Application of RhoA protein in preparation of product for enhancing treatment effect of tumor oncolytic virus

Abstract

The invention relates to the field of biomedical treatment, in particular to application of RhoA protein in preparation of a product for enhancing the treatment effect of tumor oncolytic virus. The amino acid sequence of the RhoA protein is shown as SEQ ID NO.1. Aiming at the problems that oncolytic viruses including the Newcastle disease virus are narrow in treatment spectrum and insufficient in tumor killing efficacy, the human RHOA gene is introduced through a gene recombination technology and serves as an enhancer, and the anti-tumor effect of the Newcastle disease virus is improved. The recombinant oncolytic virus disclosed by the invention has a broad-spectrum and excellent anti-tumor effect. A further research finds that the recombinant oncolytic virus can induce tumor cell acute mitochondrial active oxygen excess generation to trigger lipid peroxidation to cause autophagic cell death, which is a main mechanism for mediating efficient oncolytic of the recombinant oncolytic virus. Research results show that the recombinant oncolytic virus is a breakthrough candidate drug for overcoming the clinical treatment problem of tumor heterogeneity, and has a good clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of RhoA protein in the preparation of products that enhance the therapeutic efficacy of tumor oncolytic viruses. Background Art

[0002] Cancer poses a serious threat to human life and health. According to global cancer data released by the World Health Organization's International Agency for Research on Cancer (IARC), more than 20 million new cancer cases will be diagnosed worldwide in 2022, including 4.57 million in China. Currently, traditional cancer treatments include chemotherapy, radiotherapy, and surgery. These therapies are associated with significant side effects, lack of targeted therapy, and multidrug resistance. Therefore, the exploration of new cancer therapies remains a research hotspot in the biomedical field.

[0003] Oncolytic viruses (OVs) are natural or recombinant viruses that selectively infect and kill cancer cells while sparing normal tissue. With the recent advancement of gene editing technology, the genetic modification of OVs has become more convenient and efficient. Newcastle disease virus (NDV) is a single-stranded RNA virus belonging to the Paramyxoviridae family that primarily infects poultry. Compared with currently clinically approved oncolytic viruses, human HSV-1 and human adenovirus, NDV has a low pre-existing human exposure, and patients generally lack pre-existing antibodies against it, resulting in minimal immune interference during treatment. Furthermore, its advantages, such as being non-pathogenic to humans, not integrating into the host genome, and specifically replicating and killing only tumor cells, have led to its development as an OV. In 2015, Germany approved an NDV-activated dendritic cell vaccine for tumor treatment. Results showed that the oncolytic NDV not only lyses cancer cells but also induces long-term anti-tumor immunity. In addition, systemic administration via intravenous injection is a major difficulty in oncolytic virus therapy. Phase I clinical trials have shown that intravenous NDV is extremely safe and has demonstrated preliminary therapeutic effects in 23 patients. Although previous studies have demonstrated the great potential of NDV as an oncolytic and immunotherapeutic agent, there are still many problems to be solved, such as the efficiency of viral replication in the body, the method of viral introduction, and combined drug use. In addition, compared with FDA-approved oncolytic agents such as herpes viruses and adenoviruses, the oncolytic efficiency of NDV needs to be improved.

[0004] RhoA, a small GTPase, is encoded by the RHOA gene. RhoA participates in numerous life processes, including actin assembly, myosin contraction, cell cycle maintenance, cell proliferation and differentiation, and is closely associated with tumorigenesis and metastasis. Studies have shown that Rho GTPase expression is abnormal in various tumor cells, and altering intracellular Rho GTPase expression can significantly affect tumor invasion and metastasis. However, Rho GTPase function varies across tumor types and stages. Given that targeting Rho GTPases for cancer treatment can reduce the side effects of traditional anticancer drugs, further investigation into whether Rho GTPases could serve as novel targets for cancer drug development is of great significance. Summary of the Invention

[0005] The purpose of the present invention is to provide the use of RhoA protein in the preparation of a product that enhances the therapeutic efficacy of tumor oncolytic viruses.

[0006] To achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides the use of RhoA protein in preparing a product for enhancing the therapeutic efficacy of tumor oncolytic virus therapy.

[0007] Tumor heterogeneity is a major challenge in all current tumor treatments, including oncolytic virus therapy. Improving the efficacy of oncolytic viruses in lysing tumor cells is a key strategy for overcoming clinical bottlenecks in oncolytic virus therapy, such as tumor heterogeneity. To address this issue, the inventors applied CRISPR / Cas9 whole-genome screening technology to identify the host factor RhoA as a key regulator of NDV oncolytic efficacy. They then used reverse genetics to construct an engineered NDV carrying the RhoA gene (rNDV-RHOA). This study modulated RhoA expression levels in tumor cells using gene editing techniques and other means. In a human lung cancer cell model, overexpression of RhoA significantly enhanced the oncolytic virus's ability to kill cancer cells, while knockout of RhoA significantly inhibited the virus's ability to kill cancer cells. The recombinant virus rNDV-RHOA, generated by inserting the human RhoA encoding gene RHOA into the Newcastle disease virus (NDV) genome, contains a nucleic acid sequence expressing the RhoA protein. rNDV-RHOA significantly improved oncolytic efficacy and showed a generally significantly higher killing efficacy than wild-type oncolytic viruses NDV, HSV-1, and VSV in 35 tumor cell lines covering 13 cancer types and primary tumor cells from 6 patients, demonstrating a broad-spectrum and effective anti-tumor effect. Further studies found that rNDV-RHOA can induce acute mitochondrial reactive oxygen species (ROS) overproduction in tumor cells, triggering autophagic cell death caused by lipid peroxidation, which is the main mechanism mediating the efficient oncolysis of rNDV-RHOA. The applicant's current research results show that rNDV-RHOA is a breakthrough candidate drug for overcoming the clinical treatment difficulties of tumor heterogeneity and has good clinical application prospects.

[0008] Furthermore, the amino acid sequence of the RhoA protein is shown in SEQ ID NO.1.

[0009] Furthermore, the cancer includes lung cancer, cervical cancer, intestinal cancer, epidermal cancer, breast cancer, nervous system tumors, liver cancer, stomach cancer, ovarian cancer, prostate cancer, pancreatic cancer, esophageal cancer, thymic cancer, and lymphoma.

[0010] In a second aspect, the present invention provides a preparation for enhancing the therapeutic efficacy of tumor oncolytic virus therapy, wherein the preparation comprises the RhoA protein.

