Recombinant Newcastle disease virus as well as preparation method and anti-tumor application thereof

Abstract

The invention belongs to the technical field of tumor treatment, and particularly relates to a recombinant Newcastle disease virus as well as a preparation method and application thereof in tumor resistance. According to the recombinant gene provided by the invention, the GGTA1P coding gene can express heterologous alpha-Gal epitopes in tumor cells, so that immune escape is reduced, an immune system is enhanced, and the tumor cells are recognized and killed; cSF2, TP53 and IL12 expressed by the CSF2 coding gene, the TP53 coding gene and the IL12 coding gene respectively have the functions of immunoregulation, apoptosis promotion or angiogenesis resistance, and can enhance the anti-tumor effect by cooperating with GGTA1P. Furthermore, the Newcastle disease virus (NDV) has a natural oncolytic characteristic, the recombinant gene is inserted between the P gene and the M gene of the Newcastle disease virus, and the recombinant Newcastle disease virus obtained after transfection can infect tumor cells in a targeted manner and activate immune response.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor treatment, and specifically relates to a recombinant Newcastle disease virus, a preparation method thereof, and an application thereof in anti-tumor treatment. Background Art

[0002] Cancer is a disease caused by the loss of normal regulation and excessive proliferation of body cells, and has currently become the number one killer affecting health. For a long time, the main methods for treating cancer have been traditional surgical resection, radiotherapy, and chemotherapy. These methods can control the development of tumors to a certain extent, but the curative effect is limited, and at the same time, these methods cause serious damage to normal cells of the human body.

[0003] The technology of oncolytic viruses expressing double foreign genes has evolved from single-functional modification to synergistic enhancement. The early breakthrough began with the first oncolytic adenovirus H101 approved in China in 2005, which did not carry foreign genes but verified the feasibility of the viral vector. In 2015, the FDA approved T-VEC (carrying the GM-CSF gene), which first enhanced the immune response through a single gene. After 2020, the dual-gene design has become the mainstream: the NDV-GT virus developed by the team of Zhao Yongxiang at Guangxi Medical University integrates the porcine α1,3GT gene with the Newcastle disease virus backbone, activating hyperacute rejection and T cell infiltration; the CG0070 adenovirus of CG Oncology combines the E2F-1 tumor-specific promoter with the GM-CSF gene to achieve targeted replication and immune synergy (Hyperacute rejection-engineered oncolytic virus for interventional clinical trial in refractory cancer patients doi:10.1016 / j.cell.2024.12.010). In 2024, the team of Shumin Feng at Fudan University introduced the ICP0 gene into HSV-1 to degrade the METTL14 protein, while suppressing the host antiviral pathway and enhancing the oncolytic activity (HSV-1-induced N6-methyladenosine reprogramming via ICP0-mediated suppression of METTL14 potentiates oncolytic activity in glioma DOI:10.1016 / j.celrep.2024.114756). Currently, the dual-gene strategy significantly improves the curative effect through mechanisms such as "virus lysis + immune activation" or "targeted replication + microenvironment regulation", and has become the focus of clinical development. Summary of the Invention

[0004] The object of the present invention is to provide a recombinant Newcastle disease virus, its preparation method and application in anti-tumor, and the anti-tumor effect is remarkable.

[0005] The present invention provides a recombinant gene, and the recombinant gene includes a first gene and a second gene;

[0006] The first gene includes a GGTA1P coding gene; the GGTA1P coding gene has a sequence as shown in SEQ ID NO: 1 or a sequence having at least 80% identity therewith;

[0007] The second gene includes one or more of a CSF2 coding gene, a TP53 coding gene and an IL12 coding gene; the CSF2 coding gene has a sequence as shown in SEQ ID NO: 2 or a sequence having at least 80% identity therewith; the TP53 coding gene has a sequence as shown in SEQ ID NO: 3 or a sequence having at least 80% identity therewith; the IL12 coding gene has a sequence as shown in SEQ ID NO: 4 or a sequence having at least 80% identity therewith.

