Oncolytic recombinant Newcastle disease virus strain capable of inducing super-acute immunity as well as construction method and application of oncolytic recombinant Newcastle disease virus strain

Abstract

The invention provides an oncolytic recombinant Newcastle disease virus strain capable of inducing super-acute immunity as well as a construction method and application of the oncolytic recombinant Newcastle disease virus strain, and belongs to the technical field of gene engineering. The invention discloses an oncolytic recombinant Newcastle disease virus strain for inducing super-acute immunity. A gene participating in a glycosylation process is inserted into a genome of a host Newcastle disease virus strain; the gene involved in the glycosylation process comprises a B4GALNT2 gene and / or a CMAH gene. The natural oncolytic characteristic of the Newcastle disease virus is utilized, the gene participates in the glycosylation process to induce super-acute immune response, and the constructed recombinant Newcastle disease virus greatly improves the killing rate of cancer cells and is effective to various cancers. The invention provides a new means for cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to an oncolytic recombinant Newcastle disease virus strain capable of inducing hyperacute immunity, a construction method thereof, and an application thereof. Background Art

[0002] Newcastle disease virus (NDV) belongs to the Paramyxoviridae family and is a member of the Avian paramyxovirus genus. It is a single-stranded negative-strand RNA virus, and the full length of its genomic RNA is about 15 kb. Newcastle disease virus (NDV) has become a research hotspot for oncolytic viruses due to its natural oncolytic properties and safety (only infecting poultry and not integrating into the host DNA). It exerts 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 by neutralizing antibodies, and poor penetration of 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 capable of inducing hyperacute immunity. By recombinantly expressing genes that induce hyperacute immunity, it can induce the infiltration and penetration of immune cells into solid tumor tissues. Combining 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] The present invention provides an oncolytic recombinant Newcastle disease virus strain capable of inducing hyperacute immunity, in which a gene involved in the glycosylation process is inserted into the genome of the host Newcastle disease virus strain.

[0005] Preferably, the gene involved in the glycosylation process includes 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 positions: between the P gene and the M gene of the 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] The present invention provides a construction method of the oncolytic recombinant Newcastle disease virus strain capable of inducing hyperacute immunity, including the following steps:

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

[0010] Perform virus rescue on the recombinant Newcastle disease virus vector containing the gene involved in the glycosylation process and the helper plasmid, and harvest the oncolytic recombinant Newcastle disease virus strain capable of inducing hyperacute immunity.

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

[0012] The present invention provides the use of the oncolytic recombinant Newcastle disease virus strain that induces superacute immunity or the oncolytic recombinant Newcastle disease virus strain obtained by the construction method in the preparation of anti-cancer drugs.

[0013] Preferably, the types of cancers 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 treatment includes promoting apoptosis of cancer cells.

[0015] The present invention provides an anti-cancer drug, which includes the oncolytic recombinant Newcastle disease virus strain that induces superacute immunity or the oncolytic recombinant Newcastle disease virus strain obtained by the construction method.

[0016] The present invention provides an oncolytic recombinant Newcastle disease virus strain that induces superacute immunity, in which a gene involved in the glycosylation process is inserted into the genome of the host Newcastle disease virus strain. Through the research on the glycosylation pathway and the induction of superacute immunity, the present invention inserts a gene capable of participating in the glycosylation process into the genome of the host Newcastle disease virus. Using this gene, superacute immunity can be induced, immune cell infiltration and penetration into solid tumor tissues can be induced, and cancer cells can be killed. The oncolytic recombinant Newcastle disease virus strain provided by the present invention can not only directly kill cancer cells, but also effectively activate the immune system, further enhancing the anti-tumor immune response, providing new ideas and methods for cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the recombinant NDV genome structure formed by inserting the target gene in front of the Leader-NP gene;

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

[0019] Figure 3 Schematic diagram of the recombinant NDV genome structure formed by inserting the target gene in the P-M gene spacer region;

[0020] Figure 4 Detection result of the hemagglutination titer of the group Newcastle disease virus;

[0021] Figure 5 Detection result of the Newcastle disease virus titer;

[0022] Figure 6 It is the detection result of NDV-CMAH titer;

[0023] Figure 7 It is the cell morphology of A549 before infection;

[0024] Figure 8 It is the cytopathic morphology of A549 72 h after infection with NDV-CMAH. Specific implementation manners

[0025] The present invention provides an oncolytic recombinant Newcastle disease virus strain for inducing superacute immunity, in which a gene participating in the glycosylation process is inserted into the genome of the host Newcastle disease virus strain.