[0011] In a third aspect, the present invention provides a recombinant Newcastle disease virus genome, wherein the genome comprises a gene encoding the RhoA protein.

[0012] Furthermore, the nucleotide sequence of the recombinant Newcastle disease virus genome is sequentially connected by the sequences shown in SEQ ID NO. 3~4.

[0013] In a fourth aspect, the present invention provides a recombinant oncolytic virus expressing the RHOA gene, wherein the recombinant oncolytic virus comprises the recombinant Newcastle disease virus genome.

[0014] Furthermore, the starting strain of the recombinant oncolytic virus is selected from the Newcastle disease virus La Sota vaccine strain (GenBank: AY845400.2).

[0015] In a fifth aspect, the present invention provides a cancer treatment drug, the active ingredient of which is the recombinant oncolytic virus.

[0016] In a sixth aspect, the present invention provides a method for preparing the recombinant oncolytic virus expressing the RHOA gene, characterized in that it comprises: Construction of Newcastle disease virus genome vector; By homologous recombination, the coding gene of the RhoA protein is connected to the Newcastle disease virus genome vector, so that the coding gene of the RhoA protein is inserted into the Newcastle disease virus genome to obtain a recombinant Newcastle disease virus plasmid; The recombinant Newcastle disease virus plasmid and the helper plasmid are co-transfected into cells and the transfected cells are cultured to obtain the recombinant oncolytic virus.

[0017] Beneficial effects of the present invention: In previous basic research and clinical trials, two methods are usually used to improve the oncolytic effect of oncolytic viruses. One is to delete non-essential viral genes to improve the replication efficiency of the virus and reduce its immunogenicity; the other is to add co-stimulatory molecules, chemokines, and immune checkpoint inhibitors to promote the anti-tumor immune response of surrounding tissues. The present invention addresses the problem of insufficient tumor killing efficacy of natural Newcastle disease virus. By introducing the human RHOA gene through genetic recombination technology, it acts as an "enhancer" to enhance the anti-tumor effect of Newcastle disease virus. The results show that compared with wild-type wtNDV, the recombinant virus rNDV-RHOA has a significant inhibitory effect on the growth of tumor cells cultured in vitro and in vivo tumors in xenograft and homologous mouse models including lung cancer, colon cancer, breast cancer, and epidermal cancer. rNDV-RHOA showed rapid and efficient oncolytic effects on primary isolated tumor cells from 6 cancer patients tested, and the killing efficacy was significantly higher than that of wild-type oncolytic viruses NDV, HSV-1, and VSV. In some in vivo tumor models, more than half of the mice even completely disappeared. At the same time, the recombinant virus rNDV-RHOA showed good safety in mice and primary isolated normal tissue cells. RHOA, as a highly expressed gene in some tumor tissues, has been shown to be closely related to the proliferation and migration of lung cancer cells. The present invention introduces this gene to construct an oncolytic virus, which is completely different from the strategy adopted by predecessors to enhance the oncolytic effect, and even goes against it. On the one hand, RhoA is widely involved in the body's life activities, and its exogenous expression changes the metabolic homeostasis of the body's tumor microenvironment. On the other hand, the rapid proliferation of rNDV-RHOA in tumor cells exacerbates the imbalance of metabolic homeostasis, thereby promoting the rapid lysis of tumor tissue or tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effect of different RhoA expression levels on the oncolytic efficiency of Newcastle disease virus, including: A: Identification of RhoA knockout or overexpression A549 cell lines; B: Detection of tumor cell mortality after wild-type wtNDV infection of A549 cells and RhoA knockout or overexpression A549 cells; C: Detection of tumor cell survival rate after wild-type wtNDV infection of A549 cells and RhoA knockout or overexpression A549 cells.

[0019] Figure 2 Construction and identification of the recombinant Newcastle disease oncolytic virus rNDV-RHOA. A: Schematic diagram of the genomic site for the construction of the recombinant Newcastle disease virus rNDV-RHOA; B: Detection of RhoA protein expression levels in A549 cells infected with rNDV-RHOA; C: Detection of viral protein expression levels and genome replication levels of wtNDV and rNDV-RHOA in A549 cells.

[0020] Figure 3 To detect the killing efficiency of wtNDV and rNDV-RHOA on human lung cancer A549 cells in vitro, A: detection of tumor cell mortality after wtNDV or rNDV-RHOA infection; B: detection of tumor cell survival rate after wtNDV or rNDV-RHOA infection.

[0021] Figure 4 rNDV-RHOA demonstrated broad-spectrum therapeutic efficacy in 35 tumor cell lines covering 12 types of cancer.

[0022] Figure 5 The figure shows the broad-spectrum and superior therapeutic effect of rNDV-RHOA in primary isolated tumor cells from 6 cancer patients covering 4 types of cancer. A: patient information; B: mortality statistics of primary tumor cells 12 hours after rNDV-RHOA infection.

[0023] Figure 6 Figure 3: The in vivo tumor treatment effects of wtNDV or rNDV-RHOA in a lung cancer xenograft mouse model. A: Schematic diagram of the experimental flow (orthotopic administration); B: Ex vivo observation of subcutaneous tumor tissue from each treatment group, with the first row representing the control group, the second row representing the wtNDV-treated group, and the third row representing the rNDV-RHOA-treated group; C: Effects of wtNDV or rNDV-RHOA treatment on tumor weight in the mouse model; D: Effects of wtNDV or rNDV-RHOA treatment on tumor size in the mouse model.

[0024] Figure 7 Figure 3 shows the in vivo tumor therapeutic effect of wtNDV or rNDV-RHOA in a lung cancer homologous transplant mouse model, where: A: Schematic diagram of the experimental process (in situ administration); B: In vitro observation of subcutaneous tumor tissues of each treatment group, where the first row is the control group, the second row is the wtNDV-treated group, and the third row is the rNDV-RHOA-treated group; C: Effect of wtNDV or rNDV-RHOA treatment on tumor weight in the mouse model; D: Effect of wtNDV or rNDV-RHOA treatment on tumor size in the mouse model.

[0025] Figure 8 Figure 3 shows the in vivo tumor therapeutic effect of wtNDV or rNDV-RHOA in a breast cancer homograft mouse model, where: A: Schematic diagram of the experimental process (in situ administration); B: In vitro observation of subcutaneous tumor tissues of each treatment group, where the first row is the control group, the second row is the wtNDV-treated group, and the third row is the rNDV-RHOA-treated group; C: Effect of wtNDV or rNDV-RHOA treatment on tumor weight in the mouse model; D: Effect of wtNDV or rNDV-RHOA treatment on tumor size in the mouse model.