[0008] The present invention also provides a recombinant Newcastle disease virus vector, and the recombinant Newcastle disease virus vector includes a basic Newcastle disease virus vector and the recombinant gene described in the above technical solution inserted into the basic Newcastle disease virus vector;

[0009] The recombinant gene is located between the P gene and the M gene of the basic Newcastle disease virus genome.

[0010] Preferably, the basic Newcastle disease virus vector includes LaSota strain, HUJ strain, PV701 strain, Ulster2C strain or 73T strain.

[0011] The present invention also provides a recombinant Newcastle disease virus, and the recombinant Newcastle disease virus contains the recombinant Newcastle disease virus vector described in the above technical solution.

[0012] The present invention also provides a preparation method of the recombinant Newcastle disease virus described in the above technical solution, including the following steps: co-transfecting the recombinant Newcastle disease virus vector described in the above technical solution and an auxiliary plasmid into cells, and culturing the transfected cells to obtain the recombinant Newcastle disease virus.

[0013] Preferably, the ratio of the recombinant Newcastle disease virus vector to the cells is 5.0 μg: 1×10 6 cells;

[0014] The cells include BHK-Flag-T7opt cell line cells;

[0015] The mass ratio of the recombinant Newcastle disease virus vector to the helper plasmid is 5:3;

[0016] The helper plasmid includes pCAGGS-Ndv-L, pCAGGS-Ndv-NP, and pCAGGS-Ndv-P; the mass ratio of pCAGGS-Ndv-L, pCAGGS-Ndv-NP, and pCAGGS-Ndv-P is 2:0.5:0.5.

[0017] The present invention also provides the use of the recombinant gene described in the above technical solution, or the recombinant Newcastle disease virus vector described in the above technical solution, or the recombinant Newcastle disease virus described in the above technical solution, or the recombinant Newcastle disease virus prepared by the preparation method described in the above technical solution in the preparation of anti-tumor drugs.

[0018] Preferably, the tumors include one or more of non-small cell lung cancer, colorectal adenocarcinoma, ovarian cancer, cervical cancer, liver cancer, breast cancer, esophageal squamous cell carcinoma, and melanoma.

[0019] Preferably, the cancer cells of non-small cell lung cancer are A549; the cancer cells of colorectal adenocarcinoma are LS513; the cancer cells of ovarian cancer are HO8910 and / or OVCAR3; the cancer cells of cervical cancer are Hela; the cancer cells of liver cancer are HepG2; the cancer cells of breast cancer are MDA-MB-231 and / or MCF-7; the cancer cells of esophageal squamous cell carcinoma are TE-10; the cancer cells of melanoma are SK-MEL-28.

[0020] The present invention also provides an anti-tumor drug, which contains the recombinant gene described in the above technical solution, or the recombinant Newcastle disease virus vector described in the above technical solution, or the recombinant Newcastle disease virus described in the above technical solution, or the recombinant Newcastle disease virus prepared by the preparation method described in the above technical solution.

[0021] Beneficial effects:

[0022] The present invention provides a recombinant gene, wherein the GGTA1P coding gene can express xeno-α-Gal epitopes in tumor cells, reduce immune escape, and enhance the immune system's recognition and killing of tumor cells; the CSF2, TP53, and IL12 encoded by the CSF2 coding gene, TP53 coding gene, and IL12 coding gene respectively have immune regulation, pro-apoptotic, or anti-angiogenic functions, and can synergistically enhance the anti-tumor effect with GGTA1P.

[0023] Furthermore, Newcastle disease virus (NDV) has natural oncolytic properties. Inserting the recombinant gene between the P gene and the M gene of Newcastle disease virus, the recombinant Newcastle disease virus obtained after transfection can target and infect tumor cells and activate the immune response. Description of the drawings

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings required for the embodiments will be briefly introduced below.

