A recombinant canine distemper virus and its application in the preparation of an anti-dog breast tumor drug

Abstract

The application relates to the fields of biological medicine products and genetic engineering technology, and discloses a recombinant canine distemper virus and application of the recombinant canine distemper virus in preparation of an anti-dog breast tumor medicine. Through G7L and T9L double-site mutation on an apoptosis nucleolus retention related region of a chicken anemia virus Apoptin, Apoptin-G7L / T9L with enhanced anti-tumor activity is obtained, and a recombinant canine distemper virus rCDV-ApopG7L / T9L expressing the protein is constructed by using reverse genetic technology. Experimental results show that the recombinant virus can stably express the mutant Apoptin, has good virus proliferation capacity, has stronger killing effect on dog breast tumor cells, can significantly inhibit tumor growth of tumor-bearing mice, and has good safety. The application provides a new biological preparation and technical scheme for treatment of dog breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical products and genetic engineering technology, specifically relating to a recombinant canine distemper virus and its application in the preparation of anti-canine mammary tumor drugs. Background Technology

[0002] Canine mammary tumor (CMT) is one of the most common neoplastic diseases in dogs, with high morbidity and mortality rates. A significant proportion of these tumors can progress to canine mammary carcinoma (CMC). This disease is characterized by its aggressiveness, high recurrence rate, and tendency to metastasize, severely impacting the quality of life and lifespan of affected dogs. With the increasing importance of pet dogs in family life and the growing role of working dogs in guide dogs, drug detection, and search and rescue, the effective prevention and treatment of canine tumors has become a crucial research direction in veterinary medicine. However, current treatment options for canine malignant mammary tumors remain limited. Clinically, surgical resection is the primary treatment, but even with surgery, it is difficult to completely eliminate the risk of tumor recurrence and distant metastasis, and treatment outcomes in some cases are not ideal. Therefore, there is an urgent need to develop new, safe, and effective treatment strategies.

[0003] In recent years, oncolytic virus therapy (OVT) has attracted widespread attention as a novel anti-tumor treatment strategy. Oncolytic viruses can preferentially infect tumor cells, replicate and lyse within them, thereby producing anti-tumor effects and potentially inducing an immune response. However, the application of oncolytic viruses in the treatment of solid tumors still faces many challenges. For example, the complex tumor microenvironment, limited viral spread within tumor tissue, host immune clearance, and tumor heterogeneity can all affect therapeutic efficacy. Furthermore, relying solely on virus replication-mediated tumor lysis often fails to achieve sustained and stable anti-tumor efficacy; therefore, improving the anti-tumor efficiency of oncolytic viruses remains a significant technical challenge in this field.

[0004] Canine distemper virus (CDV) belongs to the genus Measlesvirus of the family Paramyxoviridae and possesses a strong ability to infect host cells. Previous studies have shown that some viral strains can exhibit a certain degree of replication and cytotoxicity in certain tumor cells, but its stable antitumor effect, duration of action, and safety in canine mammary cancer still require further improvement. Especially under the complex tumor microenvironment, relying solely on a single oncolytic mechanism often fails to simultaneously achieve tumor-killing efficiency and long-term immune control. Therefore, enhancing the tumor selectivity and comprehensive antitumor ability of CDV remains a significant challenge.

[0005] Apoptin, derived from chicken anemia virus, is a viral protein capable of inducing apoptosis in tumor cells. Previous studies have shown that it exhibits pro-apoptotic effects in various tumor cells. Positions 33-46 of apoptin have been identified as a nuclear retention sequence (NRS), which promotes tumor cell nuclear accumulation through hydrophobic interactions with the PML nucleosome. The proportion of hydrophobic amino acids in this native sequence is approximately 53.8%, lower than the hydrophobic intensity of typical nuclear output signals. Existing research has focused on functional identification or inactivation mutations, without any evidence of active modifications to enhance hydrophobicity. In this study, we mutated amino acids 7 and 9 of this region to leucine to enhance hydrophobic interactions and improve nuclear retention efficiency, thereby obtaining an apoptin mutant with stronger antitumor activity.