[0026] In the present invention, the recombinant Newcastle disease virus strain is genetically modified based on the host Newcastle disease virus strain. The host Newcastle disease virus strain has good safety and natural oncolytic characteristics, and can directly lyse tumor cells or cancer cells to achieve a good killing effect on tumors (or cancers). At the same time, when lysing tumor cells, antigens will be released to activate the immune system in the body, but there is a problem that the immunogenicity of the antigens is weak. In the present invention, a gene participating in the glycosylation process is inserted into the genome of the host Newcastle disease virus strain. Through the expression of the gene participating in the glycosylation process, the product can induce a superacute immune response, can induce immune cell infiltration and penetration into solid tumor tissues, and kill cancer cells, further improving the anti-tumor activity of the host Newcastle disease virus strain. The host Newcastle disease virus strain preferably includes the LaSota strain.

[0027] In the present invention, the gene participating in the glycosylation process preferably includes the B4GALNT2 gene and / or the CMAH gene. The B4GALNT2 gene encodes β-1,4-N-acetylgalactosaminyltransferase, which catalyzes the generation of the Sda antigen (such as CDw75), and can enhance the immunogenicity of tumor cells. The CMAH gene participates in the generation of non-human sialic acid Neu5Gc. The CMAH gene in humans has an inactivating mutation, resulting in these humans being unable to synthesize Neu5Gc and only being able to synthesize Neu5Ac. Neu5Gc is regarded as a xenogenic antigen, and the CMAH gene can induce the occurrence of immune rejection reactions, making the cells infected with NDV-CMAH more easily recognized by the immune system.

[0028] In the present invention, the insertion sites of the genes involved in the glycosylation process preferably include at least one of the following positions: between the P gene and the M gene of the Newcastle disease virus strain, between the Leader and the NP gene, and between the NP gene and the P gene. The results of the examples of the present invention show that different insertion sites of the genes involved in the glycosylation process result in differences in the expression levels of the genes involved in the glycosylation process. The sites with decreasing expression levels are: the insertion site between the Leader and the NP gene, the insertion site between the NP gene and the P gene, and the insertion site between the P gene and the M gene. Considering that the insertion site between the P gene and the M gene enables the genes involved in the glycosylation process to express a moderate amount of recombinant protein, which is beneficial to the replication and proliferation of the recombinant Newcastle disease virus strain, thereby enhancing the killing ability of surrounding cancer cells or tumor cells. Therefore, in the examples of the present invention, the insertion site between the P gene and the M gene was selected to construct the recombinant Newcastle disease virus strain.

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

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

[0031] Perform virus rescue on the recombinant Newcastle disease virus vector containing the genes involved in the glycosylation process and the helper plasmid, and harvest the oncolytic recombinant Newcastle disease virus strain that induces superacute immunity.

[0032] The present invention has no special limitation on the method for constructing the recombinant Newcastle disease virus vector containing the genes involved in the glycosylation process, and the well-known methods for constructing recombinant Newcastle disease virus vectors in the art can be used. In the examples of the present invention, the genes involved in the glycosylation process digested with PacI were cloned into the 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. When performing virus rescue, it is preferred to transfect the host cells with the recombinant Newcastle disease virus vector and the helper plasmid under the action of a transfection reagent, and after culturing, harvest the virus. In the examples of the present invention, the amount of host cells infected by every 5.0 μg of the recombinant Newcastle disease virus vector is preferably 1×10 6The host cell is preferably the BHK-Flag-T7opt cell line. The BHK-Flag-T7opt cell line is the stably transfected cell line BHK-Flag-T7opt described in the patent CN116855538A, with the patent title of "Preparation Method of a Cell Line for Amplifying Replication-Defective Recombinant Virus, Defective Virus and Its Application". The method for harvesting the virus is preferably centrifugation at 2000 rpm and 4 °C for 10 min.

[0033] In the present invention, through replication ability detection, the genetic modification of the present invention does not have any impact on the virus replication ability of the Newcastle disease virus strain, and there is no significant change in the replication efficiency between the recombinant Newcastle disease virus strain and the wild-type Newcastle disease virus strain.

[0034] In the present invention, hemagglutination titer detection was performed on the constructed oncolytic recombinant Newcastle disease virus strain for inducing superacute immunity. The results showed that NDV-GFP (inserted with EmGFP) obtained allantoic fluid of Newcastle disease virus with a hemagglutination titer of 6. NDV-CMAH was allantoic fluid of Newcastle disease virus with a hemagglutination titer of 6. NDV-B4GALNT2 was allantoic fluid of Newcastle disease virus with a hemagglutination titer of 6.