[0025] Figure 1 Insertion position of the gene fragment in the NDV vector in Example 1;

[0026] Figure 2 Schematic diagram of NDV-GGTA1P-CSF2 in Example 1;

[0027] Figure 3 Insertion position of the gene fragment in the NDV vector in Example 2;

[0028] Figure 4 Insertion position of the gene fragment in the NDV vector in Example 2;

[0029] Figure 5 Hemagglutination test results of allantoic fluid of Newcastle disease virus in Example 4;

[0030] Figure 6 Schematic diagram of NDV-CSF2 in Comparative Example 2;

[0031] Figure 7 Schematic diagram of NDV-IL12 in Comparative Example 3;

[0032] Figure 8 Schematic diagram of NDV-TP53 in Comparative Example 4. Detailed implementation manners

[0033] The present invention provides a recombinant gene, the recombinant gene comprising a first gene and a second gene; the first gene comprises a GGTA1P encoding gene; the GGTA1P encoding gene has a sequence as shown in SEQ ID NO:1 or a sequence having at least 80% identity therewith; the second gene comprises one or more of a CSF2 encoding gene, a TP53 encoding gene and an IL12 encoding gene; the CSF2 encoding gene has a sequence as shown in SEQ ID NO:2 or a sequence having at least 80% identity therewith; the TP53 encoding gene has a sequence as shown in SEQ ID NO:3 or a sequence having at least 80% identity therewith; the IL12 encoding gene has a sequence as shown in SEQ ID NO:4 or a sequence having at least 80% identity therewith.

[0034] As an implementation mode, the first gene and the second gene of the present invention are linked by a Newcastle disease virus promoter and a terminator. The GGTA1P coding gene of the present invention can express xeno-α-Gal epitopes in tumor cells, reduce immune escape and enhance the immune system's ability to recognize and kill tumor cells; the CSF2 expressed by the CSF2 coding gene has an immune regulatory function and can activate dendritic cells and enhance T cell responses; the TP53 expressed by the TP53 coding gene has a pro-apoptotic function and can induce apoptosis of tumor cells; the IL12 expressed by the IL12 coding gene has an anti-angiogenic function and can promote NK cell activation and inhibit angiogenesis. Recombining one or more of the GGTA1P coding gene and the CSF2 coding gene, the TP53 coding gene and the IL12 coding gene of the present invention has a synergistic effect and can enhance the anti-tumor effect.

[0035] The sequence information of SEQ ID NO: 1 to SEQ ID NO: 4 of the present invention is specifically as follows:

[0036]

[0037]

[0038]

[0039] The present invention also provides a recombinant Newcastle disease virus vector, and the recombinant Newcastle disease virus vector includes a basic Newcastle disease virus vector and the recombinant gene described in the above technical solution inserted into the basic Newcastle disease virus vector; the recombinant gene is located between the P gene and the M gene of the basic Newcastle disease virus genome.

[0040] As an implementation mode, the recombinant gene of the present invention is located between the P gene and the M gene of the basic Newcastle disease virus genome, and is ligated by PacI and KpnI digestion. As an implementation mode, the basic Newcastle disease virus vector of the present invention includes the LaSota strain, the HUJ strain, the PV701 strain, the Ulster2C strain or the 73T strain. The present invention does not have strict requirements for the preparation method of the recombinant Newcastle disease virus vector, and conventional operations in the art can be used, such as the method of enzyme digestion and ligation.

[0041] The present invention also provides a recombinant Newcastle disease virus, and the recombinant Newcastle disease virus includes the recombinant Newcastle disease virus vector described in the above technical solution.

[0042] The present invention also provides a preparation method of the recombinant Newcastle disease virus described in the above technical solution, including the following steps: co-transfecting the recombinant Newcastle disease virus vector described in the above technical solution and an auxiliary plasmid into cells, and culturing the transfected cells to obtain the recombinant Newcastle disease virus.