[0026] Figure 9 Figure 3 shows the in vivo tumor therapeutic effect of wtNDV or rNDV-RHOA in an epidermal cancer homologous transplant mouse model, where: A: Schematic diagram of the experimental process (in situ administration); B: In vitro observation of subcutaneous tumor tissues of each treatment group, where the first row is the control group, the second row is the wtNDV-treated group, and the third row is the rNDV-RHOA-treated group; C: Effect of wtNDV or rNDV-RHOA treatment on tumor weight in the mouse model; D: Effect of wtNDV or rNDV-RHOA treatment on tumor size in the mouse model.

[0027] Figure 10 Figure 3 shows the in vivo tumor therapeutic effect of wtNDV or rNDV-RHOA in a colon cancer homologous transplant mouse model, where: A: Schematic diagram of the experimental process (intravenous administration); B: In vitro observation of subcutaneous tumor tissues of each treatment group, where the first row is the control group, the second row is the wtNDV-treated group, and the third row is the rNDV-RHOA-treated group; C: Effect of wtNDV or rNDV-RHOA treatment on tumor weight in the mouse model; D: Effect of wtNDV or rNDV-RHOA treatment on tumor size in the mouse model.

[0028] Figure 11 rNDV-RHOA rapidly induces acute mitochondrial reactive oxygen species (ROS) overproduction in tumor cells, triggering autophagic cell death caused by lipid peroxidation. A: Effect of rNDV-RHOA treatment on ROS in tumor cells; B: Effect of rNDV-RHOA treatment on lipid peroxidation in tumor cells; C: Relative mortality of tumor cells under different treatments; D: Degree of lipid peroxidation in tumor cells under different treatments; E: ROS levels in tumor cells under different treatments. DETAILED DESCRIPTION

[0029] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0030] Example 1: Detection of the effect of RhoA expression level on Newcastle disease virus NDV oncolytic efficiency 1. Construction of RhoA knockout or overexpression tumor cell lines An sgRNA sequence was designed targeting the human RHOA gene (Gene ID: 387): ATCGACAGCCCTGATAGTTT. This sequence was inserted into the LentiCRISPR-v2-GFP plasmid (Addgene, Catalog No. 82416) and co-transfected with psPAX2 and pMD2.G plasmids into 293T cells. Lentivirus was then packaged into lentivirus containing the sgRNA. A549 cells were infected with the lentivirus, GFP-positive cells were sorted by flow cytometry, and monoclonal cells were cultured in 96-well plates. Western blot analysis was used to confirm the efficacy of RhoA knockout in these monoclonal cell lines.

[0031] The nucleic acid sequence expressing the human RHOA gene (SEQ ID NO. 2) was inserted into the pLVX-IRES-mCherry (Takara, Cat. No. 631237) vector and co-transfected with psPAX2 and pMD2.G plasmids into 293T cells. The lentivirus was then packaged into a lentivirus containing the RHOA sequence. A549 cells were infected with the lentivirus, and mCherry-positive cells were sorted by flow cytometry and cultured as monoclonal clones in 96-well plates. Western blot analysis was performed to identify the effect of RhoA overexpression in the monoclonal cell line. The results are shown in Figure 2. Figure 1 As shown in A.

[0032] 2. Detection of tumor cell mortality using lactate dehydrogenase (LDH) cytotoxicity assay wtA549 cells, RhoA knockout A549 cells (RhoA-KO1 / 2), and RhoA overexpressing A549 cells (RhoA-OV1 / 2) were seeded in 96-well plates. When the cells reached 70% density, wild-type NDV (AY845400) was added to infect the cells. 24 hours after infection, the cell supernatant was collected and the LDH content in the supernatant was measured according to the kit instructions (Biyuntian, Cat. No.: C0016). The tumor cell mortality rate was calculated using the formula. Figure 1 As shown in Figure 2, RhoA overexpression significantly increased the lethality of Newcastle disease virus to A549 cells from 15% to 25.11-29.13%, while RhoA knockout significantly reduced the lethality of Newcastle disease virus to A549 cells to 3.93-5.21%.

[0033] 3. Detection of tumor cell survival rate using CCK-8 assay wtA549 cells, RhoA knockout A549 cells (RhoA-KO1 / 2) and RhoA overexpressing A549 cells (RhoA-OV1 / 2) were seeded in 96-well plates, and wild-type NDV was added to infect the cells when the cells grew to 70% density. After 24 hours of infection, the cell supernatant was discarded and the cells were washed three times with PBS. CCK-8 storage solution (Biyuntian, catalog number: C0037) and DMEM culture medium were prepared in advance at a ratio of 1:10 to make CCK-8 working solution, and 100 µL of CCK-8 working solution was added to each well. The 96-well plate was placed in a 37°C cell culture incubator and incubated for 1-2 hours, and the optical density (OD) at 450 nm was measured using a microplate reader. The results of three parallel experiments were used to calculate the survival rate of tumor cells in different treatment groups according to the CCK-8 instructions. Figure 1 As shown in Figure C, RhoA knockout significantly increased the survival rate of A549 cells after infection with Newcastle disease virus from 66.55% to 82.39%-83.3%, while RhoA overexpression significantly reduced the survival rate of A549 cells after infection with Newcastle disease virus to 36.88%-42.8%.

[0034] Example 2: Preparation of a NDV recombinant oncolytic virus expressing the RHOA gene 1. Construction of NDV oncolytic virus expressing RHOA gene using homologous recombination First, the genomic cDNA of the Newcastle disease virus La Sota vaccine strain (GenBank: AY845400.2) was divided into four segments, namely segments A, B, C, and D. When designing the amplification primers for each segment, the sequence of the adjacent segment junction of about 25bp was added as the homologous sequence for subsequent segment connection. The specific primer sequences are as follows: AF: AGCTCGGAAGCGGCCGCTAATACGACTCACTATAGGGACCAAACAGAGAATCCGTGAGT.

[0035] AR: AGCTAGATTAATTACGGTTACGCGATCATTCAGTGGGGCTGAAGAAGCGAG.

[0036] BF: TCGCTTCTTCAGCCCCACTGAATGATCGCGTAACCGTAATTAATCTAGCT.

[0037] BR: GCTCGCCATGTTCTACCCGTATTATTTACCATGAGCTGTTTTGCCTTGTA.

