A recombinant oncolytic Newcastle disease virus strain that induces hyperacute immunity, its construction method and application

Abstract

This invention provides an oncolytic recombinant Newcastle disease virus strain for inducing hyperacute immunity, its construction method, and its applications, belonging to the field of genetic engineering technology. An oncolytic recombinant Newcastle disease virus strain for inducing hyperacute immunity involves inserting genes involved in the glycosylation process into the genome of a host Newcastle disease virus strain; these genes include the B4GALNT2 gene and / or the CMAH gene. This invention utilizes the natural oncolytic properties of Newcastle disease virus and the gene involvement in the glycosylation process to induce a hyperacute immune response. The constructed recombinant Newcastle disease virus significantly improves the killing rate of cancer cells and is effective against various cancers. This invention provides a new approach for cancer treatment.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to an oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity, its construction method, and its application. Background Technology

[0002] Newcastle disease virus (NDV) belongs to the Paramyxoviridae family and the genus Avian mumpsvirus. It is a single-stranded negative-sense RNA virus with a genome RNA length of approximately 15 kb. NDV has become a hot topic in oncolytic virus research due to its natural oncolytic properties and safety (infecting only birds and not integrating into host DNA). It achieves a dual anti-tumor effect by directly lysing tumor cells and releasing antigens to activate the immune system (such as recruiting T cells and dendritic cells), but faces challenges such as weak immunogenicity, interference from neutralizing antibodies, and poor penetration into solid tumors. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide an oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity. By recombinantly expressing genes that induce hyperacute immunity, immune cells can be induced to infiltrate and penetrate solid tumor tissues. Combined with the natural oncolytic properties of Newcastle disease virus, it can not only directly kill cancer cells, but also effectively activate the immune system and further enhance the anti-tumor immune response.

[0004] This invention provides an oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity, wherein a gene involved in the glycosylation process is inserted into the genome of the host Newcastle disease virus strain.

[0005] Preferably, the genes involved in the glycosylation process include the B4GALNT2 gene and / or the CMAH gene.

[0006] Preferably, the insertion site of the gene involved in the glycosylation process includes at least one of the following locations: between the P gene and the M gene of Newcastle disease virus strain, between the Leader and the NP gene, and between the NP gene and the P gene.

[0007] Preferably, the host Newcastle disease virus strain includes the LaSota strain.

[0008] This invention provides a method for constructing the oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity, comprising the following steps:

[0009] Construct a recombinant Newcastle disease virus vector containing genes involved in the glycosylation process;

[0010] The recombinant Newcastle disease virus vector containing genes involved in the glycosylation process and the helper plasmid were used for virus rescue to harvest oncolytic recombinant Newcastle disease virus strains that induce hyperacute immunity.

[0011] Preferably, the auxiliary plasmids include pCAGGS-Ndv-P, pCAGGS-Ndv-NP, and pCAGGS-Ndv-L.

[0012] This invention provides the application of the oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity, or the oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity obtained by the construction method, in the preparation of anticancer drugs.

[0013] Preferably, the types of cancer in the anti-cancer treatment include at least one of the following: liver cancer, breast cancer, non-small cell lung cancer, ovarian cancer, rectal cancer, esophageal cancer, melanoma, and cervical cancer.

[0014] Preferably, the anti-cancer effect includes promoting apoptosis of cancer cells.

[0015] The present invention provides an anticancer drug comprising the oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity or the oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity obtained by the construction method.