[0006] Furthermore, the function of apoptin depends on entering the target cell, while the transmembrane delivery efficiency of large protein molecules is limited. Therefore, inserting apoptin into a canine distemper virus vector to construct a biological agent that can stably function in tumor tissue is a treatment strategy that can improve both therapeutic efficacy and safety. Summary of the Invention

[0007] The purpose of this invention is to provide a recombinant canine distemper virus and its application in the preparation of anti-canine mammary tumor drugs, specifically a recombinant canine distemper virus expressing the mutated apoptin Apoptin-G7L / T9L, constructed using reverse genetics technology.

[0008] This invention first provides a nucleic acid fragment for preparing recombinant canine distemper virus, wherein the amino acid sequence of the fusion protein encoded by the nucleic acid fragment is SEQ ID NO:1.

[0009] MNALQEDTPPGPSTVFRPPTSSRPLETPHCREIRIGIA L I L ITLSLCGCANARAPTLRSATADNSESTGFKNVPDLRTDQPKPPSKKRSCDPSEYRVSELKESLITTTPSRPRTARRRRIRL;

[0010] Furthermore, the nucleic acid fragment, specifically the nucleotide sequence of one of its nucleotide fragments, is SEQ ID NO:2;

[0011] ATGAACGCTCTCCAAGAAGATACTCCACCCGGACCATCAACGGTGTTCAGGCCACCAACAAGTTCACGGCCGTTGGAAACCCTCACTGCAGAGAGATCCGGATTGGTATCGCT CTA ATT CTGATCACTCTATCGCTGTGTGGCTGCGCGAATGCTCGCGCTCCCACGCTAAGATTCTGCAACTGCGGACAATTCAGAAAGCACTGGTTTCAAGAATGTGCCGGACTTGAGGACCGATCAACCCAAGCCTCCCTCGAAGAAGCGATCCTGCGACCCCTCCGAGTACAGGGTAAGCGAGCTAAAAGAAAGCTTGATTACCACTACTCCCAGCCGACCCGAACCGCAAGAAGGCGTATAAGACTGTAA.

[0012] The present invention also provides an application of the aforementioned nucleic acid fragment in the preparation of recombinant canine distemper virus.

[0013] In another aspect, the present invention also provides an insert expression cassette for recombinant expression of the above-mentioned nucleic acid fragment, one specific nucleotide sequence of which is SEQ ID NO:3.

[0014] The present invention also provides a recombinant expression vector for recombinant virus rescue, wherein the recombinant expression vector is an insertion expression cassette into which the above-mentioned nucleic acid fragment for preparing recombinant canine distemper virus is inserted.

[0015] In another aspect, the present invention provides a recombinant canine distemper virus, which is constructed by reverse genetics technology and is capable of expressing the above-mentioned nucleic acid fragment.

[0016] The present invention also provides an application of the recombinant canine distemper virus in the preparation of a biological agent for treating canine mammary cancer.

[0017] The present invention also provides a biological agent for the treatment of canine mammary cancer, comprising the above-mentioned recombinant canine distemper virus.

[0018] This invention constructs a recombinant canine distemper virus rCDV-ApopG7L / T9L expressing the mutant apoptin-G7L / T9L using reverse genetics. Compared with the parent virus and recombinant viruses expressing wild-type apoptin, this invention has the following beneficial effects: First, the G7L / T9L double mutation enhances the hydrophobicity and nuclear retention capacity of the apoptin-related region, which is beneficial to increasing its aggregation level in the tumor cell nucleus, thereby enhancing the selective killing effect on tumor cells; Second, the recombinant virus can proliferate well in Vero cells, and the insertion of the foreign gene does not significantly weaken the viral replication ability, indicating that the construct has good virological stability; Third, it can effectively express the foreign protein in canine mammary tumor cells CIPp, and its in vitro cytotoxicity is significantly enhanced, showing stronger tumor-suppressing activity; Fourth, it can significantly inhibit tumor growth in tumor-bearing mice without causing significant weight loss, suggesting that the recombinant virus has good tolerability and safety while exerting anti-tumor effects. Attached Figure Description

