Preparation of chicken anemia virus and circle virus human I-type VP3 recombinant adenovirus and application of recombinant adenovirus in anti-tumor aspect
By preparing recombinant adenoviruses rAd5-CAV-VP3 and rAd5-GyH1-VP3, the problem of precise targeting of tumor cells in existing tumor treatment strategies has been solved, achieving highly efficient inhibition of tumor growth and induction of apoptosis, with significant anti-tumor activity and low side effects.
Patent Information
- Application Number
- CN202511378184.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-25
- Publication Date
- 2025-12-19
AI Technical Summary
Existing technologies make it difficult to develop anti-tumor treatment strategies that can precisely target tumor cells, are highly effective, and have few side effects, especially for the application of chicken anemia virus VP3 and human type I VP3 of circovirus.
Recombinant adenoviruses rAd5-CAV-VP3 and rAd5-GyH1-VP3 were prepared. The chicken anemia virus VP3 and human type I VP3 gene of circle virus were amplified and cloned by PCR into a shuttle plasmid vector. Subsequently, they were co-transfected with the adenovirus backbone vector into 293T cells and packaged to obtain high-titer recombinant adenoviruses for inhibiting tumor cell growth and inducing apoptosis.
Recombinant adenovirus can significantly inhibit the growth of human ovarian cancer and mouse melanoma cells and induce their apoptosis. Both in vivo and in vitro experiments showed strong anti-tumor activity with few side effects.
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Abstract
Description
Technical Field
[0001] This invention belongs to the fields of genetic engineering and tumor treatment technology, specifically involving two novel recombinant adenovirus preparation methods and their applications in the field of anti-tumor therapy, particularly involving recombinant adenoviruses carrying chicken anemia virus VP3 (CAV-VP3) or human type I VP3 (GyH1-VP3) circovirus genes and their use as anti-tumor drugs. Background Technology
[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.
[0003] Malignant tumors, as one of the leading causes of death worldwide, continue to pose a serious challenge to human health. Currently, standard clinical treatments for tumors mainly include surgical resection, radiotherapy, and chemotherapy. While these traditional therapies have controlled tumor progression to some extent, their inherent limitations are becoming increasingly apparent. Against this backdrop, tumor gene therapy, particularly the strategy of delivering therapeutic genes using viral vectors, has shown great promise. The core of this strategy lies in safely and efficiently introducing exogenous genes capable of specifically killing tumor cells or inhibiting their growth into tumor tissue via gene vectors. Among numerous candidate therapeutic genes, those capable of selectively inducing programmed cell death (apoptosis) in tumor cells are highly favored due to their unique mode of action.
[0004] Oncolytic adenoviruses are adenoviruses with tumor-specific replication capabilities. They can replicate extensively within tumor cells and lyse the cells, releasing more viral particles to infect surrounding tumor cells. This self-amplifying property makes oncolytic adenoviruses a powerful tool for veterinary oncology treatment. Currently, several gene therapy programs based on oncolytic adenoviruses have entered clinical trials and have shown promising therapeutic effects in various tumor models.
[0005] Chicken anemia virus (CAV) and human gyrovirus homsa1 (GyH1) both belong to the genus Gyrovirus within the family Anelloviridae. Both viruses contain three main open reading frames: VP1, VP2, and VP3 genes, which encode the capsid protein VP1, the backbone protein VP2, and the apoptosis protein VP3 (also known as apoptin), respectively. The VP3 protein, commonly referred to as apoptin, is a widely studied molecule in this field. Numerous studies have confirmed that the VP3 protein, derived from avian viruses, can efficiently induce apoptosis in human tumor cells from various sources (such as liver cancer, lung cancer, colon cancer, and ovarian cancer cells), but is almost non-toxic to normal human diploid cells (such as fibroblasts and endothelial cells). This tumor-specific killing ability is thought to be related to its subcellular localization and phosphorylation status differences in different cell types, making it a near-ideal candidate molecule for anticancer drugs. Therefore, developing a novel treatment strategy that can precisely target tumor cells, is highly effective, and has few side effects is a key technical problem that urgently needs to be solved in the field of modern oncology research. Summary of the Invention
[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide a method for preparing recombinant adenoviruses of chicken anemia virus and circovirus type I VP3 (apoptin) and their application in anti-tumor treatment.