[0016] This invention provides an oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity, by inserting a gene involved in glycosylation into the genome of the host Newcastle disease virus strain. Through research on the glycosylation pathway and the induction of hyperacute immunity, this invention inserts a gene capable of participating in glycosylation into the host Newcastle disease virus genome. This gene can be used to induce hyperacute immunity, induce immune cell infiltration and penetration into solid tumor tissue, and kill cancer cells. The oncolytic recombinant Newcastle disease virus strain provided by this invention not only directly kills cancer cells but also effectively activates the immune system, further enhancing the anti-tumor immune response, providing new ideas and methods for cancer treatment. Attached Figure Description

[0017] Figure 1 A schematic diagram of the recombinant NDV genome structure formed by inserting the target gene before the Leader-NP gene;

[0018] Figure 2 A schematic diagram of the recombinant NDV genome structure formed by inserting the target gene into the NP-P gene spacer region;

[0019] Figure 3 A schematic diagram of the recombinant NDV genome structure formed by inserting the target gene into the PM gene spacer region;

[0020] Figure 4 To obtain the hemagglutination titer test results for Newcastle disease virus;

[0021] Figure 5 This is the result of Newcastle disease virus titer testing;

[0022] Figure 6 The results are for NDV-CMAH titer detection.

[0023] Figure 7 Cell morphology before A549 infection;

[0024] Figure 8 The cytopathic morphology of A549 infected with NDV-CMAH 72 hours later. Detailed Implementation

[0025] This invention provides an oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity, wherein a gene involved in the glycosylation process is inserted into the genome of the host Newcastle disease virus strain.

[0026] In this invention, the recombinant Newcastle disease virus strain is genetically modified from a host Newcastle disease virus strain. This host Newcastle disease virus strain possesses good safety and natural oncolytic properties, allowing it to directly lyse tumor cells or cancer cells to achieve a good tumor (or cancer) killing effect. However, while lysing tumor cells releases antigens that activate the body's immune system, the immunogenicity of these antigens is relatively weak. To address this issue, this invention inserts a gene involved in glycosylation into the genome of the host Newcastle disease virus strain. Through the expression of this gene, the product can induce a hyperacute immune response, inducing immune cell infiltration and penetration into solid tumor tissue and killing cancer cells, further enhancing the antitumor activity of the host Newcastle disease virus strain. The host Newcastle disease virus strain preferably includes the LaSota strain.

[0027] In this invention, the genes involved in the glycosylation process preferably include the B4GALNT2 gene and / or the CMAH gene. The B4GALNT2 gene encodes β-1,4-N-acetylgalactosamine transferase, which catalyzes the generation of Sda antigens (such as CDw75), enhancing the immunogenicity of tumor cells. The CMAH gene is involved in the generation of non-human sialic acid Neu5Gc. In humans, the CMAH gene has undergone inactivation mutations, preventing these humans from synthesizing Neu5Gc and allowing them to synthesize Neu5Ac instead. Neu5Gc is considered a xenoantigen, and the CMAH gene can induce an immune rejection response, making cells infected with NDV-CMAH more easily recognized by the immune system.

[0028] In this invention, the insertion sites of the genes involved in the glycosylation process preferably include at least one of the following positions: between the P and M genes of the Newcastle disease virus strain, between the Leader and NP genes, and between the NP and P genes. The results of the embodiments of this invention show that different insertion sites of the genes involved in the glycosylation process lead to differences in the expression levels of these genes. The sites with the highest to lowest expression levels are: the insertion site between the Leader and NP genes, the insertion site between the NP and P genes, and the insertion site between the P and M genes. Considering that the insertion site between the P and M genes results in the expression of a moderate amount of recombinant protein in the genes involved in the glycosylation process, it is beneficial to the replication and proliferation of the recombinant Newcastle disease virus strain, thereby increasing the killing power of surrounding cancer cells or tumor cells. Therefore, in the embodiments of this invention, the insertion site between the P and M genes is selected to construct the recombinant Newcastle disease virus strain.

[0029] This invention provides a method for constructing the oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity, comprising the following steps:

[0030] Construct a recombinant Newcastle disease virus vector containing genes involved in the glycosylation process;

[0031] The recombinant Newcastle disease virus vector containing genes involved in the glycosylation process and the helper plasmid were used for virus rescue to harvest oncolytic recombinant Newcastle disease virus strains that induce hyperacute immunity.