[0019] Figure 1 Schematic diagram of pB-CDV plasmid construction;

[0020] Figure 2 : Construction diagram of pB-CDV-ApopG7L / T9L plasmid;

[0021] Figure 3 : RT-PCR identification image of rCDV-ApopG7L / T9L recombinant virus, where M: marker; 1: rCDV-ApopG7L / T9L; 2: rCDV-Apop;

[0022] Figure 4 WB identification image of rCDV-ApopG7L / T9L recombinant virus, where M: marker; 1: rCDV-ApopG7L / T9L; 2: rCDV-Apop; 3: rCDV;

[0023] Figure 5 Growth curve of recombinant virus on Vero cells;

[0024] Figure 6 Immunofluorescence assay of recombinant virus on CIPp cells, where A: rCDV-Apop; B: rCDV-ApopG7L / T9L; C: rCDV; D: negative control;

[0025] Figure 7 : Diagram illustrating the cytotoxic effect of recombinant viruses on CIPp cells;

[0026] Figure 8 : Changes in tumor volume on the surface of mice;

[0027] Figure 9 : Graph showing changes in mouse body weight. Detailed Implementation

[0028] This invention provides a fusion protein expressing a mutant apoptin and a canine distemper virus constructed using reverse genetics technology.

[0029] A method for constructing canine distemper virus expressing the fusion protein Apoptin-G7L / T9L, comprising the following steps:

[0030] 1) Construct a transcription plasmid comprising the genomic cDNA sequence of canine distemper virus in which a nucleic acid fragment (SEQ ID NO:1) encoding the fusion protein Apoptin-G7L / T9L is inserted.

[0031] 2) Construct multiple helper plasmids, including the gene sequence encoding the phosphoprotein (P) of the canine distemper CDV-L strain, the gene sequence encoding the nucleoprotein (NP) of the canine distemper CDV-L strain, and the gene sequence encoding the large polymerase protein (L) of the canine distemper CDV-L strain.

[0032] 3) Co-transfect the transcription plasmid and helper plasmid into host cells that are permitted to replicate canine distemper virus, and culture the transfected host cells.

[0033] 4) Rescue recombinant canine distemper virus from cell suspensions of transfected host cells.

[0034] This invention uses reverse genetics technology to rescue the recombinant canine distemper virus rCDV-ApopG7L / T9L expressing Apoptin-G7L / T9L from the recombinant canine distemper virus CDV-L strain. This recombinant canine distemper virus has the following effects:

[0035] ① Viral growth curve: rCDV-ApopG7L / T9L proliferated well on Vero cells, reaching its peak at 48h.

[0036] ② Killing effect on tumor cells: CCK8 assay results of canine mammary tumor cells CIPp showed that rCDV-ApopG7L / T9L had high cytotoxicity to CIPp cells.

[0037] In this invention, pBluScript II SK (+) was purchased from Shanghai Jingfeng Biotechnology Co., Ltd.; pB-CDV plasmid and helper plasmids (pEMC-N, pEMC-P, and pEMC-L) were constructed and preserved in our laboratory; BHK-T7 cells and Vero cells were preserved in our laboratory; PrimeSTAR® Max DNA Polymerase was purchased from TaKaRa Corporation, Japan; agarose gel extraction kit was purchased from Shanghai Sangon Biotech Co., Ltd.; restriction endonuclease Sac II was purchased from NEB Corporation, USA; T4 DNA ligase was purchased from TaKaRa Corporation, Japan; E. coli HST08 competent cells were purchased from TaKaRa Corporation, Japan; plasmid mini-prep kit and endotoxin-free plasmid large-scale prep kit were purchased from Beijing Jianshi Biotechnology Co., Ltd.; HighGene plus Transfection reagent was purchased from ABclonal; and PrimeScript II High Fidelity OneStep RT-PCR was used. Kit was purchased from TAKARA Corporation, Japan; apoptin protein (positive control) was stored in our laboratory; apoptin rabbit polyclonal antibody was purchased from Abcam; HRP goat anti-rabbit secondary antibody was purchased from Proteintech; CCK-8 Cell Counting Kit was purchased from Novizan Biotechnology Co., Ltd.