[0038] C-F: TACAAGGCAAAACAGCTCATGGTAAATAATACGGGTAGAACATGGCGAGC。

[0039] C-R: ATCAGGATTAGATACACCCAGTATTTTCCTGCCTCCCGTCAAAGGTGACC。

[0040] D-F: GGTCACCTTTGACGGGAGGCAGGAAAATACTGGGTGTATCTAATCCTGAT。

[0041] D-R: AGCGAGGAGGCTGGGACCATGCCGGCCACCAAACAAAGATTTGGTGAATGACGAG。

[0042] Each fragment was amplified using a high-fidelity amplification enzyme (Accor Bio, Catalog No. AG12207) in a 50 µL amplification system. The amplification program was as follows: 94°C for 1 min; 98°C for 10 s; 68°C for 1 min (adjusted to 1 min / 1 kb); and 4°C storage for 35 cycles. The fragments were recovered using a gel recovery kit (Tiangen, Catalog No. DP209), and the concentration of the recovered product was measured using a NanoDrop. The four gel-recovered fragments were ligated with the linearized vector pBlueScript SK (+ / -) (Miaoling, Catalog No. P37442) obtained by PCR (primer sequences are as follows: pB-F: CTCGTCATTCACCAAATCTTTGTTTGGTggccggcatggtcccagcctcctcgct; pB-R: ACTCACGGATTCTCTGTTTGGTccctatagtgagtcgtattaGCGGCCGCTTCCGAGCT) using homologous recombination enzyme (Novozymes, Catalog No. C115) according to the manufacturer's instructions. The ligation system and procedure are as follows: vector and each fragment amount = [0.02 × number of base pairs per fragment] ng (0.03 pmol), enzyme: 5 µL; vector and fragment total: 3 µL; water: 2 µL. Ligation was performed in a PCR instrument at 50°C for 30 min. The resulting ligation products were transformed into stbl2 competent Escherichia coli cells, and the transformed competent cells were plated on LB plates containing ampicillin. After incubation at 30°C for 16 hours, a single colony was picked and inoculated into ampicillin-resistant liquid LB medium. The culture was shaken at 170 rpm and incubated at 35°C for 16-18 hours for amplification. After amplification, the bacteria were collected by centrifugation at 12,000 rpm. Plasmids were extracted according to the instructions of the Plasmid Extraction Kit (OMEGA, Cat. No. D6915). The resulting plasmids were sequenced and confirmed (Shanghai Biotech). The correctly identified plasmids were stored at -20°C until further use and named pB-LaSota.

[0043] The human RHOA gene (NM_001313941.2) was searched through Genebank, and amplification primers were designed according to the six-base principle. The specific primer sequences are as follows: pB-LaSota-RHOA-F: AGTTTAGTTGATAGTTGTAGCCACCatggctgccatccggaag; pB-LaSota-RHOA-R: GGGCTTAGTACACGGTAGTTACACACtcacaagacaaggcacccagatt. The amplification system was 50 μL, and the amplification program was as follows: 94°C, 1 min; 98°C, 10 s; 68°C, 1 min (adjusted according to 1 min / 1 kb); stored at 4°C for 35 cycles, and homologous bases were added according to their insertion position in the NDV genome for subsequent homologous recombination ( Figure 1 A). After successful fragment amplification, the fragments were recovered from the gel and subsequently ligated using homologous recombinase. The ligation system and procedure were as follows: vector and each fragment amount = [0.02 × number of base pairs per fragment] ng (0.03 pmol), enzyme: 5µL; total fragment: 3µL; water: 2µL. Ligation was performed in a PCR instrument at 50°C for 50 minutes. The ligated plasmids were sequenced, and the resulting recombinant viral genome sequence was a concatenation of the sequences shown in SEQ ID NOs. 3 and 4, where the single underline represents the P gene; the double underline represents the RHOA gene with added homology arms; and the dotted underline represents the M gene.

[0044] The same method was used to construct auxiliary plasmids, namely pVAX1-NP, pVAX1-P and pcDNA-L. The specific primer sequences are as follows: pVAX1-F: GAGACCCAAGCTGGCTAGC; pVAX1-R: CCTATAGTGAGTCGTATTAATTTCGATAAGCCAG.

[0045] NP-F: CGAAATTAATACGACTCACTATAGGATTGTCTTCCGTATTTGATGAGTACGAAC.

[0046] NP-R:TTAAACGCTAGCCAGCTTGGGTCTCTTTTTTTCTAATTCCCTCGGCCTCTGTTTTGA.

[0047] PF: CGAAATTAATACGACTCACTATAGGACCGGGTAGAAGAGGGATATTCAGAGA.

[0048] PR: TTAAACGCTAGCCAGCTTGGGTCTCCACAATTGGGGCACTCCAATTCTAC.

[0049] pcDNA-F: GAGACCCAAGCTGGCTAGC; pcDNA-R: CCTATAGTGAGTCGTATTAATTTCGATAAGCCAG.

[0050] LF: CGAAATTAATACGACTCACTATAGGACGGGTAGAACATGGCGAG.

[0051] LR: TTAAACGCTAGCCAGCTTGGGTCTCACCAAACAAAGATTTGGTGAATGACGAG.

[0052] Amplification was performed using a high-fidelity enzyme in a 50µL amplification system. The amplification program was as follows: 94°C for 1 minute; 98°C for 10 seconds; 68°C for 1 minute (adjusted to 1 minute / kb); and 4°C storage for 35 cycles. After successful amplification of the vector and fragments, gel extraction was performed and ligation was performed using homologous recombinase. The ligation system and program were as follows: vector and fragment usage = [0.02 × number of base pairs per fragment] ng (0.03 pmol), enzyme: 5µL; total fragment: 3µL; water: 2µL. Ligation was performed in a PCR instrument at 50°C for 50 minutes. The gene sequences of the three target fragments NP, P, and L are shown in SEQ ID NOs. 5-7, respectively.

[0053] SEQ ID NO.1: MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPTVFENYVADIEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVKHFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKTKDGVREFFEMATRAALQARRGKKKSGCLVL.