[0032] This invention does not impose any particular limitation on the method for constructing a recombinant Newcastle disease virus vector containing genes involved in the glycosylation process; any method well-known in the art for constructing a recombinant Newcastle disease virus vector can be used. In this embodiment, the gene involved in the glycosylation process, digested with PacI, is cloned into a linear pNDV vector treated with the same enzyme. The helper plasmid preferably includes pCAGGS-Ndv-P, pCAGGS-Ndv-NP, and pCAGGS-Ndv-L. The mass ratio of the recombinant Newcastle disease virus vector, pCAGGS-Ndv-P, pCAGGS-Ndv-NP, and pCAGGS-Ndv-L is 1:0.1:0.1:0.4. For virus rescue, it is preferred to transfect host cells with the recombinant Newcastle disease virus vector and helper plasmids using a transfection reagent, followed by culture and virus harvesting. In this embodiment, the amount of host cells infected by each 5.0 μg of recombinant Newcastle disease virus vector is preferably 1 × 10⁻⁶. 6The host cell is preferably the BHK-Flag-T7opt cell line. The BHK-Flag-T7opt cell line is a stable cell line described in patent CN116855538A, entitled "Preparation Method of Cell Line for Amplification of Replication-Defective Recombinant Virus and Defective Virus and Its Application". The preferred method for harvesting the virus is centrifugation at 2000 rpm and 4°C for 10 min.

[0033] In this invention, replication ability testing showed that the genetic modification did not affect the viral replication ability of the Newcastle disease virus strain, and the replication efficiency of the recombinant Newcastle disease virus strain and the wild-type Newcastle disease virus strain did not show significant changes.

[0034] In this invention, the hemagglutination titer of the constructed oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity was detected. The results showed that NDV-GFP (with EmGFP insertion) yielded Newcastle disease virus allantoic fluid with a hemagglutination titer of 6. NDV-CMAH and NDV-B4GALNT2 were also Newcastle disease virus allantoic fluids with a hemagglutination titer of 6.

[0035] Given that oncolytic recombinant Newcastle disease virus strains that induce hyperacute immunity have good killing effects on tumor cells or cancer cells, the present invention provides the application of the oncolytic recombinant Newcastle disease virus strains that induce hyperacute immunity or the oncolytic recombinant Newcastle disease virus strains that induce hyperacute immunity obtained by the construction method in the preparation of anticancer drugs.

[0036] In this invention, the cancer types in the anti-cancer treatment preferably include at least one of the following: liver cancer, breast cancer, non-small cell lung cancer, ovarian cancer, rectal cancer, esophageal cancer, melanoma, and cervical cancer. The anti-cancer treatment preferably includes promoting apoptosis of cancer cells. In one embodiment of this invention, experimental results show that, compared with wild-type NDV, treatment with oncolytic recombinant Newcastle disease virus strains that induce hyperacute immunity increases the apoptosis rate by 20%–30%.

[0037] The present invention provides an anticancer drug comprising the oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity or the oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity obtained by the construction method.

[0038] This invention does not impose any particular limitation on the preparation method of the anticancer drug; any method well-known in the art for preparing viral drugs may be used. The preferred effective titer of the oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity is 5 × 10⁻⁶. 8 PFU / mL ~100×10 8 PFU / mL. The preferred dosage form of the drug is an injection.

[0039] The following detailed description, in conjunction with embodiments, illustrates a recombinant oncolytic Newcastle disease virus strain that induces hyperacute immunity, its construction method, and its applications. However, these descriptions should not be construed as limiting the scope of protection of this invention.

[0040] Example 1

[0041] 1. Three methods for constructing recombinant Newcastle disease viruses

[0042] Using EmGFP as the target gene, the target gene is inserted between the Leader-NP genes in the NDV gene. Figure 1 NDV-NP-EmGFP was prepared by inserting the target gene between the NP and P genes in the NDV gene. Figure 2 Prepare NDV-NPP-EmGFP; insert the target gene between the PM gene and the NDV gene. Figure 3 ), to prepare NDV-PM-EmGFP.