[0038] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0039] Example 1: Construction of CDV-L backbone plasmid and helper plasmid for canine distemper virus

[0040] To ensure accurate end structures of the full-length viral RNA transcript, several important elements need to be added to both ends of the viral genome: The 3' end of the viral genome is seamlessly linked to the core sequence of the T7 RNA polymerase promoter, enabling T7 RNA polymerase to initiate transcription from the first base at the 3' end of the genome; the 5' end of the genome contains a self-splicing dilhexane ribozyme sequence and a T7 terminator. Once transcribed, the dilhexane ribozyme sequence performs its self-splicing function, releasing the 5' end of the genome. This results in a complete and accurate viral genome.

[0041] 1. Modification of cloning vectors

[0042] Using PCR, the multiple cloning site and the adjacent T3 and T7 promoter sequences of the cloning vector pBluScript II SK (+) were removed and replaced with a short linker fragment containing only three restriction sites: BssHII, SnaBI, and MluI.

[0043] Vector modification primers:

[0044] Upstream: 5'GATCTACGTAACGCGTCATGGTCATAGCTGTTTCCTGTGTG3'

[0045] Downstream: 5'CCAATACGTAGCGCGCTCACTGGCCGTCGTTTTACAACGTC3'

[0046] Using the pBluScript II SK (+) vector as a template, PCR amplification was performed with the aforementioned primer pair. After gel recovery of the amplification product, it was digested with SnaBI, and then the digested product was ligated with T4 DNA ligase. The ligation product was transformed into competent cells to obtain the successfully modified vector: the vector can be digested by BssHII, SnaBI, and MluI, but cannot be digested by the original restriction site KpnI. The newly modified vector was named pB-BSM.

[0047] 2. Construction of the backbone plasmid pB-CDV

[0048] The canine CDV-L gene (sequence number: KM926612.1) was obtained from NCBI, and synthesized into five fragments. Using in-fusion technology, these five fragments were ligated into the vector pB-BSM in two steps.

[0049] Table 1: Primer sequence listing for amplification

[0050]

[0051] Note: Italics indicate the 15 bp homologous sequence required for in-fusion ligation; the bold text in the FL1 upstream primer represents the 17-nucleotide T7 RNA polymerase promoter core sequence; the blue text in the FL3 downstream primer represents the SnaBI restriction site required for the next cloning step.

[0052] The pB-BSM vector was linearized by double digestion with restriction enzymes BssHII and SnaBI. Fragments FL1, FL2, and FL3 were amplified by PCR. Specifically, the T7 promoter sequence and a 15 bp sequence homologous to the left of the BssHII restriction site were introduced into the upstream primer of fragment FL1 using PCR technology, and a 15 bp sequence homologous to the right of the SnaBI restriction site was introduced into the downstream primer of fragment FL3. The fragments were then infused into the vector to form the vector pB-CDV-1.

[0053] The vector pB-CDV-1 was linearized by double digestion with restriction enzymes SnaBI and MluI. PCR amplification of fragments FL4, FL5-1, and FL5-2 was performed, followed by in-fusion ligation of each fragment into the vector, forming pB-CDV(…). Figure 1 ).

[0054] 3. Construction of helper plasmids pEMC-N, pEMC-P, and pEMC-L

[0055] The carrier is derived from the excision of MV-L by pEMC-MV-L.

[0056] The CDV-L strain N protein gene contains an NcoI restriction site and is constructed into a vector in two steps.

[0057] This fragment is formed by double digestion with NcoI and EcoRI and ligation into the vector pTM-1 to create pEMC-N1.

[0058] The MluI restriction site is a restriction site not found in vector pTM-1, proteins N, P, and L. Its introduction is to facilitate the construction of expression vectors for P and L proteins.

[0059] Step 2: Amplify a large fragment of the N protein from position 76 to the termination site.