[0054] SEQ ID NO.2:ATGGCTGCCATCCGGAAGAAACTGGTGATTGTTGGTGATGGAGCCTGTGGAAAGACATGCTTGCTCATAGTCTTCAGCAAGGACCAGTTCCCAGAGGTGTATGTGCCCACAGTGTTTGAGAACTATGTGGCAGATATCGAGGTGGATGGAAAGCAGGTAGAGTTGGCTTTGTGGGACACAGCTGGGCAGGAAGATTATGATCGCCTGAGGCCCCTCTCCTACCCAGATACCGATGTTATACTGATGTGTTTTTCCATCGACAGCCCTGATAGTTTAGAAAACATCCCAGAAAAGTGGACCCCAGAAGTCAAGCATTTCTGTCCCAACGTGCCCATCATCCTGGTTGGGAATAAGAAGGATCTTCGGAATGATGAGCACACAAGGCGGGAGCTAGCCAAGATGAAGCAGGAGCCGGTGAAACCTGAAGAAGGCAGAGATATGGCAAACAGGATTGGCGCTTTTGGGTACATGGAGTGTTCAGCAAAGACCAAAGATGGAGTGAGAGAGGTTTTTGAAATGGCTACGAGAGCTGCTCTGCAAGCTAGACGTGGGAAGAAAAAATCTGGGTGCCTTGTCTTGTGA。

[0055] ACGGGTAGAAGAGGGATATTCAGAGATCAGGGCAAGTCTCCCGAGTCTCTGCTCTCTCCT CTACCTGATAGACCAGGACAAACATGGCCACCTTTACAGATGCAGAGATCGACGAGCTATTTGAGACAAGTGGAAC TGTCATTGACAACATAATTACAGCCCAGGGTAAACCAGCAGAGACTGTTGGAAGGAGTGCAATCCCACAAGGCAAG ACCAAGGTGCTGAGCGCAGCATGGGAGAAGCATGGGAGCATCCAGCCACCGGCCAGTCAAGGCAACCCCGATCGAC AGGACAGATCTGACAAACAACCATCCACACCCGGGCAAACGACCCCGCATGACAGCCCGCCGGCCACATCCGCCGA CCAGCCCCCCACCCAGGCCACAGACGAAGCCGTCGACACACAGCTCAGGACCGGAGCAAGCAACTCTCTGCTGTTG ATGCTTGACAAGCTCAGCAATAAATCGTCCAATGCTAAAAAGGGCCCATGGTCGAGCCCCCAAGAGGGGAATCACC AACGTCCGACTCAACAGCAGGGGAGTCAACCCAGTCGCGGAAACAGTCAGGAAAGACCGCAGAACCAAGTCAAGGC CGCCCCTGGAAACCAGGGCACAGACGTGAACACAGCATATCATGGACAATGGGAGGAGTCACAACTATCAGCTGGT GCAACCCCTCATGCTCTCCGATCAAGGCAGAGCCAAGACAATACCCTTGTATCTGCGGATCATGTCCAGCCACCTG TAGACTTTGTGCAAGCGATGATGTCTATGATGGAGGCGATATCACAGAGAGTAAGTAAGGTCGACTATCAGCTAGA TCTTGTCTTGAAACAGACATCCTCCATCCCTATGATGCGGTCCGAAATCCAACAGCTGAAAACATCTGTTGCAGTC ATGGAAGCCAACTTGGGAATGATGAAGATTCTGGATCCCGGTTGTGCCAACATTTCATCTCTGAGTGATCTACGGG CAGTTGCCCGATCTCACCCGGTTTTAGTTTCAGGCCCTGGAGACCCCTCTCCCTATGTGACACAAGGAGGCGAAAT GGCACTTAATAAACTTTCGCAACCAGTGCCACATCCATCTGAATTGATTAAACCCGCCACTGCATGCGGGCCTGAT ATAGGAGTGGAAAAGGACACTGTCCGTGCATTGATCATGTCACGCCCAATGCACCCGAGTTCTTCAGCCAAGCTCC TAAGCAAGTTAGATGCAGCCGGGTCGATCGAGGAAATCAGGAAAATCAAGCGCCTTGCTCTAAATGGCTAATTACT ACTGCCACACGTAGCGGGTCCCTGTCCACTCGGCATCACACGGAATCTGCACCGAGTTCCCCCCCGCAGACCCAAG GTCCAACTCTCCAAGCGGCAATCCTCTCTCGCTTCTTCAGCCCCACTGAATGATCGCGTAACCGTAATTAATCTAG CTACATTAAGGATTAAGAAAAAATACGGGTAGAATTGGAGTGCCCCAATTGTG AGTTTAGTTGATAGTTGTAGCCACCATGGCTGCCATCCGGAAGAAACTGGTGATTGTTGGTGATGGAGCCTGTGGAAAGACATGCTTGCTCATAGTCTTCAGCAAGGACCAGTTCCCAGAGGTGTATGTGCCCACAGTGTTTGAGAACTATGTGGCAGATATCGAGGTGGATGGAAAGCAGGTAGAGTTGGCTTTGTGGGACACAGCTGGGCAGGAAGATTATGATCGCCTGAGGCCCCTCTCCTACCCAGATACCGATGTTATACTGATGTGTTTTTCCATCGACAGCCCTGATAGTTTAGAAAACATCCCAGAAAAGTGGACCCCAGAAGTCAAGCATTTCTGTCCCAACGTGCCCATCATCCTGGTTGGGAATAAGAAGGATCTTCGGAATGATGAGCACACAAGGCGGGAGCTAGCCAAGATGAAGCAGGAGCCGGTGAAACCTGAAGAAGGCAGAGATATGGCAAACAGGATTGGCGCTTTTGGGTACATGGAGTGTTCAGCAAAGACCAAAGATGGAGTGAGAGAGGTTTTTGAAATGGCTACGAGAGCTGCTCTGCAAGCTAGACGTGGGAAGAAAAAATCTGGGTGCCTTGTCTTGTGAGTGTGTAACTACCGTGTACTAAGCCC。

[0056]

[0057]

[0058]

[0059]

[0060] After all plasmids were successfully constructed, the recombinant NDV was rescued and identified. First, 1500 ng of pB-rNDV-RHOA and three helper plasmids—pVAX-NP (1000 ng), pVAX-P (500 ng), and pcDNA-L (100 ng)—were co-transfected into BSR cells at a density of 70% using Lip3000 (Thermo Fisher Scientific, Cat. No. L3000001). 72 hours after transfection, six-well plates were sealed with sealing film and placed in a -80°C freezer, followed by three freeze-thaw cycles. 200 µL of the cell mixture was inoculated into 9- to 12-day-old SPF chicken embryos. The inoculated embryos were incubated in an incubator for 72 hours. Allantoic fluid was collected for hemagglutination titer, and the successfully rescued recombinant virus was designated rNDV-RHOA.

[0061] A549 cells were infected with wild-type NDV and rNDV-RHOA, protein samples were collected, and the expression level of RhoA was detected by western blot. Figure 2 As shown in B, the expression level of RhoA protein in the rNDV-RHOA infection group was significantly increased, indicating that the NDV recombinant virus expressing the RHOA gene was successfully constructed.