[0043] As an embodiment, the ratio of the recombinant Newcastle disease virus vector to the cells in the present invention is 1.0 - 10.0 μg: 1×10 5 - 1×10 7 cells; as another embodiment, the ratio of the recombinant Newcastle disease virus vector to the cells in the present invention is 5.0 μg: 1×10 6 cells. As an embodiment, the cells in the present invention include BHK-Flag-T7opt cell line cells. As an embodiment, the mass ratio of the recombinant Newcastle disease virus vector to the helper plasmid in the present invention is 5:3. As an embodiment, the helper plasmid in the present invention includes pCAGGS-Ndv-L, pCAGGS-Ndv-NP, and pCAGGS-Ndv-P. As an embodiment, the mass ratio of pCAGGS-Ndv-L, pCAGGS-Ndv-NP, and pCAGGS-Ndv-P in the present invention is 2:0.5:0.5.

[0044] The present invention also provides the use of the recombinant gene described in the above technical solution, or the recombinant Newcastle disease virus vector described in the above technical solution, or the recombinant Newcastle disease virus described in the above technical solution, or the recombinant Newcastle disease virus prepared by the preparation method described in the above technical solution in the preparation of anti-tumor drugs.

[0045] As an embodiment, the tumors in the present invention include one or more of non-small cell lung cancer, colon adenocarcinoma, ovarian cancer, cervical cancer, liver cancer, breast cancer, esophageal squamous cell carcinoma, and melanoma; as another embodiment, the tumors in the present invention include non-small cell lung cancer, colon adenocarcinoma, ovarian cancer, cervical cancer, liver cancer, breast cancer, esophageal squamous cell carcinoma, and melanoma.

[0046] As an embodiment, the cancer cells of the non-small cell lung cancer in the present invention are A549. As an embodiment, the cancer cells of the colon adenocarcinoma in the present invention are LS513. As an embodiment, the cancer cells of the ovarian cancer in the present invention are HO8910 and / or OVCAR3. As an embodiment, the cancer cells of the cervical cancer in the present invention are Hela. As an embodiment, the cancer cells of the liver cancer in the present invention are HepG2. As an embodiment, the cancer cells of the breast cancer in the present invention are MDA-MB-231 and / or MCF-7. As an embodiment, the cancer cells of the esophageal squamous cell carcinoma in the present invention are TE-10. As an embodiment, the cancer cells of the melanoma in the present invention are SK-MEL-28.

[0047] The present invention also provides an anti-tumor drug, which comprises the recombinant gene described in the above technical solution or the recombinant Newcastle disease virus vector described in the above technical solution or the recombinant Newcastle disease virus described in the above technical solution or the recombinant Newcastle disease virus prepared by the preparation method described in the above technical solution.

[0048] As an embodiment, the anti-tumor drug of the present invention further comprises a pharmaceutically acceptable excipient.

[0049] In order to further illustrate the present invention, a recombinant Newcastle disease virus and its preparation method and application in anti-tumor provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1

[0051] Construction of recombinant Newcastle disease virus (NDV-GGTA1P-CSF2)

[0052] 1. Design of a fragment containing the porcine GGTA1 gene and the human CSF2 gene

[0053] The porcine GGTA1 gene and the human CSF2 gene are connected by the promoter (GS) and terminator (GE) of the Newcastle disease virus genome; wherein, the accession number of the porcine GGTA1 gene in NCBI is NM_213810, and the specific nucleotide sequence is shown in SEQ ID NO:1; the accession number of the human CSF2 gene in NCBI is NM_000758, and the specific nucleotide sequence is shown in SEQ ID NO:2.

[0054] 2. The synthesized gene fragment was cloned into the genome of Newcastle disease virus (NDV) LaSota strain (NCBI No. AF077761) between the P gene and the M gene by PacI and KpnI restriction enzyme ligation. Figure 1 ), and obtain NDV-GGTA1P-CSF2 virus vector, the schematic diagram is as follows Figure 2 shown.

