttgg 180

tgtgcgccgg tgtacacagg aagtgacaat tttcgcgcgg ttttaggcgg atgttgtagt 240

aaatttgggc gtaaccgagt aagatttggc cattttcgcg ggaaaactga ataagaggaa 300

gtgaaatctg aataattttg tgttactcat agcgcgtaat atctcgagac ctgcccctca 360

ctccaccccc atcctagctt tggtatgggg gagagggcac agggccagac aaacctgtga 420

gactttggct ccatctctgc aaaagggcgc tctgtgagtc agcctgctcc cctccaggct 480

tgctcctccc ccaccagct ctcgtttcca atgcacgtac agcccgtaca caccgtgtgc 540

tgggaacaccc cacagtcagc cgcatgtact acgtaatgag acatattatc tgccacggag 600

gtgttattac cgaagaaatg gccgccagtc ttttggacca gctgatcgaa gaggtactgg 660

ctgataatct tccacctcct agccattttg aaccacctac ccttcacgaa ctgtatgatt 720

tagacgtgac ggcccccgaa gatcccaacg aggaggcggt ttcgcagatt tttcccgact 780

ctgtaatgtt ggcggtgcag gaagggattg acttactcac ttttccgccg gcgcccggtt 840

ctccggagcc gcctcacctt tcccggcagc ccgagcagcc ggagcagaga gccttgggtc 900

cggtttctat gccaaacctt gtaccggagg tgatcgatct tacctgccac gaggctggct 960

ttccacccag tgacgacgag gatgaagagg gtgaggagtt tgtgtgttagat tatgtggagc 1020

accccggggca cggttgcagg tcttgtcatt atcaccggag gaatacgggg gaccgata 1080

ttatgtgttc gctttgctat atgaggacct gtggcatgtt tgtctacagt aagtgaaaat 1140

tatgggcagt gggtgataga gtggtgggtt tggtgtggta attttttttt taatttttac 1200

agttttgtgg tttaaagaat tttgtattgt gattttttta aaaggtcctg tgtctgaacc 1260

tgagcctgag cccgagccag aaccggagcc tgcaagacct acccgccgtc ctaaaatggc 1320

gcctgctatc ctgagacgcc cgacatcacc tgtgtctaga gaatgcaata gtagtacgga 1380

tagctgtgac tccggtcctt ctaacacacc tcctgagata cacccggtgg tcccgctgtg 1440

ccccattaaa ccagttgccg tgagagttgg tgggcgtcgc caggctgtgg aatgtatcga 1500

ggacttgctt aacgagcctg ggcaaccttt ggacttgagc tgtaaacgcc ccaggccata 1560

aggtgtaaac ctgtgattgc gtgtgtggtt aacgcctttg tttgctgaat gagttgatgt 1620

aagtttaata aagggtgaga taatgtttaa cttgcatggc gtgttaaatg gggcggggct 1680

taaagggtat ataatgcgcc gtgggctaat cttggttaca tctgacctca tggaggcttg 1740

ggagtgtttg gaagattttt ctgctgtgcg taacttgctg gaacagagct ctaacagtac 1800

ctcttggttt tggaggtttc tgtggggctc atcccaggca aagttagtct gcagaattaa 1860

ggaggattac aagtgggaat ttgaagagct tttgaaatcc tgtggtgagc tgtttgattc 1920

tttgaatctg ggtcaccagg cgcttttcca agagaaggtc atcaagactt tggatttttc 1980

cacaccgggg cgcgctgcgg ctgctgttgc ttttttgagt tttataaagg ataaatggag 2040

cgaagaaacc catctgagcg gggggtacct gctggatttt ctggccatgc atctgtggag 2100

agcggttgtg agacacaaga atcgcctgct actgttgtct tccgtccgcc cggcgataat 2160

accgacggag gagcagcagc agcagcagga ggaagccagg cggcggcggc aggagcagag 2220

cccatggaac ccgagagccg gcctggaccc tcgggaatga atgttgtaca ggtggctgaa 2280

ctgtatccag aactgagacg cattttgaca attacagagg atgggcaggg gctaaagggg 2340

gtaaagagggg agcggggggc ttgtgaggct acagaggagg ctaggaatct agcttttagc 2400

ttaatgacca gacaccgtcc tgagtgtatt acttttcaac aagatctaat tccctggcat 2460

tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc 2520

atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt 2580

gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 2640

caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 2700

ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc 2760

gcctggagac gccatccagcg ctgttttgac ctccatagaa gacaccggga ccgatccagc 2820

ctggggatca gtcttcgagt cgacaagctt atggagagta aatacaagga gatactcttg 2880

ctaacaggcc tggataacat cactgatgag gaactggata ggtttaagtt ctttctttca 2940

gacgagttta atattgccac aggcaaacta catactgcaa acagaataca agtagctacc 3000

ttgatgattc aaaatgctgg ggcggtgtct gcagtgatga agaccattcg tatttttcag 3060

aagttgaatt atatgctttt ggcaaaacgt cttcaggagg agaaggagaa agttgataag 3120

caatacaaat cggtaacaaa accaaagcca ctaagtcaag ctgaaatgag tcctgctgca 3180

tctgcagcca tcagaaatga tgtcgcaaag caacgtgctg caccaaaagt ctctcctcat 3240

gttaagcctg aacagaaaca gatggtggcc cagcaggaat ctatcagaga agggtttcag 3300

aagcgctgtt tgccagttat ggtactgaaa gcaaagaagc ccttcacgtt tgagacccaa 3360

gaaggcaagc aggagatgtt tcatgctaca gtggctacag aaaaggaatt cttctttgta 3420

aaagttttta atacactgct gaaagataaa ttcattccaa agagaataat tataatagca 3480

agatattatc ggcacagtgg tttcttagag gtaaatagcg cctcacgtgtgttagatgct 3540