[0062] 2. Determination of the proliferation capacity of A549 cells after infection with wild-type NDV and rNDV-RHOA A549 cells were seeded in six-well plates and infected with wild-type NDV or rNDV-RHOA when cells reached 70% confluence. Samples were collected at 3, 6, 9, 12, 24, and 36 hours. The viral genome was extracted according to the kit instructions (Takara, Catalog No. 9796). The extracted RNA was reverse transcribed to generate cDNA according to the kit instructions (Abclonal, Catalog No. RK21203). Real-time quantitative analysis of the reverse-transcribed cDNA was performed using a kit (Abclonal, Catalog No. RK20404), and statistical analysis was performed based on a curve drawn from a serially diluted standard plasmid.

[0063] Following the above steps, A549 cells were infected with wild-type NDV or rNDV-RHOA, and protein samples and cell supernatants were collected. The expression level of viral HN protein was detected by western blot. Figure 2 As shown in Figure C, there was no significant difference in the genome replication ability and HN protein expression level between the recombinant rNDV-RHOA and wild-type NDV in A549 cells. These results indicate that the introduction of the RHOA gene does not affect the proliferation ability of the recombinant Newcastle disease virus.

[0064] Example 3: Detection of the killing effect of recombinant Newcastle disease virus rNDV-RHOA on tumor cells 1. Detection of the mortality rate of A549 tumor cells using lactate dehydrogenase (LDH) cytotoxicity assay A549 cells were seeded in a 96-well plate and infected with wild-type NDV or rNDV-RHOA when the cells reached 70% density. Cell supernatants were collected 12, 16, 24, 36, and 48 hours after infection. The LDH content in the supernatant was determined according to the kit instructions (Biyuntian, Cat. No.: C0016), and the tumor cell mortality rate was calculated using the formula. Figure 3 As shown in A, the recombinant Newcastle disease virus rNDV-RHOA-treated group had the highest tumor cell lethality. Compared with the wild-type NDV-treated group, the tumor cell lethality increased from 32.30% to 91.10% 48 hours after infection.

[0065] 2. Detecting the survival rate of tumor cells using CCK-8 assay A549 cells were seeded in a 96-well plate, and when the cells grew to a density of 70%, wild-type NDV or rNDV-RHOA was added to infect the cells. The cell supernatant was discarded at 12h, 16h, 24h, 36h and 48h after infection, and the cells were washed three times with PBS. CCK-8 storage solution (Biyuntian, product number: C0037) and DMEM culture medium were prepared in advance at a ratio of 1:10 to make CCK-8 working solution, and 100 µL of CCK-8 working solution was added to each well. The 96-well plate was placed in a 37°C cell culture incubator and incubated for 1~2 h. The optical density (OD) at 450 nm was measured using a microplate reader. The results of three parallel experiments were used to calculate the survival rate of tumor cells in different treatment groups according to the CCK-8 instructions. Figure 3 As shown in Figure B, the tumor cell survival rate in the rNDV-RHOA-treated group was the lowest. Compared with the wild-type NDV-treated group, the tumor cell survival rate decreased from 68.77% to 5.49% 48 hours after infection.

[0066] Example 4: Detection of the broad spectrum of oncolytic effects of recombinant Newcastle disease virus rNDV-RHOA 35 tumor cell lines (caco2, HCT116, sw620, HepG2, huh7, huh7.5, BOPX3, MIA, Primary cells from six cancer patient tumor tissues (Luad1, Luad2, SCC, Thymoma, CRC, and GC) were seeded in 96-well plates. When the cells reached 70% confluence, wtNDV, rNDV-RHOA, HSV-1, and VSV were added to infect the cells. Cell supernatants were collected 12 hours after infection and the LDH content in the supernatants was determined according to the kit instructions (Biyuntian, Cat. No. C0016). The tumor cell mortality rate was calculated using the formula: Figure 4 As shown, rNDV-RHOA induced cell death in all tumor cells, significantly better than wtNDV, and significantly higher than wild-type oncolytic viruses HSV-1 and VSV in 2 / 3 cell lines. Figure 5 As shown in Figure 2, rNDV-RHOA induced 69.56-88.65% cell death in all primary tumor cell samples from patients within 12 hours of infection, significantly outperforming wild-type oncolytic viruses wtNDV, HSV-1, and VSV. These results indicate that rNDV-RHOA has a broad-spectrum anti-tumor effect.

[0067] Example 5: Therapeutic Effect of Recombinant Newcastle Disease Virus rNDV-RHOA on Tumors in Lung Cancer Xenograft Mouse Model BALB / c-nu immunodeficient mice were subcutaneously inoculated with 2*10 6 A549 cells were used to construct a lung cancer xenograft mouse model. When the subcutaneous tumor volume of the mouse reached 80-120 mm 3 The mice were randomly divided into groups of 6 each. Every 3 days, 50 μL of PBS, rNDV-RHOA or wtNDV suspension was injected into the tumor of each group of mice. The dose of rNDV-RHOA and wtNDV was 1*10 7 PFU. Thereafter, the tumor volume was measured and recorded daily using a vernier caliper. The volume calculation formula was: V = (width * width * length) / 2. The effect of each treatment group on the tumor in the mouse body was evaluated by plotting the change curve of the mouse tumor volume. After 24 days, the tumor in the mouse body was isolated and weighed. The results are shown in the figure below. Figure 6As shown in BD, compared with the PBS control group, both rNDV-RHOA and wtNDV significantly inhibited tumor growth in mice, and rNDV-RHOA had a more significant tumor inhibitory effect.

[0068] Example 6: Therapeutic Effect of Recombinant Newcastle Disease Virus rNDV-RHOA on Tumors in Lung Cancer Homograft Mouse Model C57BL / 6J mice were subcutaneously inoculated with 1*10 6 LLC cells were used to construct a lung cancer homograft mouse model. When the subcutaneous tumor volume of the mouse reached 80-120 mm 3 The mice were randomly divided into groups of 5. Every 3 days, 50 μL of PBS, rNDV-RHOA or wtNDV suspension was injected into the tumor of each group of mice. The dose of rNDV-RHOA and wtNDV was 1*10 7 PFU. Thereafter, the tumor volume was measured and recorded daily using a vernier caliper. The volume calculation formula was: V = (width * width * length) / 2. The effect of each treatment group on the tumor in the mouse body was evaluated by plotting the change curve of the mouse tumor volume. After 18 days, the tumor in the mouse body was isolated and weighed. The results are shown in the figure below. Figure 7 As shown in BD, compared with the PBS control group, both rNDV-RHOA and wtNDV significantly inhibited tumor growth in mice, and rNDV-RHOA had a more significant tumor inhibitory effect. 60% of the tumors in the rNDV-RHOA treatment group completely regressed.