[0055] 3. Transfection

[0056] (1) Mix 5.0 μg of Newcastle disease virus vector obtained in step 2 and helper plasmids (2.0 μg pCAGGS-Ndv-L, 0.5 μg pCAGGS-Ndv-NP and 0.5 μg pCAGGS-Ndv-P), and use the transfection reagent ExFect Transfection Reagent (No. T101-01 of Norveg) according to the instructions to transfect 1×10 6BHK-Flag-T7opt cell line cells (published Chinese patent CN116855538A) were placed in a 37°C, 5% CO2 cell culture incubator after transfection, and new cell culture medium was replaced after 8 to 12 hours, and cultured overnight.

[0057] (2) 24 h after transfection, the cells were placed in a 32°C 5% CO2 cell culture incubator. 48 h after transfection, the culture medium was replaced with serum-free DMEM medium containing 1.0 μg / mL TPCK-treated trypsin. 96 h after transfection, the virus was harvested and centrifuged at 2000 rpm at 4°C for 10 min to obtain the supernatant of the recombinant Newcastle disease virus (NDV-GGTA1P-CSF2).

[0058] Example 2

[0059] Construction of recombinant Newcastle disease virus (NDV-GGTA1P-IL12)

[0060] 1. Design of a fragment containing the porcine GGTA1 gene and the human IL12 gene

[0061] The porcine GGTA1 gene and the human IL12 gene are connected by the promoter (GS) and terminator (GE) of the Newcastle disease virus genome; wherein, the accession number of the porcine GGTA1 gene in NCBI is NM_213810, and the specific nucleotide sequence is shown in SEQ ID NO: 1; the human IL12 gene is a fusion gene of the IL12B gene and the IL12A gene, and the accession numbers of the IL12B gene and the IL12A gene in NCBI are NM_002187 and NM_000882 respectively, and the nucleotide sequence of the human IL12 gene is shown in SEQ ID NO: 3.

[0062] 2. The synthesized gene fragment was cloned into the genome of Newcastle disease virus (NDV) LaSota strain (NCBI No. AF077761) between the P gene and the M gene by PacI and KpnI restriction enzyme ligation ( Figure 3 ) to obtain the NDV-GGTA1P-IL12 virus vector.

[0063] 3. According to the method of Example 1, the NDV-GGTA1P-IL12 virus vector and the helper plasmid were co-transfected into BHK-Flag-T7opt cell line cells to finally obtain the recombinant Newcastle disease virus (NDV-GGTA1P-IL12) supernatant.

[0064] Example 3

[0065] Construction of recombinant Newcastle disease virus (NDV-GGTA1P-TP53)

[0066] 1. Design a fragment containing porcine GGTA1 gene and human TP53 gene

[0067] Connect the porcine GGTA1 gene and the human TP53 gene through the promoter (GS) and terminator (GE) carried by the genome of Newcastle disease virus; among them, the accession number of the porcine GGTA1 gene in NCBI is NM_213810, and the specific nucleotide sequence is shown in SEQ ID NO:1; the accession number of human TP53 in NCBI is NM_000546, and the specific nucleotide sequence is shown in SEQ ID NO:4.

[0068] 2. Clone the synthesized gene fragment into the region between the P gene and the M gene of the genome of Newcastle disease virus (NDV) LaSota strain (NCBI number AF077761) by restriction enzyme digestion and ligation with PacI and KpnI Figure 4 ) to obtain the NDV-GGTA1P-TP53 viral vector.

[0069] 3. Co-transfect the NDV-GGTA1P-TP53 viral vector and the helper plasmid into BHK-Flag-T7opt cell line cells in the manner of Example 1, and finally obtain the supernatant of recombinant Newcastle disease virus (NDV-GGTA1P-TP53).

[0070] Comparative Example 1

[0071] Construct a control virus (NDV-GGTA1P) that only expresses the porcine GGTA1 gene of Example 1 in the manner of Example 1 without inserting the human gene, and finally obtain the supernatant of the GGTA1 control virus.

[0072] Example 4

[0073] Inoculate the supernatant of Newcastle disease virus obtained in Examples 1 to 3 and the supernatant of the GGTA1 control virus obtained in Comparative Example 1 into 9-day-old SPF chicken embryos at a ratio of 0.2 mL per egg, incubate at 35 °C for 72 h, then place them in the refrigerator overnight, take them out of the refrigerator after 96 h, and use a needle to extract the allantoic fluid.