[0060] This fragment is ligated into the vector pEMC-N1 by double digestion with BglII and MluI, forming pEMC-N.

[0061] The P and L protein genes were ligated into the vector pEMC-N by double digestion with NcoI and MluI, forming pEMC-P and pEMC-L, respectively.

[0062] Example 2: Acquisition and sequence design of the apoptin gene

[0063] 1. Obtaining the apoptin gene from chicken anemia virus

[0064] The chicken anemia virus apoptin gene (sequence number: AY171617.1) was obtained from NCBI, and its amino acid fragment is as follows:

[0065] MNALQEDTPPGPSTVFRPPTSSRPLETPHCREIRIGIAGITITLSLCGCANARAPTLRSATADNSESTGFKNVPDLRTDQPKPPSKKRSCDPSEYRVSELKESLITTTPSRPRTARRRIRL

[0066] The nucleotide fragments are as follows:

[0067] ATGAACGCTCTCCAAGAAGATACTCCACCCGGACCATCAACGGTGTTCAGGCCACCAACAAGTTCACGGCCGTTGGAAACCCTCACTGCAGAGAGATCCGGATTGGTATCGCTGGAATTACAATCACTCTATCGCTGTGTGGCTGCGCGAATGCTCGCGCTCCCACGCTAAGATCTGCAACT GCGGACAATTCAGAAAGCACTGGTTTCAAGAATGTGCCGGACTTGAGGACCGATCAACCCAAGCCTCCCTCGAAGAAGCGATCCTGCGACCCCTCCGAGTACAGGGTAAGCGAGCTAAAAGAAAGCTTGATTACCACTACTCCCAGCCGACCCGAACCGCAAGAAGGCGTATAAGACTGTAA

[0068] 2. Apoptin sequence design

[0069] Previous studies have shown that positions 33-46 of the apoptin amino acid sequence constitute a nuclear retention sequence, with hydrophobic amino acids comprising 57.1%. In this invention, glycine (G) at position 7 and threonine (T) at position 9 are simultaneously mutated to leucine (L), resulting in a G7L and T9L double mutation. This mutation significantly enhances the hydrophobicity and leucine enrichment in this region, thereby strengthening the hydrophobic interaction between the mutant protein and the promyelocytic leukemia nucleosomes (PML-NBs) within the tumor cell nucleus. This significantly increases the mutant's retention capacity and aggregation level within the tumor cell nucleus compared to wild-type apoptin, thereby enhancing its selective killing activity against tumor cells. The mutated apoptin sequence is named ApopG7L / T9L.

[0070] The original amino acid and nucleotide sequence from 33 to 46 is as follows:

[0071] IRIGIA G I T ITLSL

[0072] ATCCGGATTGGTATCGCT GGA ATT ACA ATCACTCTATCGCTG

[0073] The mutated amino acid and nucleotide sequences of amino acids 33-46 are as follows:

[0074] IRIGIA L I L ITLSL

[0075] ATCCGGATTGGTATCGCT CTA ATT CTG ATCACTCTATCGCTG

[0076] Note: Underlined lines indicate the amino acid and nucleotide sequences at the mutation sites.

[0077] The mutated amino acid sequence is as follows:

[0078] MNALQEDTPPGPSTVFRPPTSSRPLETPHCREIRIGIA L I L ITLSLCGCANARAPTLRSATADNSESTGFKNVPDLRTDQPKPPSKKRSCDPSEYRVSELKESLITTTPSRPRTARRRIRL

[0079] The mutated nucleotide sequence is as follows:

[0080] ATGAACGCTCTCCAAGAAGATACTCCACCCGGACCATCAACGGTGTTCAGGCCACCAACAAGTTCACGGCCGTTGGAAACCCTCACTGCAGAGAGATCCGGATTGGTATCGCT CTA ATT CTG ATCACTCTATCGCTGTGTGGCTGCGCGAATGCTCGCGCTCCCACGCTAAGATTCTGCAACTGCGGACAATTCAGAAAGCACTGGTTTCAAGAATGTGCCGGACTTGAGGACCGATCAACCCAAGCCTCCCTCGAAGAAGCGATCCTGCGACCCCTCCGAGTACAGGGTAAGCGAGCTAAAAGAAAGCTTGATTACCACTACTCCCAGCCGACCCGAACCGCAAGAAGGCGTATAAGACTGTAA.