[0069] Example 7: Therapeutic Effect of Recombinant Newcastle Disease Virus rNDV-RHOA on Tumors in a Breast Cancer Homograft Mouse Model BALB / c mice were subcutaneously inoculated with 1*10 6 4T1 cells were used to construct a breast cancer homograft mouse model. When the subcutaneous tumor volume of the mouse reached 80-120 mm 3 The mice were randomly divided into groups of 5. Every 3 days, 50 μL of PBS, rNDV-RHOA or wtNDV suspension was injected into the tumor of each group of mice. The dose of rNDV-RHOA and wtNDV was 1*10 7 PFU. Thereafter, the tumor volume was measured and recorded daily using a vernier caliper. The volume calculation formula was: V = (width * width * length) / 2. The effect of each treatment group on the tumor in the mouse body was evaluated by plotting the change curve of the mouse tumor volume. After 15 days, the tumor in the mouse body was isolated and weighed. The results are shown in the figure below. Figure 8As shown in BD, compared with the PBS control group, both rNDV-RHOA and wtNDV significantly inhibited tumor growth in mice, and rNDV-RHOA had a more significant tumor inhibitory effect. 60% of the tumors in the rNDV-RHOA treatment group completely regressed.

[0070] Example 8: Therapeutic Effect of Recombinant Newcastle Disease Virus rNDV-RHOA on Tumors in Epidermal Cancer Homograft Mouse Model C57BL / 6J mice were subcutaneously inoculated with 1*10 6 B16F10 cells were used to construct epidermal cancer homologous transplant mouse models. When the subcutaneous tumor volume of the mouse reached 80-120 mm 3 The mice were randomly divided into groups of 5. Every 3 days, 50 μL of PBS, rNDV-RHOA or wtNDV suspension was injected into the tumor of each group of mice. The dose of rNDV-RHOA and wtNDV was 1*10 7 PFU. Thereafter, the tumor volume was measured and recorded daily using a vernier caliper. The volume calculation formula was: V = (width * width * length) / 2. The effect of each treatment group on the tumor in the mouse body was evaluated by plotting the change curve of the mouse tumor volume. After 10 days, the tumor in the mouse body was isolated and weighed. The results are shown in the figure below. Figure 9 As shown in BD, compared with the PBS control group, both rNDV-RHOA and wtNDV significantly inhibited tumor growth in mice, and rNDV-RHOA had a more significant tumor inhibitory effect.

[0071] Example 9: Therapeutic Effect of Intravenous Injection of Recombinant Newcastle Disease Virus rNDV-RHOA on Tumors in a Colon Cancer Homograft Mouse Model C57BL / 6J mice were subcutaneously inoculated with 1*10 6 MC38 cells were used to construct a colon cancer homograft mouse model. When the subcutaneous tumor volume of the mouse reached 80-120 mm 3 The mice were randomly divided into groups of 5 each. Every 3 days, 100 μL of PBS, rNDV-RHOA or wtNDV suspension was injected into the tail vein of each group of mice. The dose of rNDV-RHOA and wtNDV was 1*10 9 PFU / kg. Tumor volume was measured and recorded daily using a vernier caliper. The volume calculation formula was: V = (width * width * length) / 2. The effect of each treatment group on the tumor in mice was evaluated by plotting the change curve of mouse tumor volume. After 14 days, the tumors in the mice were isolated and weighed. The results are shown in the figure below. Figure 10As shown in BD, compared with the PBS control group, intravenous injection of wtNDV failed to show a significant tumor inhibitory effect, but intravenous injection of rNDV-RHOA treatment still significantly inhibited tumor growth in mice, indicating that rNDV-RHOA significantly improved the anti-tumor therapeutic efficacy of intravenous administration of oncolytic viruses.

[0072] Example 10: Exploring the Mechanism of Acute Tumor Cell Death Induced by Recombinant Newcastle Disease Virus rNDV-RHOA 1. Use the reactive oxygen species assay to detect the effect of oncolytic virus infection on the expression level of reactive oxygen species in A549 cells A549 cells were seeded in a six-well plate, and when the cells grew to a density of 70%, wild-type NDV or rNDV-RHOA was added to infect the cells. Samples were collected at 9h and 12h, respectively, and DCFH-DA (Biyuntian, Product No.: S0033M) was diluted with PBS at a ratio of 1:1000 in advance to a final concentration of 10 micromoles / liter. The cells were suspended in the diluted DCFH-DA and incubated in a cell culture incubator at 37°C for 20 min. Invert and mix every 3-5 minutes to ensure full contact between the probe and the cells. After the incubation, the cells were washed three times with PBS to fully remove the DCFH-DA that did not enter the cells. After the last wash, the cells were resuspended in 500 µL of PBS and collected in a flow tube through a 300-mesh cell sieve. The fluorescence intensity was detected using a flow cytometer at an excitation wavelength of 488nm. The results are shown in Figure 2. Figure 11 As shown in A, rNDV-RHOA rapidly induced excessive ROS production in A549 tumor cells.

[0073] 2. Lipid peroxidation assay to detect the effect of oncolytic virus infection on lipid peroxidation in A549 cells A549 cells were seeded in a six-well plate, and when the cells grew to a density of 70%, wild-type NDV or rNDV-RHOA was added to infect the cells. Samples were collected at 9h and 12h, respectively, and an appropriate volume of BODIPY 581 / 591 C11 staining working solution (Biyuntian, catalog number: S0043M) was added, and incubated in a 37°C cell culture incubator for 20 min. After the incubation, the cells were washed twice with pre-cooled PBS. After the last wash, the cells were resuspended in 500 μL of PBS and collected in a flow tube through a 300-mesh cell sieve. The fluorescence intensity was detected using a flow cytometer at an excitation wavelength of 488 / 510nm. The results are shown in Figure 2. Figure 11 As shown in B, rNDV-RHOA rapidly induced lipid peroxidation in A549 tumor cells.