[0074] 1. Hemagglutination experiment

[0075] Prepare a 96-well V-shaped hemagglutination plate, add 50 μL of PBS to each well, add 50 μL of the allantoic fluid sample to the first well, mix well and perform two-fold dilution until the last well. Add 50 μL of 1% SPF chicken red blood cell suspension to each well, and gently shake and mix. Let stand at room temperature for 45 minutes, observe the results, and perform 2 replicates. The results are as Figure 5 shown; among them, the allantoic fluid concentration decreases from left to right, and the negative control is PBS without adding virus solution.

[0076] When Newcastle disease virus binds to chicken red blood cells, the red blood cells will completely agglutinate, showing a uniform thin layer formed at the bottom of the well and not flowing after tilting; the unagglutinated red blood cells will precipitate into dots and flow in a line after tilting, indicating that the virus is not sufficient to cause red blood cell agglutination.

[0077] According to Figure 5 It can be seen that the allantoic fluid of chicken embryos prepared with the Newcastle disease virus supernatant (recombinant Newcastle disease virus) obtained in Examples 1 to 3 has hemagglutination activity.

[0078] 2. Virus titer detection

[0079] In a 12-well plate, 1 μL of Newcastle disease virus allantoic fluid was used to infect 5×10 5 BHK-21 cells, and cultured at 32 °C in a 5% CO2 environment for 72 h. The infected BHK-21 cells were digested into a cell suspension, resuspended with 100 μL of 0.5% BSA PBS, 1 μg of anti-NDV rabbit IgG was added, and incubated on ice for 30 minutes. After incubation, the cells were washed with 1 mL of 0.5% BSA PBS, centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the washing was repeated 2 times. Fluorescently labeled rabbit secondary antibody was added. Incubated on ice for 30 min, protected from light. After incubation, the cells were washed with 1 mL of 0.5% BSA PBS, centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the washing was repeated 2 times. The cells were resuspended in 200 μL of 0.5% BSA PBS and detected using a flow cytometer. The virus titer was calculated using the Poisson distribution: MOI (multiplicity of infection) = -1×ln(1 - proportion of positive cells); virus titer (pfu / mL) = (1 mL / 1 μL) × MOI. The detection results are shown in Table 1.

[0080] Table 1 Virus titer detection results

[0081] Virus name Virus titer (pfu / mL) NDV-GGTA1P <![CDATA[8.82×10 8 > NDV-GGTA1P-CSF2 <![CDATA[4.86×10 8 > NDV-GGTA1P-IL12 <![CDATA[3.54×10 8 > NDV-GGTA1P-TP53 <![CDATA[3.08×10 8 >

[0082] It can be seen from Table 2 that the virus allantoic fluids prepared with the Newcastle disease virus supernatant (recombinant Newcastle disease virus) obtained in Examples 1 to 3 all have infectious activity, and there is no difference in infectious activity between the double-gene expression group and the single-gene expression group.

[0083] Comparative Example 2

[0084] Construct a control virus (NDV-CSF2, Figure 6 ) that only expresses the human CSF2 gene of Example 1 in the manner of Example 1, without inserting the porcine GGTA1 gene, and finally obtain the CSF2 control virus supernatant.

[0085] Comparative Example 3

[0086] Construct a control virus (NDV-IL12, Figure 7 ) that only expresses the human IL12 gene of Example 1 in the manner of Example 1, without inserting the porcine GGTA1 gene, and finally obtain the supernatant of the IL12 control virus.

[0087] Comparative Example 4

[0088] Construct a control virus (NDV-TP53, Figure 8 ) that only expresses the human TP53 gene of Example 1 in the manner of Example 1, without inserting the porcine GGTA1 gene, and finally obtain the supernatant of the TP53 control virus.