[0043] First, a PacI restriction enzyme was inserted between the Leader and NP genes in the pNDV vector to create the first pNDV vector. Then, a PacI restriction enzyme was inserted between the NP and P genes in the pNDV vector to create the second pNDV vector. Finally, a PacI restriction enzyme was inserted between the P and M genes in the pNDV vector to create the third pNDV vector. The site-directed mutagenesis method used the Novizan Point Mutagenesis Kit (catalog number C214-02) according to the kit instructions, adding PacI restriction endonuclease sites at the designated locations. The pNDV vector plasmid amplification products were digested with DpnI, recombinantly circularized with ClonExpress, and then directly transformed to complete the site-directed mutagenesis.

[0044] EmGFP (SEQ ID NO:1, synthesized by gene)

[0045] The fragment was cloned into three pNDV vectors via PacI digestion and ligation to form the recombinant vector pNDV-EmGFP. 5.0 μg of the recombinant vector pNDV-EmGFP was mixed with helper plasmids (pCAGGS-Ndv-P 0.5 μg, pCAGGS-Ndv-NP 0.5 μg, pCAGGS-Ndv-L 2.0 μg) and co-transfected 1×10⁶ cells using ExFectTransfection Reagent (Novazia product number T101-01). 6BHK-Flag-T7opt cell lines were transfected and placed in a 37°C, 5% CO2 cell culture incubator. The culture medium was replaced with fresh medium after 8–12 hours, and the cells were cultured overnight. 24 hours after transfection, the cells were placed in a 32°C, 5% CO2 cell culture incubator. 48 hours after transfection, the medium was replaced with serum-free DMEM containing trypsin treated with 1.0 μg / mL TPCK. The virus was harvested after 96 hours. The Newcastle disease virus supernatant was obtained by centrifugation at 2000 rpm, 4°C for 10 minutes.

[0046] 2. Determination of cell viability infected with three recombinant Newcastle disease viruses

[0047] The three recombinant Newcastle disease virus strains prepared above were used to infect BHK-21 cell lines with viruses at MOI=1, with wild-type NDV as a negative control and no virus as a blank control. 5 × 10⁵ cells were added to each well of a 48-well plate. 4 Cells were used to infect six wells at a time, and the fluorescence signal of each well was detected on a Tecan Spark microplate reader 48 hours after infection.

[0048] The three sets of recombinant Newcastle disease virus test results were normalized, and the results are shown in Table 1.

[0049] Table 1. Detection results of recombinant Newcastle disease virus at different insertion sites.

[0050] Group Normalized fluorescence signal (%) Standard error (%) NDV-NP-EmGFP 100 ±4.08 NDV-NPP-EmGFP 67 ±2.45 NDV-PM-EmGFP 39 ±1.22 NDV wild type <1 - No virus control <1 -

[0051] Note: The standard error is calculated as SD / sqrt(n), where n = 6.

[0052] NDV-PM-EmGFP showed the weakest normalized fluorescence signal intensity, but in subsequent experiments, the cells survived the longest under infection conditions. NDV-NP-EmGFP showed the fastest cell death rate: 60% at 48 h, 30% at 72 h, 10% at 96 h, and 0% at 120 h. NDV-NPP-EmGFP showed a moderate cell death rate: 70% at 48 h, 40% at 72 h, 20% at 96 h, and 0% at 120 h. NDV-PM-EmGFP showed the highest cell survival rate: 80% at 48 h, 70% at 72 h, 60% at 96 h, and 40% at 120 h, indicating the weakest cytotoxicity.

[0053] Considering that recombinant Newcastle disease virus (NDV) strains require a certain amount of time to replicate and proliferate in cells after infection in order to effectively infect surrounding target cells, and that rapid cell death affects the replication and proliferation process of recombinant NDV strains, which is not conducive to achieving the goal of inducing cell death in more target cells through a single inoculation, the construction strategy with the highest cell viability was selected for constructing a recombinant NDV strain that kills tumors, and NDV-PM-EmGFP was designated as NDV-GFP for subsequent detection.