[0074] 3. Lactate dehydrogenase (LDH) cytotoxicity assay to examine the effect of inhibiting key biological events on the oncolytic efficacy of rNDV-RHOA A549 cells were seeded in 96-well plates, and different inhibitors were added when the cells grew to 70% density, including CQ (autophagy inhibitor, Sigma, Catalog No.: C6628), Fer-1 (lipid peroxidation inhibitor, Sigma, Catalog No.: SML0583), Mito-Q (mitochondrial ROS inhibitor, selleck, Catalog No.: S8978), and DFOM (iron ion chelator, MCE, Catalog No.: HY-B0988). After 6 hours of action, wild-type NDV or rNDV-RHOA was infected. Cell supernatants were collected 12 hours after infection, and the LDH content in the supernatant was detected according to the kit instructions (Biyuntian, Catalog No.: C0016), and the tumor cell mortality rate was calculated using the formula. Figure 11 As shown in C, the three inhibitors CQ, Fer-1, and Mito-Q significantly inhibited the high tumor cell mortality caused by rNDV-RHOA infection, while DFOM had no significant effect, suggesting that the acute burst of mitochondrial ROS caused by rNDV-RHOA infection and the subsequent rapid lipid peroxidation and autophagy have an important influence on the oncolytic efficacy of rNDV-RHOA.

[0075] 4. Lipid peroxidation assay to examine the effects of inhibiting key biological events on lipid peroxidation in rNDV-RHOA-infected cells A549 cells were seeded in six-well plates and treated with various inhibitors (CQ (autophagy inhibitor, Sigma, Catalog No. C6628), Fer-1 (lipid peroxidation inhibitor, Sigma, Catalog No. SML0583), Mito-Q (mitochondrial ROS inhibitor, selleck, Catalog No. S8978), and DFOM (iron chelator, MCE, Catalog No. HY-B0988)) for 6 hours. The cells were then infected with wild-type NDV or rNDV-RHOA. Twelve hours after infection, samples were harvested and an appropriate volume of BODIPY 581 / 591C11 staining solution (Biyuntian, Catalog No. S0043M) was added. The cells were then incubated at 37°C in a cell culture incubator for 20 minutes. After the incubation, the cells were washed twice with pre-cooled PBS. After the last wash, the cells were resuspended in 500 μL of PBS and collected in a flow cytometer through a 300-mesh cell sieve. The fluorescence intensity was detected by flow cytometry at an excitation wavelength of 488 / 510 nm. Figure 11 As shown in D, Fer-1 and Mito-Q significantly inhibited the degree of lipid peroxidation induced by rNDV-RHOA in A549 tumor cells, while CQ and DFOM had no significant effect, indicating that the rapid lipid peroxidation caused by rNDV-RHOA infection was controlled by the acute burst of mitochondrial ROS.

[0076] 5. Use the reactive oxygen species assay to detect the effects of different ROS sources on the reactive oxygen species levels in rNDV-RHOA infected cells A549 cells were seeded in six-well plates and treated with various inhibitors (Mito-Q (mitochondrial ROS inhibitor, selleck, Catalog No. S8978), Setanaxib (NADPH oxidase inhibitor, selleck, Catalog No. S7171), UAS (NADPH oxidase inhibitor, MCE, Catalog No. HY-B2130A), DFOM (iron chelator, MCE, Catalog No. HY-B0988), DFX (iron chelator, MCE, Catalog No. HY-17359), and DFO (iron chelator, MCE, Catalog No. HY-B0568)) for 6 hours. The cells were then infected with wild-type NDV or rNDV-RHOA. Samples were harvested 12 hours post-infection and pre-treated with DCFH-DA (Biyuntian, Catalog No. S0033M) diluted 1:1000 in PBS to a final concentration of 10 μmol / L. The cells were suspended in diluted DCFH-DA and incubated in a 37°C cell culture incubator for 20 min. The tubes were inverted and mixed every 3-5 min to ensure full contact between the probe and the cells. After the incubation, the cells were washed three times with PBS to fully remove the DCFH-DA that had not entered the cells. After the last wash, the cells were resuspended in 500 μL of PBS and passed through a 300-mesh cell sieve into a flow cytometer. The fluorescence intensity was detected using a flow cytometer at an excitation wavelength of 488 nm. The results are shown in Figure 2. Figure 11 As shown in E, Mito-Q significantly inhibited the high level of ROS produced by A549 tumor cells induced by rNDV-RHOA, while other inhibitors had no significant effect, indicating that the acute burst of ROS caused by rNDV-RHOA infection originated from the mitochondrial pathway.

[0077] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0078] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. Application of RhoA protein in the preparation of products that enhance the therapeutic efficacy of oncolytic tumor viruses.

2. The use of the RhoA protein according to claim 1 in preparing a product for enhancing the therapeutic efficacy of tumor oncolytic virus therapy, characterized in that: The amino acid sequence of the RhoA protein is shown in SEQ ID NO.

1.

3. The use of the RhoA protein according to claim 1 in preparing a product for enhancing the therapeutic efficacy of tumor oncolytic virus therapy, characterized in that: The cancers include lung cancer, cervical cancer, intestinal cancer, epidermal cancer, breast cancer, nervous system tumors, liver cancer, stomach cancer, ovarian cancer, prostate cancer, pancreatic cancer, esophageal cancer, thymic cancer, and lymphoma.

4. A preparation for enhancing the therapeutic efficacy of oncolytic virus therapy, characterized in that: The preparation comprises the RhoA protein according to claim 1.

5. A recombinant Newcastle disease virus genome, characterized in that The genome comprises the gene encoding the RhoA protein according to claim 1.

6. The recombinant Newcastle disease virus genome according to claim 5, wherein The nucleotide sequence of the recombinant Newcastle disease virus genome is sequentially connected by the sequences shown in SEQ ID NO. 3-4.

7. A recombinant oncolytic virus expressing the RHOA gene, characterized in that: The recombinant oncolytic virus comprises the recombinant Newcastle disease virus genome according to any one of claims 5 to 6.

8. The recombinant oncolytic virus expressing the RHOA gene according to claim 7, characterized in that The starting strain of the recombinant oncolytic virus is selected from the Newcastle disease virus La Sota vaccine strain.

9. A cancer treatment drug, characterized in that: The active ingredient is the recombinant oncolytic virus according to claim 7.

10. The method for preparing the recombinant oncolytic virus expressing the RHOA gene according to claim 8, characterized in that: include: Construction of Newcastle disease virus genome vector; By homologous recombination, the coding gene of the RhoA protein is connected to the Newcastle disease virus genome vector, so that the coding gene of the RhoA protein is inserted into the Newcastle disease virus genome to obtain a recombinant Newcastle disease virus plasmid; The recombinant Newcastle disease virus plasmid and the helper plasmid are co-transfected into cells and the transfected cells are cultured to obtain the recombinant oncolytic virus.

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