[0089] Example 5

[0090] Apoptosis detection

[0091] 1. Test cancer cells

[0092] HepG2 cells, liver cancer, purchased from Wuhan Pusai Biotechnology Co., Ltd., product number: CL-0103;

[0093] MCF-7 cells, breast cancer, purchased from Wuhan Pusai Biotechnology Co., Ltd., product number: CL-0149;

[0094] A549 cells, lung cancer, purchased from Wuhan Pusai Biotechnology Co., Ltd., product number: CL-0016;

[0095] OVCAR3 cells, ovarian cancer, purchased from Wuhan Pusai Biotechnology Co., Ltd., product number: CL-0178;

[0096] LS513 cells, colon cancer, purchased from Wuhan Pusai Biotechnology Co., Ltd., product number: CL-0640;

[0097] TE-10 cells, esophageal cancer, purchased from Wuhan Pusai Biotechnology Co., Ltd., product number: CL-0453;

[0098] SK-MEL-28 cells, melanoma, purchased from Wuhan Pusai Biotechnology Co., Ltd., product number: CL-0717;

[0099] Hela cells, cervical cancer, purchased from Wuhan Pusai Biotechnology Co., Ltd., product number: CL-0101;

[0100] HO-8910PM, ovarian cancer, Catalogue number of the cell line information of the Cell Bank of the Chinese Academy of Sciences: TCHu 25, purchased from the Cell Bank of the Chinese Academy of Sciences Committee for Type Culture Collection or the Cell Resource Center of the Shanghai Institute of Life Sciences, Chinese Academy of Sciences;

[0101] MDA-MB-231, breast cancer, Catalogue No. of the Cell Bank of the Institute of Microbiology, Chinese Academy of Sciences: SCSP-5043, purchased from the Cell Bank of the Institute of Microbiology, Chinese Academy of Sciences or the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0102] 2. Test samples

[0103] The supernatant of Newcastle disease virus obtained in Examples 1-3 and the control virus supernatant obtained in Comparative Examples 1-4 were respectively diluted to 0.1 MOI with complete medium to obtain virus dilutions.

[0104] 3. Take the test cells in the logarithmic growth phase, digest them with trypsin, and suspend them in complete medium at a density of 5×10 4 / mL. Take the cell suspension and add it to a 12-well plate, 1 mL per well, and incubate at 37 °C for 24 h. After incubation, discard the medium, and add 1 mL of virus dilution to each well. The control group was added 1 mL of complete medium. After 1 h of infection, wash the cells with fresh PBS, add 1 mL of complete medium containing 5% human serum and 1×10 5 / mL PBMCs. After 72 h, discard the medium, and digest the cells with 0.25% trypsin without EDTA. Collect the cells, wash them twice with PBS, and resuspend them in 200 μL of 0.5% BSA PBS. Then add 10 μL of Annexin V-FITC and incubate at room temperature in the dark at 4 °C for 10 min. Finally, add 200 μL of 0.5% BSA PBS and 5 μL of PI, mix gently, and incubate at room temperature in the dark for 5 min, and perform flow cytometry to calculate the proportion of apoptotic cells. Each well was repeated 4 times and the average value was taken. The results are shown in Table 2.

[0105] Table 2 Proportion of apoptotic cells of cancer cells induced by different Newcastle disease viruses (%)

[0106]

[0107] Note: In Comparative Examples 2-4, ** indicates significant difference compared with NDV, p<0.01; in Examples 1-3, ** indicates significant difference compared with Comparative Example 1, p<0.01; in Comparative Example 1, ## indicates significant difference compared with Comparative Examples 2-4, p<0.01;

[0108] It can be seen from Table 2 that compared with the wild-type Newcastle disease (NDV empty vector) group, the recombinant Newcastle disease expressing GGTA1P, CSF2, IL12, and TP53 (Comparative Examples 1-4) can significantly increase the apoptosis rate of cancer cells and enhance the anti-tumor effect of Newcastle disease virus; after co-expression of CSF2, IL12 or TP53 with GGTA1P (Examples 1-3), the anti-tumor effect is significantly improved compared with single-gene expression.