[0054] Example 2

[0055] Construction method of recombinant Newcastle disease virus strain NDV-CMAH

[0056] 6 BHK-Flag-T7opt cell lines were transfected and placed in a 37°C, 5% CO2 cell culture incubator. After 8–12 hours, the culture medium was replaced with fresh medium, and the cells were cultured overnight. 24 hours after transfection, the cells were placed in a 32°C, 5% CO2 cell culture incubator. 48 hours after transfection, the medium was replaced with serum-free DMEM containing trypsin treated with 1.0 μg / mL TPCK. The virus was harvested after 96 hours.

[0057] Newcastle disease virus supernatant was obtained by centrifugation at 2000 rpm and 4℃ for 10 min, and the recombinant Newcastle disease virus strain NDV-CMAH was obtained.

[0058] Example 3

[0059] Construction method of recombinant Newcastle disease virus strain NDV-B4GALNT2

[0060] 6 BHK-Flag-T7opt cell lines were transfected and placed in a 37°C, 5% CO2 cell culture incubator. The culture medium was replaced with fresh medium after 8–12 hours, and the cells were cultured overnight. 24 hours after transfection, the cells were placed in a 32°C, 5% CO2 cell culture incubator. 48 hours after transfection, the medium was replaced with serum-free DMEM containing trypsin treated with 1.0 μg / mL TPCK. The virus was harvested after 96 hours. The Newcastle disease virus supernatant was obtained by centrifugation at 2000 rpm and 4°C for 10 minutes, yielding the recombinant Newcastle disease virus strain NDV-B4GALNT2.

[0061] Example 4

[0062] Recombinant Newcastle disease virus strain chicken embryo amplification and hemagglutination titer detection

[0063] The recombinant Newcastle disease virus supernatant prepared in Examples 1-3 was inoculated into 9-day-old SPF chicken embryos at a ratio of 0.2 mL per embryo. After 72 hours, the embryos were placed in a refrigerator overnight. After 96 hours, the embryos were removed from the refrigerator, and the allantoic fluid was extracted using a needle to obtain the Newcastle disease virus.

[0064] Prepare a 96-well V-type microplate, adding 50 μL of PBS to each well. Alternatively, prepare another 96-well V-type microplate, adding 50 μL of physiological saline to each well. Add 50 μL of allantoic fluid sample to the first well, mix well, and then dilute twofold until the last well is reached. Add 50 μL of 1% SPF chicken red blood cell suspension to each well, and gently vortex to mix. Incubate at room temperature for 45 min, then observe the results.

[0065] See results Figure 4 Hemagglutination results showed that NDV-GFP (with EmGFP insertion) yielded Newcastle disease virus allantoic fluid with a hemagglutination titer of 6. NDV-CMAH and NDV-B4GALNT2 were also Newcastle disease virus allantoic fluid samples with a hemagglutination titer of 6. All samples tested positive for hemagglutination activity.

[0066] Example 5

[0067] Recombinant Newcastle disease virus strain titer detection

[0068] Three groups of 1 μL Newcastle disease virus allantoic fluid prepared in Example 4 will be used in 12-well plates for cell infection at 5 × 10⁻⁶ cells / well. 5BHK-21 cells were cultured at 32°C and 5% CO2 for 72 h. Infected BHK-21 cells were digested into a cell suspension, resuspended in 100 μL of 0.5% BSAPBS, and 1 μg of anti-NDV rabbit IgG was added. The cells were incubated on ice for 30 min. After incubation, the cells were washed with 1 mL of 0.5% BSAPBS, centrifuged (800 rpm, 5 min), and the supernatant was discarded. This washing process was repeated twice. Fluorescently labeled rabbit secondary antibody was added. The cells were incubated on ice for 30 min in the dark. After incubation, the cells were washed with 1 mL of 0.5% BSAPBS, centrifuged (800 rpm, 5 min), and the supernatant was discarded. This washing process was repeated twice. The cells were resuspended in 200 μL of 0.5% BSAPBS. Flow cytometry was used for analysis.

[0069] The test results are shown in Table 1. The negative control results are shown in... Figure 5 The detection results for NDV-CMAH can be found in [link to relevant documentation]. Figure 6 .