gaatctgacc aaaaggttaa tgtcccgctg aacattatca gaaaagctgg tgaaaccccg 3600

aagatcaaca cgcttcaaac tcagcccctt ggaacaattg tgaatggttt gtttgtagtc 3660

cagaaggtaa cagaaaagaa gaaaaacata ttatttgacc taagtgacaa cactgggaaa 3720

atggaagtac tgggggttag aaacgaggac acaatgaaat gtaaggaagg agataaggtt 3780

cgacttacat tcttcacact gtcaaaaaat ggagaaaaac tacagctgac atctggagtt 3840

catagcacca taaaggttat taaggccaaa aaaaaaacat ctagactcga gggatccatc 3900

gagcaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt 3960

cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt 4020

atcttatcat gtctggatcg tctagcatcg aagatctgga taattgcgct aatgagcttg 4080

atctgctggc gcagaagtat tccatagagc agctgaccac ttactggctg cagccagggg 4140

atgattttga ggaggctatt agggtatatg caaaggtggc acttaggcca gattgcaagt 4200

acaagatcag caaacttgta aatatcagga attgttgcta catttctggg aacggggccg 4260

aggtggagat agatacggag gatagggtgg cctttagatg tagcatgata aatatgtggc 4320

cgggggtgct tggcatggac ggggtggtta ttatgaatgt aaggtttact ggccccaatt 4380

ttagcggtac ggttttcctg gccaatacca accttatcct acacggtgta agcttctatg 4440

ggtttaacaa tacctgtgtg gaagcctgga ccgatgtaag ggttcggggc tgtgcctttt 4500

actgctgctg gaagggggtg gtgtgtcgcc ccaaaagcag ggcttcaatt aagaaatgcc 4560

tctttgaaag gtgtaccttg ggtatcctgt ctgagggtaa ctccagggtg cgccacaatg 4620

tggcctccga ctgtggttgc ttcatgctag tgaaaagcgt ggctgtgatt aagcataaca 4680

tggtatgtgg caactgcgag gacagggcct ctcagatgct gacctgctcg gacggcaact 4740

gtcacctgct gaagaccatt cacgtagcca gccactctcg caaggcctgg ccagtgtttg 4800

agcataacat actgacccgc tgttccttgc atttgggtaa caggaggggg gtgttcctac 4860

cttaccaatg caatttgagt cacactaaga tattgcttga gcccgagagc atgtccaagg 4920

tgaacctgaa cggggtgttt gacatgacca tgaggaaggt gctgaggtac gatgagaccc 4980

gcaccaggtg cagaccctgc gagtgtggcg gtaaacatat taggaaccag cctgtgatgc 5040

tggatgtgac cgaggagctg aggcccgatc acttggtgct ggcctgcacc cgcgctgagt 5100

ttggctctag cgatgaagat acagatgag gtactgaaat gtgtgggcgt ggcttaaggg 5160

tgggaaagaa tatataaggt gggggtctta tgtagttttg tatctgtttt gcagcagccg 5220

ccgccgccat gagcaccaac tcgtttgatg gaagcattgt gagctcatat ttgacaacgc 5280

gcatgccccc atgggccggg gtgcgtcaga atgtgatggg ctccagcatt gatggtcgcc 5340

ccgtcctgcc cgcaaactct actaccttga cctacgagac cgtgtctgga acgccgttgg 5400

agactgcagc ctccgccgcc gcttcagccg ctgcagccac cgcccgcggg attgtgactg 5460

actttgcttt cctgagcccg cttgcaagca gtgcagcttc ccgttcatcc gcccgcgatg 5520

acaagttgac ggctcttttg gcacaattgg attctttgac ccgggaactt aatgtcgttt 5580

ctcagcagct gttggatctg cgccagcagg tttctgccct gaaggcttcc tcccctccca 5640

atgcggttta aaacataaat aaaaaaccag actctgtttg gatttggatc aagcaagtgt 5700

cttgctgtct ttatttaggg gttttgcgcg cgcggtaggc ccgggaccag cggtctcggt 5760

cgttgagggt cctgtgtatt ttttccagga cgtggtaaag gtgactctgg atgttcagat 5820

acatgggcat aagcccgtct ctggggtgga ggtagcacca ctgcagagct tcatgctgcg 5880

gggtggtgtt gtagatgatc cagtcgtagc aggagcgctg ggcgtggtgc ctaaaaatgt 5940

ctttcagtag caagctgatt gccaggggca ggcccttggt gtaagtgttt acaaagcggt 6000

taagctggga tgggtgcata cgtggggata tgagatgcat cttggactgt atttttaggt 6060

tggctatgtt cccagccata tccctccggg gattcatgtt gtgcagaacc accagcacag 6120

tgtatccggt gcacttggga aatttgtcat gtagcttaga aggaaatgcg tggaagaact 6180

tggagacgcc cttgtgacct ccaagatttt ccatgcattc gtccataatg atggcaatgg 6240

gcccacgggc ggcggcctgg gcgaagatat ttctgggatc actaacgtca tagttgtgtt 6300

ccaggatgag atcgtcatag gccatttta caaagcgcgg gcggagggtg ccagactgcg 6360

gtataatggt tccatccggc ccaggggcgt agttaccctc acagatttgc atttcccacg 6420

ctttgagttc agatgggggg atcatgtcta cctgcggggc gatgaagaaa acggtttccg 6480

gggtagggga gatcagctgg gaagaaagca ggttcctgag cagctgcgac ttaccgcagc 6540

cggtgggccc gtaaatcaca cctattaccg ggtgcaactg gtagttaaga gagctgcagc 6600

tgccgtcatc cctgagcagg ggggccactt cgttaagcat gtccctgact cgcatgtttt 6660

ccctgaccaa atccgccaga aggcgctcgc cgcccagcga tagcagttct tgcaaggaag 6720

caaagttttt caacggtttg agaccgtccg ccgtaggcat gcttttgagc gtttgaccaa 6780

gcagttccag gcggtcccac agctcggtca cctgctctac ggcatctcga tccagcatat 6840