[0081] 3. Insertion of expression box design and synthesis

[0082] GS and KozaK sequences were introduced before the ATG start codon upstream of the ApopG7L / T9L gene, and KozaK and GE sequences were introduced after the stop codon downstream. CTT was added before the upstream GS sequence as a spacer nucleotide between the ApopG7L / T9L and P genes, and CTT was added after the downstream GE sequence as a spacer nucleotide between the ApopG7L / T9L and M genes. Each of the P and M genes contains one SacII restriction site. The sequence between the two SacII restriction sites (containing parts of the P gene sequence, ApopG7L / T9L sequence, part of the M gene sequence, and the GS, GE, and KozaK sequences), totaling 1507 bp, was synthesized by Sangon Biotech Co., Ltd.

[0083] The synthesized sequence is as follows: SEQ ID NO:3:

[0084]

[0085] Primers were designed for amplification of the target gene.

[0086] Table 2: Primer Information Table for Amplification

[0087]

[0088] 4. Construction of full-length cDNA of pB-CDV-ApopG7L / T9L recombinant viral gene

[0089] The synthesized gene was amplified using CDV-Sac II-F and CDV-Sac II-R primers, and the 1507 bp fragment was recovered after digestion with Sac II. pB-CDV was digested with Sac II, and the 17604 bp fragment was recovered. The two recovered fragments were ligated using T4 DNA ligase and incubated overnight at 4°C. Afterwards, the cells were transformed into HST08 competent cells. Single colonies were picked, and after PCR and sequencing confirmation, plasmids were extracted and used for transfection.

[0090] Example 3: Recombinant Virus Rescue

[0091] BHK-T7 cells were seeded in a six-well plate. When the cells grew to 70%-80% monolayer, the transcription plasmid and helper plasmids pEMC-N, pEMC-P and pEMC-L were co-transfected into BHK-T7 cells at 5 μg, 1 μg, 0.5 μg and 1 μg respectively using ABclonal's HighGene plus Transfection reagent. The cells were cultured at 37°C with 5% CO2. The specific operation was performed according to the kit instructions: (1) BHK-T7 cells were seeded into a six-well plate and the cell density was controlled at 70%-90%. (2) On the second day, take 7.5 μg of transcription plasmid and helper plasmids pEMC-N, pEMC-P and pEMC-L and add them to a centrifuge tube containing 200 μL of serum-free DMEM basal medium. Mix well by pipetting. Then add 15 μL of HighGene transfection reagent, mix by pipetting, and let stand at room temperature for 20-30 min before transfecting the cells. (3) Add 200 μL of plasmid / HighGene transfection reagent complex evenly to the wells of a 6-well cell culture plate and gently shake the cell culture plate to distribute it evenly. (4) 4-6 hours after cell transfection, replace half of the medium with fresh complete medium. (5) 4 days later, scrape off the cells, mix the cell suspension, and take 300 μL of cell suspension to seed a monolayer of Vero cells that have grown overnight and have a density of about 60% to 70%. After 1 hour of infection, add DMEM containing 5% fetal bovine serum, and culture at 37°C with 5% CO2 for 4-6 days. Observe the cell pathogenesis under a microscope. When cytopathic effects appeared, the cell suspension was harvested and stored as a seed virus at -70°C. The recombinant canine distemper viruses expressing the infectious ApopG7L / T9L and Apop proteins were rescued and named rCDV-ApopG7L / T9L and rCDV-Apop, respectively.