[0070] Table 1. Titer test results of the three strains

[0071] Newcastle disease virus types Viral titer (PFU / mL) NDV-GFP <![CDATA[12.75×10 8 ]]> NDV-CMAH <![CDATA[8.62×10 8 ]]> NDV-B4GALNT2 <![CDATA[5.54×10 8 ]]>

[0072] Example 6

[0073] Replication kinetics of NDV-B4GALNT2 and NDV-CMAH in BHK-21 cell line

[0074] BHK-21 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and maintained at 37°C in a 5% CO2 incubator. 24 hours before infection, cells were sputtered at a rate of 1×10⁻⁶ cells / mL. 6 Cells were seeded into 6-well plates, ensuring 90% confluence at infection. Viral titers of NDV (wild-type), NDV-B4GALNT2, and NDV-CMAH allantoic fluid were pre-confirmed using the method in Example 3. BHK-21 cells were infected at an MOI of 0.1. The virus was diluted with DMEM medium, and 1 mL of virus solution was added to each well. Adsorption was initiated at 37°C for 1 h, with gentle shaking every 15 min. After adsorption, the virus solution was discarded, and the cells were washed twice with DMEM. 2 mL of DMEM containing 1 μg / mL TPCK-treated trypsin was added for further culture. Cell supernatant was collected at 0, 24, 48, 72, and 96 h post-infection. Three replicate wells were set up for each time point, with independent sampling, and viral titers were detected using the method in Example 5.

[0075] The experimental results are shown in Table 2. As can be seen from the results in Table 2, the titers of each virus strain gradually increased with the extension of infection time, reaching the highest level at 72h. Moreover, compared with the wild-type Newcastle disease virus strain, the replication ability of the two recombinant Newcastle disease viruses did not show significant changes.

[0076] Table 2. Results of titer determination of recombinant Newcastle disease virus at different time points.

[0077]

[0078] Example 7

[0079] NDV-B4GALNT2 and NDV-CMAH were used to detect apoptosis in various cancer cell lines.

[0080] HepG2 cells (liver cancer, purchased from Wuhan Pronosun Pharmaceutical Co., Ltd., catalog number: CL-0103), MCF-7 cells (breast cancer, purchased from Wuhan Pronosun Pharmaceutical Co., Ltd., catalog number: CL-0149), A549 cells (lung cancer, purchased from Wuhan Pronosun Pharmaceutical Co., Ltd., catalog number: CL-0016), OVCAR3 cells (ovarian cancer, purchased from the company, catalog number: CL-0178), LS513 cells (colon cancer, purchased from Wuhan Pronosun Pharmaceutical Co., Ltd., catalog number: CL-0640), TE-10 cells (esophageal cancer, purchased from Wuhan Pronosun Pharmaceutical Co., Ltd., catalog number: CL-0453), SK-MEL-28 cells (melanoma, purchased from Wuhan Pronosun Pharmaceutical Co., Ltd., catalog number: CL-0717), and HeLa cells (cervical cancer, purchased from Wuhan Pronosun Pharmaceutical Co., Ltd., catalog number: CL-0101) in logarithmic growth phase were digested with trypsin at a concentration of 5 × 10⁻⁶. 4 Cells were suspended in complete culture medium at a density of 1 mL / mL. 1 mL of the cell suspension was added to each well of a 12-well plate and incubated at 37°C for 24 h. After incubation, the culture medium was discarded, and 1 mL of diluted NDV, NDV-B4GALNT2, or NDV-CMAH (0.1 MOI) complete culture medium was added to each well. 1 mL of complete culture medium was added to the control group. One h after infection, cells were washed with fresh PBS, and 1 mL of a solution containing 5% human serum and 1×10⁻⁶ ppm of NDV was added. 5 Cells were inoculated with complete medium at a concentration of 1 / mL for 72 hours. After 72 hours, the medium was discarded, and the cells were digested with 0.25% trypsin (EDTA-free). Cells were collected, washed twice with PBS, and resuspended in 200 μL of 0.5% BSA PBS. Then, 10 μL of Annexin V-FITC was added, and the cells were incubated at 4°C in the dark for 10 minutes. Finally, 200 μL of 0.5% BSA PBS and 5 μL of PI were added, gently mixed, and incubated at room temperature in the dark for 5 minutes before flow cytometry analysis.