ctcctcgttt cgcgggttgg ggcggctttc gctgtacggc agtagtcggt gctcgtccag 6900

acgggccagg gtcatgtctt tccacgggcg cagggtcctc gtcagcgtag tctgggtcac 6960

ggtgaagggg tgcgctccgg gctgcgcgct ggccagggtg cgcttgaggc tggtcctgct 7020

ggtgctgaag cgctgccggt cttcgccctg cgcgtcggcc aggtagcatt tgaccatggt 7080

gtcatagtcc agcccctccg cggcgtggcc cttggcgcgc agcttgccct tggaggaggc 7140

gccgcacgag gggcagtgca gacttttgag ggcgtagagc ttgggcgcga gaaataccga 7200

ttccggggag taggcatccg cgccgcaggc cccgcagacg gtctcgcatt ccacgagcca 7260

ggtgagctct ggccgttcgg ggtcaaaaac caggtttccc ccatgctttt tgatgcgttt 7320

cttacctctg gtttccatga gccggtgtcc acgctcggtg acgaaaaggc tgtccgtgtc 7380

cccgtataca gacttgagag gcctgtcctc ggcctgtcct cgaccgatgc ccttgagagc 7440

cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat 7500

gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct gggtcatttt 7560

cggcgaggac cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg 7620

aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac gtttcggcga 7680

gaagcaggcc attatcgccg gcatggcggc cgacgcgctg ggctacgtct tgctggcgtt 7740

cgcgacgcga ggctggatgg ccttccccat tatgattctt ctcgcttccg gcggcatcgg 7800

gatgcccgcg ttgcaggcca tgctgtccag gcaggtagat gacgaccatc agggacagct 7860

tcaaggatcg ctcgcggctc ttaccagcct aacttcgatc actggaccgc tgatcgtcac 7920

ggcgattat gccgcctcgg cgagcacatg gaacgggttg gcatggattg taggcgccgc 7980

cctatacctt gtctgcctcc ccgcgttgcg tcgcggtgca tggagccggg ccacctcgac 8040

ctgaatggaa gccggcggca cctcgctaac ggattcacca ctccaagaat tggagccaat 8100

caattcttgc ggagaactgt gaatgcgcaa accaaccctt ggcagaacat atccatcgcg 8160

tccgccatct ccagcagccg cacgcggcgc atctcgggca gcgttgggtc ctggccacgg 8220

gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact 8280

ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg 8340

tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa 8400

acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg 8460

ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt 8520

tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac 8580

cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc 8640

attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa 8700

accgccctta acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc 8760

aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat 8820

gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg 8880

cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt 8940

cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc 9000

gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 9060

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct 9120

cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 9180

cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 9240

acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 9300

ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 9360

ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 9420

gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 9480

gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 9540

ccaagctggg ctgtgtgcac gaacccccccg ttcagcccga ccgctgcgcc ttatccggta 9600

actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 9660

gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 9720

ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta 9780

ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 9840

gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 9900

tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 9960

tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 10020

aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 10080

aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 10140

tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 10200

gagaccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 10260

agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 10320

aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctgcag 10380

gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 10440

caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 10500

cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 10560

ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 10620

ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac 10680

gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 10740

cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 10800

gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 10860

caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 10920

tactcttcct ttttcaatat tattgaagca tttatcaggg ttaattgtctc atgagcggat 10980

acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 11040

aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc 11100

gtatcacgag gccctttcgt cttcaagaat tctcatgttt gacagcttat catcgataag 11160

ctttaatgcg gtagtttatc acagttaaat tgctaacgca gtcaggcacc gtgtatgaaa 11220

tctaacaatg cgctcatcgt catcctcggc accgtcaccc tggatgctgt aggcataggc 11280

ttggttatgc cggtactgcc gggcctcttg cgggatatcg tccattccga cagcatcgcc 11340

agtcactatg gcgtgctgct agcgctatat gcgttgatgc aatttctatg cgcacccgtt 11400

ctcggagcac tgtccgaccg ctttggccgc cgcccagtcc tgctcgcttc gctacttgga 11460

gccactatcg actacgcgat catggcgacc acacccgtcc tgtggatccg ggcccccatt 11520

tcccct 11526

Claims (4)

1. A method for constructing an oncolytic adenovirus carrying the AIM2 gene, comprising the steps of: (1) Construction of CAIX promoter vector pZD55-CAIX Through PCR reaction and cloning technology, the 218bp CAIX core promoter fragment was obtained from the plasmid pGL3-CAIX, and then inserted into the adenovirus plasmid pZD55 to obtain pZD55-CAIX; (2) Construction of adenovirus shuttle plasmid pZD55-CAIX-AIM2 Using pEF-BOS-AIM2 as a template, AIM2 sequence without terminator 1029bp was amplified by PCR and cloned into the HindIII and XbaI sites of the proliferation-deficient adenovirus vector pCA13 to obtain pCA13-AIM2 plasmid; from the pCA13-AIM2 plasmid The CMV+AIM2+PolyA expression cassette was obtained from , and inserted into pZD55-CAIX to obtain the recombinant adenovirus shuttle plasmid pZD55-CAIX-AIM2; (3) Preparation of target oncolytic adenovirus The identified plasmid pZD55-CAIX-AIM2 and the adenovirus backbone plasmid pBHGE3 were co-transfected into HEK293 cells. After the cells appeared viral plaques, the target oncolytic adenovirus Ad-CAIX-AIM2 was obtained. The sequence of the adenovirus shuttle plasmid pZD55-CAIX-AIM2 is shown in SEQ ID NO.:6. 2. The construction method of the oncolytic adenovirus carrying the AIM2 gene according to claim 1, characterized in that, in step (1), the specific method for obtaining the 218bp CAIX core promoter fragment from the plasmid pGL3-CAIX is: design Primer 1, and introduce the Xho I restriction site in the upstream primer of primer 1, and introduce the SnaBI restriction site in the downstream primer, wherein the sequence of the upstream primer is: 5'-TTGTCTCGAGACCTGCCCCTCACTC-3', and the sequence of the downstream primer is: 5 '-TTGCTACGTACATGCGGCTGACTG-3'; Use the designed primer 1 and plasmid pGL3-CAIX to amplify the CAIX promoter fragment by PCR to obtain the PCR product, use XhoI and SnaBI restriction endonucleases to double-enzyme digest the PCR product, and carry out after incubation 1.5% agarose gel electrophoresis, gel recovery to obtain the 218bp CAIX core promoter fragment. 3. The method for constructing an oncolytic adenovirus carrying the AIM2 gene according to claim 1 or 2, wherein in step (2), the sequence of the upstream primer used for PCR amplification is 5'-GATAAGCTTATGGAGAGTAAATACAAGGAGATACTCTT-3' , The sequence of the downstream primer is 5'-TGCTCTAGACTATGTTTTTTTTTTGGCCTTAATAA-3'; the PCR cycle conditions parameters are: 94°C 3min, 94°C 1min, 55°C 1min, 72°C 1min 30s, 32 cycles, 72°C 10min. 4. The application of the oncolytic adenovirus carrying the AIM2 gene prepared by the method of claim 1 or 2 or 3 in the preparation of drugs for treating kidney cancer.

Images