[0109] As can be seen from the above, co-expression of any one or more of the CSF2-encoding gene, the TP53-encoding gene, and the IL12-encoding gene with the GGTA1P-encoding gene can synergistically enhance the anti-tumor effect.

[0110] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A recombinant gene, characterized in that, The recombinant gene includes a first gene and a second gene; The first gene includes a GGTA1P coding gene; the GGTA1P coding gene has the sequence shown in SEQ ID NO:1 or a sequence having at least 80% identity thereto; The second gene includes one or more of a CSF2 coding gene, a TP53 coding gene, and an IL12 coding gene; the CSF2 coding gene has the sequence shown in SEQ ID NO:2 or a sequence having at least 80% identity thereto; the TP53 coding gene has the sequence shown in SEQ ID NO:3 or a sequence having at least 80% identity thereto; the IL12 coding gene has the sequence shown in SEQ ID NO:4 or a sequence having at least 80% identity thereto.

2. A recombinant Newcastle disease virus vector, characterized in that, The recombinant Newcastle disease virus vector includes a basic Newcastle disease virus vector and the recombinant gene as claimed in claim 1 inserted into the basic Newcastle disease virus vector; The recombinant gene is located between the P gene and the M gene of the basic Newcastle disease virus genome.

3. The recombinant Newcastle disease virus vector according to claim 2, wherein The basic Newcastle disease virus vector includes the LaSota strain, the HUJ strain, the PV701 strain, the Ulster 2C strain, or the 73T strain.

4. A recombinant Newcastle disease virus, characterized in that, The recombinant Newcastle disease virus contains the recombinant Newcastle disease virus vector as claimed in claim 2 or 3.

5. The preparation method of the recombinant Newcastle disease virus according to claim 4, characterized in that, Comprising the following steps: co-transfecting the recombinant Newcastle disease virus vector as claimed in claim 2 or 3 and an auxiliary plasmid into cells, culturing the transfected cells to obtain the recombinant Newcastle disease virus.

6. The preparation method according to claim 5, wherein, The ratio of the recombinant Newcastle disease virus vector to the cells is 5.0 μg: 1×10 6 cells; The cells include BHK-Flag-T7opt cell line cells; The mass ratio of the recombinant Newcastle disease virus vector to the auxiliary plasmid is 5:3; The auxiliary plasmid includes pCAGGS-Ndv-L, pCAGGS-Ndv-NP, and pCAGGS-Ndv-P; the mass ratio of pCAGGS-Ndv-L, pCAGGS-Ndv-NP, and pCAGGS-Ndv-P is 2:0.5:0.

5.

7. Use of the recombinant gene as claimed in claim 1, or the recombinant Newcastle disease virus vector as claimed in claim 2 or 3, or the recombinant Newcastle disease virus as claimed in claim 4, or the recombinant Newcastle disease virus prepared by the preparation method as claimed in any one of claims 4 to 6 in the preparation of an anti-tumor drug.

8. The application according to claim 7, characterized in that, The tumors include one or more of non-small cell lung cancer, colon adenocarcinoma, ovarian cancer, cervical cancer, liver cancer, breast cancer, esophageal squamous cell carcinoma, and melanoma.

9. The application according to claim 8, characterized in that, The cancer cells of the non-small cell lung cancer are A549; the cancer cells of the colon adenocarcinoma are LS513; the cancer cells of the ovarian cancer are HO8910 and / OVCAR3; the cancer cells of the cervical cancer are Hela; the cancer cells of the liver cancer are HepG2; the cancer cells of the breast cancer are MDA-MB-231 and / or MCF-7; the cancer cells of the esophageal squamous cell carcinoma are TE-10; the cancer cells of the melanoma are SK-MEL-28.

10. An antitumor drug, characterized in that, The anti-tumor drug comprises the recombinant gene described in claim 1, or the recombinant Newcastle disease virus vector described in claim 2 or 3, or the recombinant Newcastle disease virus described in claim 4, or the recombinant Newcastle disease virus prepared by the preparation method described in any one of claims 4 to 6.

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