[0081] The test results are shown in Table 3.

[0082] Table 3. Effects of NDV-B4GALNT2 and NDV-CMAH on apoptosis in different cancer cells.

[0083] Processing group PBS (control) NDV NDV-B4GALNT2 NDV-CMAH HepG2 apoptosis rate (%) 5.2±1.1 35.5±2.4 56.7±3.1** 58.3±2.9** MCF-7 apoptosis rate (%) 4.8±0.9 28.6±2.1 49.2±2.8** 51.4±3.2** A549 apoptosis rate (%) 6.0±1.3 42.3±3.0 64.8±3.5** 66.5±4.0** OVCAR3 apoptosis rate (%) 8.7±1.9 36.8±2.5 66.8±2.2** 86.5±2.9** LS513 apoptosis rate (%) 6.9±2.4 30.3±1.9 68.3±5.1** 75.2±3.8** TE-10 apoptosis rate (%) 7.2±1.2 37.5±4.0 79.2±2.3** 92.4±4.0** Apoptosis rate of SK-MEL-28 (%) 8.3±2.2 34.9±3.5 72.6±3.5** 86.7±3.3** Hela apoptosis rate (%) 5.9±1.8 43.2±2.1 75.0±3.1** 83.0±1.2**

[0084] Note: ** indicates that the t-test corresponds to p<0.01, indicating that there is a significant difference between the experimental group and the NDV group.

[0085] Pre-infection NDV-CMAH cell morphology of A549 is shown in [insert cell morphology here]. Figure 7 Before viral infection, the cells exhibited normal morphology. Cell morphology 72 hours after A549 infection with NDV-CMAH was observed... Figure 8 After viral infection, cells fuse together, exhibiting obvious cytopathic morphology.

[0086] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An oncolytic recombinant Newcastle disease virus strain inducing hyperacute immunity, characterized in that, The host Newcastle disease virus strain has a gene involved in the glycosylation process inserted in its genome; the gene involved in the glycosylation process is a B4GALNT2 gene and / or a CMAH gene.

2. The oncolytic recombinant Newcastle disease virus strain inducing hyperacute immunity according to claim 1, characterized in that, The insertion site of the gene involved in the glycosylation process includes between the P gene and the M gene or between the NP gene and the P gene of the Newcastle disease virus strain.

3. The oncolytic recombinant Newcastle disease virus strain inducing hyperacute immunity according to claim 1, characterized in that, The host Newcastle disease virus strain includes the LaSota strain.

4. A method for constructing a recombinant oncolytic Newcastle disease virus strain inducing hyperacute immunity according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: Constructing a recombinant Newcastle disease virus vector containing a gene involved in the glycosylation process; Performing virus rescue on the recombinant Newcastle disease virus vector containing the gene involved in the glycosylation process and a helper plasmid, and harvesting the oncolytic recombinant Newcastle disease virus strain inducing superacute immunity.

5. The method of claim 4, wherein, The helper plasmid includes pCAGGS-Ndv-P, pCAGGS-Ndv-NP, and pCAGGS-Ndv-L.

6. Use of the oncolytic recombinant Newcastle disease virus strain inducing superacute immunity according to any one of claims 1-3 or the oncolytic recombinant Newcastle disease virus strain obtained by the construction method of claim 4 or 5 in the preparation of an anticancer drug.

7. Use according to claim 6, characterized in that, The types of cancer against which the anticancer drug is effective include at least one of the following: liver cancer, breast cancer, non-small cell lung cancer, ovarian cancer, rectal cancer, esophageal cancer, melanoma, and cervical cancer.

8. Use according to claim 6 or 7, characterized in that, The anticancer includes promoting apoptosis of cancer cells.

9. An anticancer drug, characterized by, The oncolytic recombinant Newcastle disease virus strain inducing superacute immunity according to any one of claims 1-3 or the oncolytic recombinant Newcastle disease virus strain obtained by the construction method of claim 4 or 5.

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