[0035] In view of the good killing effect of the oncolytic recombinant Newcastle disease virus strain for inducing superacute immunity on tumor cells or cancer cells, the present invention provides the application of the oncolytic recombinant Newcastle disease virus strain for inducing superacute immunity or the oncolytic recombinant Newcastle disease virus strain obtained by the construction method in the preparation of anti-cancer drugs.

[0036] In the present invention, the types of cancer in anti-cancer 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 preferably includes promoting apoptosis of cancer cells. In one embodiment of the present invention, the experimental results show that compared with wild-type NDV, the apoptosis rate of cells increases by 20% - 30% after treatment with the oncolytic recombinant Newcastle disease virus strain for inducing superacute immunity.

[0037] The present invention provides an anti-cancer drug, including the oncolytic recombinant Newcastle disease virus strain for inducing superacute immunity or the oncolytic recombinant Newcastle disease virus strain obtained by the construction method.

[0038] The present invention has no special limitation on the preparation method of the anti-cancer drug, and the well-known preparation methods of viral drugs in the art can be used. The effective titer of the oncolytic recombinant Newcastle disease virus strain for inducing superacute immunity is preferably 5×10 8 PFU / mL - 100×10 8 PFU / mL. The dosage form of the drug is preferably an injection.

[0039] The following is a detailed description of an oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity provided by the present invention, its construction method and application in combination with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] Example 1

[0041] 1. Construction methods of three recombinant Newcastle disease viruses

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

[0043] First, PacI digestion was inserted between the Leader and NP genes in the pNDV vector by site-directed mutagenesis to make the first pNDV vector; PacI digestion was inserted between the NP gene and the P gene in the pNDV vector by site-directed mutagenesis to make the second pNDV vector; PacI digestion was inserted between the P gene and the M gene in the pNDV vector by site-directed mutagenesis to make the third pNDV vector. The method of site-directed mutagenesis was to use the Novoprotein site-directed mutagenesis kit (product number C214-02) according to the kit operation instructions to add PacI restriction endonuclease cleavage sites at the corresponding positions. The amplified product of the pNDV vector plasmid was digested with DpnI, circularized by ClonExpress recombination, and then directly transformed to complete site-directed mutagenesis.

[0044] The gene-synthesized EmGFP (SEQ ID NO:1,

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

[0046] 2. Determination of the survival rate of cells infected with three recombinant Newcastle disease viruses

[0047] The three recombinant Newcastle disease virus strains prepared above were used to infect the BHK-21 cell line with a virus MOI of 1. The NDV wild type was used as a negative control, and no virus was added as a blank control. 5×10 4 cells were added to each well in a 48-well plate, and a group of 6 wells were infected. 48 h after infection, the fluorescence signal of each well was detected on a multifunctional microplate reader (Tecan Spark).

[0048] The detection results of the three groups of recombinant Newcastle disease viruses were normalized, and the results are shown in Table 1.

[0049] Table 1 Detection results of recombinant Newcastle disease viruses with 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 - Virus-free control <1 -

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

[0052] The normalized fluorescence signal intensity of NDV-PM-EmGFP was the weakest, but in subsequent experiments, the cells had the longest survival time under infection conditions. For NDV-NP-EmGFP, at 48 h after infecting the cells, the cell survival rate was 60%; at 72 h, the cell survival rate was 30%; at 96 h, the cell survival rate was 10%; at 120 h, the cell survival rate was 0%, and the cells died the fastest. For NDV-NPP-EmGFP, at 48 h after infecting the cells, the cell survival rate was 70%; at 72 h, the cell survival rate was 40%; at 96 h, the cell survival rate was 20%; at 120 h, the cell survival rate was 0%, with a medium death rate. For NDV-PM-EmGFP, at 48 h after infecting the cells, the cell survival rate was 80%; at 72 h, the cell survival rate was 70%; at 96 h, the cell survival rate was 60%; at 120 h, the cell survival rate was 40%, with the highest cell survival rate and the weakest cytotoxicity.

[0053] Considering that the recombinant Newcastle disease virus strain needs a certain time to replicate and proliferate in cells after infection in order to effectively infect surrounding target cells, and the rapid death of cells affects the replication and proliferation process of the recombinant Newcastle disease virus strain, which is not conducive to achieving the purpose of inducing death of more target cells through a single virus inoculation. Therefore, the construction strategy with the highest cell survival rate was selected for the construction of the recombinant Newcastle disease virus strain for killing tumors, and NDV-PM-EmGFP was denoted as NDV-GFP for subsequent detection.