[0092] 1. Identification of recombinant virus by RT-PCR

[0093] Genomic RNA was extracted from the rescued recombinant viruses rCDV-ApopG7L / T9L and rCDV-Apop using the Trizol method. The ApopG7L / T9L and Apop genes were amplified using the PrimeScript II High Fidelity One Step RT-PCR Kit and Apop-F and Apop-R primers. The amplification products were then sequenced to confirm the presence of the mutated apoptin gene in the rescued recombinant virus genome. As shown in the figure below, both rCDV-ApopG7L / T9L and rCDV-Apop showed distinct bands between 250bp and 500bp, similar in size to the apoptin gene (366bp), indicating successful construction of the pB-CDV-ApopG7L / T9L plasmid. Transfection of BHK-T7 cells successfully rescued the recombinant virus rCDV-ApopG7L / T9L. Figure 3 Sequencing results showed that all sequences were correct and no other mutations were found.

[0094] 2. Western blot identification of rCDV-ApopG7L / T9L recombinant virus

[0095] After seeding Vero cells with rCDV-ApopG7L / T9L, rCDV-Apop, and rCDV, samples were prepared and Western blotted together. The results showed that rCDV-ApopG7L / T9L and rCDV-Apop both showed bands consistent with the size of APOP protein (13.3KD) at around 15KD, while rCDV showed no band, indicating that APOP protein was successfully expressed in Vero cells. Figure 4 ).

[0096] 3. Growth kinetics of recombinant virus on Vero cells

[0097] The rescued recombinant viruses rCDV-ApopG7L / T9L and rCDV-Apop, along with the parent rCDV virus, were inoculated into Vero cells at an MOI of 0.01. The virus-infected cells were harvested at 24 h, 48 h, 72 h, 96 h, and 120 h post-infection, and the viral titer (TCID) was measured. 50 The study also plotted viral growth kinetics curves to evaluate the growth kinetics of the three viruses on Vero cells.

[0098] The recombinant virus rCDV-ApopG7L / T9L reached its peak viral titer 72 hours after inoculation. Figure 5 The rCDV and rCDV-Apop viruses reached their peak values ​​within 48 hours of inoculation, indicating that the recombinant virus rCDV-ApopG7L / T9L grew well on Vero cells, and the insertion of foreign genes did not reduce the viral proliferation ability.

[0099] 4. Results of indirect immunofluorescence identification of recombinant virus

[0100] CIPp cells were infected with rCDV-ApopG7L / T9L, rCDV-Apop, and rCDV recombinant viruses, and indirect immunofluorescence was performed using an anti-apoptin antibody. The results showed that green fluorescence was observed in CIPp cells infected with rCDV-ApopG7L / T9L and rCDV-Apop recombinant viruses, and the green fluorescence signal of rCDV-ApopG7L / T9L was stronger, while rCDV and the negative control showed no green fluorescence. This indicates that apoptin was successfully expressed in CIPp cells and that rCDV-ApopG7L / T9L accumulated more in CIPp cells.

[0101] Example 4: Verification of the in vitro killing effect of recombinant virus on canine mammary tumor cells

[0102] 1. Direct killing effect on canine mammary tumor cells (CCK8 assay)

[0103] The working principle of CCK-8 is that it can be reduced by dehydrogenases in the mitochondria of cells to produce a highly water-soluble, orange-yellow formazan product. The intensity of the color is directly proportional to cell proliferation and inversely proportional to cytotoxicity. The OD value is measured at a wavelength of 450 nm using a microplate reader, indirectly reflecting the number of viable cells.

[0104] In the experiment, the cell viability of CIPp cells 72 hours after inoculation with the recombinant virus was measured and calculated. The results are as follows: Figure 7 The results showed that all three recombinant viruses significantly inhibited cell viability. Compared with the negative control, the OD of the rCDV-treated group was significantly lower. 450 The value decreased significantly to approximately 0.9 (P < 0.001). This indicates that rCDV infection itself has a significant inhibitory effect on CIPp cells. rCDV-APOP and rCDV-ApopG7L / T9L further demonstrated stronger CIPp cell inhibitory capabilities than rCDV alone, with rCDV-ApopG7L / T9L exhibiting a stronger inhibitory effect on CIPp cells.