[0054] Example 2

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

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

[0057] The supernatant of Newcastle disease virus was obtained by centrifugation at 2000 rpm at 4 °C 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 The BHK-Flag-T7opt cell line was transfected and then placed in a cell incubator at 37 °C with 5% CO2. After 8 - 12 h, the cell culture medium was replaced with fresh medium and cultured overnight. At 24 h post-transfection, it was placed in a cell incubator at 32 °C with 5% CO2. At 48 h post-transfection, it was replaced with serum-free DMEM medium containing 1.0 μg / mL TPCK-treated trypsin, and the virus was harvested after 96 h. The supernatant of Newcastle disease virus was obtained by centrifugation at 2000 rpm for 10 min at 4 °C, and the recombinant Newcastle disease virus strain NDV-B4GALNT2 was obtained.

[0061] Example 4

[0062] Chicken embryo amplification and hemagglutination titer detection of recombinant Newcastle disease virus strain

[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 h, it was placed in the refrigerator overnight and taken out of the refrigerator after 96 h. The allantoic fluid was taken out using a needle to obtain Newcastle disease virus.

[0064] Prepare a 96-well V-shaped microplate, and add 50 μL of PBS to each well. Prepare a 96-well V-shaped microplate, and add 50 μL of normal saline to each well. Add 50 μL of the allantoic fluid sample to the first well, mix well and then 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 well. Let it stand at room temperature for 45 min and observe the results.

[0065] The results are shown in Figure 4 . For the hemagglutination results, the allantoic fluid of Newcastle disease virus with a hemagglutination titer of 6 was obtained for NDV-GFP (inserted with EmGFP). The allantoic fluid of Newcastle disease virus with a hemagglutination titer of 6 was obtained for NDV-CMAH. The allantoic fluid of Newcastle disease virus with a hemagglutination titer of 6 was obtained for NDV-B4GALNT2. The allantoic fluid samples of the virus with positive hemagglutination activity detection.

[0066] Example 5

[0067] Titer detection of recombinant Newcastle disease virus strain

[0068] Three groups of 1 μL of the allantoic fluid of Newcastle disease virus prepared in Example 4 were used in a 12-well plate for cell infection at 5×10 5BHK-21 cells were cultured in an environment of 32 °C and 5% CO2 for 72 h. The infected BHK-21 cells were digested into cell suspensions, resuspended with 100 μL of 0.5% BSA PBS, and 1 μg of anti-NDV rabbit IgG was added, followed by incubation on ice for 30 min. After incubation, the cells were washed with 1 mL of 0.5% BSA PBS, centrifuged (800 rpm, 5 min), the supernatant was discarded, and the washing was repeated 2 times. Fluorescently labeled rabbit secondary antibody was added. Incubate on ice for 30 min in the dark. After incubation, the cells were washed with 1 mL of 0.5% BSA PBS, centrifuged (800 rpm, 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. Detection was performed using a flow cytometer.

[0069] The detection results are shown in Table 1. The results of the negative control are shown in Figure 5 , and the detection results of NDV-CMAH are shown in Figure 6 .

[0070] Table 1 Detection results of the titers of three strains

[0071] Newcastle disease virus type Virus 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] Detection of the replication kinetics of NDV-B4GALNT2 and NDV-CMAH in the BHK-21 cell line

[0074] BHK-21 cells were used and cultured in DMEM medium containing 10% fetal bovine serum (FBS), maintained in an incubator at 37 °C and 5% CO2. 24 h before infection, the cells were seeded into 6-well plates at 1×10 6 / well to ensure that the cell confluence reached 90% at the time of infection. The virus titers of NDV (wild type), NDV-B4GALNT2, and NDV-CMAH allantoic fluid were confirmed in advance by the method of Example 3. BHK-21 cells were infected at an MOI of 0.1. The virus was diluted with DMEM medium, 1 mL of virus solution was added to each well, adsorbed at 37 °C for 1 h, and gently shaken and mixed every 15 min. After adsorption, the virus solution was discarded, washed 2 times with DMEM, and 2 mL of 1 μg / mL TPCK-treated trypsin DMEM was added for continued culture. Cell supernatants were collected at 0, 24, 48, 72, and 96 h after infection. Three replicate wells were set at each time point, sampled independently, and the virus titers were detected in the manner of Example 5.