[0105] 2. Verification of the in vivo killing effect of recombinant virus on canine mammary tumors

[0106] Forty nude mice were divided into 5 groups of 8 mice each. Except for the blank control group, mice in the treatment group 1-3 were injected subcutaneously into the right axilla with 0.2 ml of CIPp cell suspension (containing 2×10⁻⁶ cells). 6 (cells), observed subcutaneous nodules approximately 50 mm in length. 3 At that time, oncolytic virus injection was performed.

[0107] Mice were weighed every 7 days, and the long diameter (a) and short diameter (b) of the tumor were measured with calipers. The tumor volume was calculated using the formula V = a × b² / 2, and growth curves of body weight and tumor volume over time were plotted for each group.

[0108] The grouping, tumor formation, and drug administration information are shown in Table 3.

[0109] Table 3: Dosing Information for In Vivo Tumor Therapy Trials

[0110]

[0111] 3. Monitoring of tumor growth in mice

[0112] like Figure 8 As shown, the tumor volume in the PBS control group increased rapidly over time, reaching nearly 50 mm³ on day 28. In contrast, all groups receiving viral therapy (rCDV, rCDV-APOP, and rCDV-ApopG7L / T9L) exhibited significant tumor growth inhibition. Among them, the rCDV-ApopG7L / T9L and rCDV-APOP groups showed the strongest inhibitory effects, with the rCDV-ApopG7L / T9L group showing even stronger inhibitory effects, maintaining a lower tumor volume on day 28, significantly lower than the rCDV-APOP group. The rCDV group also showed some inhibitory effect, but its tumor volume was higher than that of the rCDV-ApopG7L / T9L and rCDV-APOP groups.

[0113] 4. Changes in mouse body weight

[0114] as follows Figure 9 As shown, the mouse body weight of the rCDV, rCDV-APOP, rCDV-ApopG7L / T9L groups and the PBS control group all showed similar growth trends over 28 days. From day 1 to day 21, the body weight of mice in each group steadily increased from approximately 17g to over 19g. The body weight growth curves of the PBS control group and the treatment groups (rCDV, rCDV-APOP, rCDV-ApopG7L / T9L) basically overlapped, with no obvious separation or abnormal decrease.

[0115] Although there were minor numerical differences between the groups at certain time points, such as day 7 or day 14, the overall range of fluctuation was consistent. There was no statistically significant difference in body weight between the treatment groups and the PBS control group. This indicates that these recombinant viruses did not cause significant weight loss or growth inhibition in mice, demonstrating good tolerability and safety.

[0116] In summary, this invention successfully constructed a recombinant canine distemper virus expressing the mutant apoptin-G7L / T9L and verified its in vitro and in vivo antitumor effects in canine mammary tumors. This recombinant virus possesses viral replication capacity, selective tumor cell killing ability, and good biocompatibility, providing a new candidate drug and technical solution for the biotherapy of canine mammary cancer, and has high application value and promising prospects.

Claims

1. A nucleic acid fragment for preparing recombinant canine distemper virus, characterized in that, The nucleic acid fragment encoded by the fusion protein has the amino acid sequence SEQ ID NO:

1.

2. The nucleic acid fragment as described in claim 1, characterized in that, The nucleic acid fragment has the nucleotide sequence SEQ ID NO:

2.

3. The use of the nucleic acid fragment according to claim 1 in the preparation of recombinant canine distemper virus.

4. An insert expression cassette for recombinant expression of the nucleic acid fragment of claim 1, characterized in that, The nucleotide sequence of the inserted expression cassette is SEQ ID NO:

3.

5. A recombinant expression vector for recombinant virus rescue, characterized in that, The recombinant expression vector is the insertion expression cassette of claim 4, which inserts the nucleic acid fragment of claim 1.

6. A recombinant canine distemper virus, characterized in that, The recombinant canine distemper virus described herein is constructed using reverse genetics technology and is capable of expressing the nucleic acid fragment described in claim 1.

7. The use of the recombinant canine distemper virus according to claim 6 in the preparation of a biological agent for treating canine mammary cancer.

8. A biological agent for treating canine mammary cancer, characterized in that, The biological agent contains the recombinant canine distemper virus as described in claim 7.

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