[0075] The experimental results are shown in Table 2. From the results in Table 2, it can be seen that as the infection time extended, the titers of each virus strain gradually increased, reached the highest at 72 h, and compared with the wild-type Newcastle disease virus strain, the replication ability of the two recombinant Newcastle disease virus strains did not show significant changes.

[0076] Table 2 Results of titration of recombinant Newcastle disease virus at different time points

[0077]

[0078] Example 5

[0079] Detection of apoptosis of NDV-B4GALNT2 and NDV-CMAH in various cancer cell lines

[0080] Take HepG2 cells (liver cancer, purchased from Wuhan Punosai Co., Ltd., product number: CL-0103), MCF-7 cells (breast cancer, purchased from Wuhan Punosai Co., Ltd., product number: CL-0149), A549 cells (lung cancer, purchased from Wuhan Punosai Co., Ltd., product number: CL-0016), OVCAR3 cells (ovarian cancer, purchased from the company, product number: CL-0178), LS513 cells (colon cancer, purchased from Wuhan Punosai Co., Ltd., product number: CL-0640), TE-10 cells (esophageal cancer, purchased from Wuhan Punosai Co., Ltd., product number: CL-0453), SK-MEL-28 cells (melanoma, purchased from Wuhan Punosai Co., Ltd., product number: CL-0717) and Hela cells (cervical cancer, purchased from Wuhan Punosai Co., Ltd., product number: CL-0101) in the logarithmic growth phase, digest them with trypsin, and suspend them in complete medium at a density of 5×10 4 / mL. Add 1 mL of the cell suspension to each well of a 12-well plate and incubate at 37 °C for 24 h. After incubation, discard the medium, and add 1 mL of diluted NDV, NDV-B4GALNT2, and NDV-CMAH (0.1 MOI) complete medium to each well of each group. Add 1 mL of complete medium to the control group. 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, 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 in the dark at 4 °C for 10 min at room temperature. Finally, add 200 μL of 0.5% BSA PBS and 5 μL of PI, mix gently, and incubate in the dark at room temperature for 5 min for flow cytometry detection.

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

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

[0083] Treatment 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** SK-MEL-28 apoptosis rate (%) 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 p-value corresponding to the t-test is <0.01, indicating a significant difference between the experimental group and the NDV group.

[0085] The cell morphology of A549 before infection with NDV-CMAH is shown in Figure 7 , and before virus infection, the cells showed a normal morphology. The cell morphology of A549 72 h after infection with NDV-CMAH is shown in Figure 8 . After virus infection, cell fusion occurred and obvious cytopathic morphology was presented.

[0086] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A recombinant Newcastle disease virus strain that induces hyperacute immunity, characterized in that: Genes involved in the glycosylation process are inserted into the genome of the host Newcastle disease virus strain.

2. The oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity according to claim 1, characterized in that: The genes involved in the glycosylation process include B4GALNT2 gene and / or CMAH gene.

3. The oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity according to claim 1, characterized in that: The insertion site of the gene involved in the glycosylation process includes at least one of the following positions: between the P gene and the M gene of the Newcastle disease virus strain, between the Leader and the NP gene, and between the NP gene and the P gene.

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

5. A method for constructing an oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity according to any one of claims 1 to 4, characterized in that: The following steps are involved: constructing a recombinant Newcastle disease virus vector containing genes involved in the glycosylation process; The recombinant Newcastle disease virus vector containing the gene involved in the glycosylation process and the auxiliary plasmid are subjected to virus rescue to harvest the oncolytic recombinant Newcastle disease virus strain that induces hyperacute immunity.

6. The construction method according to claim 5, characterized in that: The auxiliary plasmids include pCAGGS-Ndv-P, pCAGGS-Ndv-NP and pCAGGS-Ndv-L.

7. Use of the oncolytic recombinant Newcastle disease virus strain inducing hyperacute immunity according to any one of claims 1 to 4 or the oncolytic recombinant Newcastle disease virus strain inducing hyperacute immunity obtained by the construction method according to claim 5 or 6 in the preparation of anticancer drugs.

8. The use according to claim 7, characterized in that: The types of cancer in the anti-cancer 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.

9. The use according to claim 7 or 8, characterized in that: The anti-cancer effect includes promoting apoptosis of cancer cells.

10. An anticancer drug, characterized in that: It comprises the oncolytic recombinant Newcastle disease virus strain inducing hyperacute immunity as described in any one of claims 1 to 4 or the oncolytic recombinant Newcastle disease virus strain inducing hyperacute immunity obtained by the construction method according to claim 5 or 6.

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