[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a recombinant adenovirus for inhibiting tumor cell growth and inducing tumor cell apoptosis, wherein the recombinant adenovirus is rAd5-CAV-VP3 recombinant adenovirus or rAd5-GyH1-VP3 recombinant adenovirus; Preparation method of rAd5-CAV-VP3 recombinant adenovirus: Chicken anemia virus VP3 (CAV-VP3) target gene was obtained by PCR amplification gel recovery. Using seamless cloning technology, the target gene was cloned into the shuttle plasmid vector pcADV-EF1-mNeonGreen-CMV-MCS to obtain the pcADV-EF1-mNeonGreen-CMV-CAV-VP3 shuttle plasmid. The obtained shuttle plasmid and adenovirus backbone vector were co-transfected into 293T cells for packaging and recombination to obtain rAd5-CAV-VP3 recombinant adenovirus. Preparation method of rAd5-GyH1-VP3 recombinant adenovirus: The target gene of human type I VP3 (GyH1-VP3) of circle virus was obtained by PCR amplification gel recovery. Using seamless cloning technology, the target gene was cloned into the shuttle plasmid vector pcADV-EF1-mNeonGreen-CMV-MCS to obtain the pcADV-EF1-mNeonGreen-CMV-GyH1-VP3 shuttle plasmid. The obtained shuttle plasmid and adenovirus backbone vector were co-transfected into 293T cells for packaging and recombination to obtain rAd5-GyH1-VP3 recombinant adenovirus.
[0008] The nucleotide sequence of CAV-VP3 (VP3 sequence in GenBank: OL448984.1) is shown in SEQ ID NO:1, with a full length of 366 bp, as follows: ATGAACGCTCTCCAAGAAGATACTCCACCCGGACCATCAACGGGGTTCAGGCCACCAACAAGTTCACGGCCGTTGGAAACCCTCACTGCAGAGAGATCCGGATTGGTATCGCTGGAATTACAATCACTCTATCGCTGTGTGGCTGCGCGAATGCTCGCGCTCCCACGCTAAGATCTGCAACT GCGGACAATTCAGAAAGCACTGGTTTCAAGAATGTGCCGGACTTGAGGACCGATCAACCCAAGCCTCCCTCGAAGAAGCGATCCTGCGACCCCTCCGAGTACAGGGTAAGCGAGCTAAAAGAAAGCTTGATTACCACTACTCCCAGCCGACCCGAACCGCAAGAAGGGGTATAAGACTGTAA.
[0009] The nucleotide sequence of GyH1-VP3 (VP3 sequence in GenBank: OL448985.1) is shown in SEQ ID NO:2, with a full length of 378 bp, as follows: ATGGAACCGGGACTTGGACACCAGACCCCAAGAACTACAGAAACATCCAGGTCGGTGATATTCGAGCATCCAATAAGTTCGTCGGAGTCGGTTGGGACTCTCTCCAAAGAGATCCAAATTGGGCTCGGGTCAACTATAATTACCGTATCGCTTCCTGGCTTCGCGAGTGTTCGCGTACTCACGACGCGA TCTGCAACTGCGGGGGCTTCAGACGCCACTGGTTCCAGGAGGCAGCAGGACTGTCCACACAGGAGACCCAGACGGACCCGGTCGCCAGAGATCTCGATCGCCTGGTCGTGCGTGGAAACGCAGCAAAAAGAAAATTGGATTACATCGCGAACAGAAAAACTCCCAAAAAGAAAAAGGCTAAGACTGTAA.
[0010] The amino acid sequence of the protein encoded by the CAV-VP3 gene is shown in SEQ ID NO:3, with a full length of 122 aa, as detailed below: MNALQEDTPPGPSTGFRPPTSSRPLETPHCREIRIGIAGITITLSGCANARAPTLRSATADNSESTGFKNVPDLRTDQPKPPSKKRSCDPSEYRVSELKESLITTTPSRPRTARRGIRL The amino acid sequence of the protein encoded by the GyH1-VP3 gene is shown in SEQ ID NO:4, with a full length of 126 aa, as detailed below: MEPGLGHQTPRTTETSRSVIFEHPISSSESVGTLSKEIQIGLGSTIITVSLPGFASVRVLTTRSATAGASDATGSRRQQDCPHRRPRRTRSPEISIAWSCVETQQKENWITSRTEKLPKRKRLRL.
[0011] Preferably, the adenovirus backbone vector is a human replication-defective adenovirus type 5.
[0012] The viral titer of the rAd5-CAV-VP3 recombinant adenovirus was 1.58 × 10⁻⁶. 10 The viral titer of the rAd5-GyH1-VP3 recombinant adenovirus was 3.95 × 10⁻⁶ PFU / mL. 10 PFU / mL.
[0013] In a second aspect, the present invention provides the use of the above-described recombinant adenovirus in (1) or (2) as follows: (1) Prepare products for inhibiting the growth of tumor cells; (2) Prepare products for inducing tumor cell apoptosis.
[0014] Preferably, the tumor cells include human ovarian cancer cells or mouse melanoma cells.
[0015] Preferably, the product includes a vaccine or a drug with the above-mentioned recombinant adenovirus as the active ingredient.
[0016] The beneficial effects of this invention are: This invention prepared two recombinant adenoviruses, rAd5-CAV-VP3 and rAd5-GyH1-VP3, capable of inhibiting tumor cell growth and inducing tumor cell apoptosis. Experimental verification showed that the recombinant adenoviruses rAd5-CAV-VP3 and rAd5-GyH1-VP3 significantly inhibited the growth and apoptosis of human ovarian adenocarcinoma cells A2780 and cutaneous melanoma cells B16. In vitro experiments confirmed that both recombinant adenoviruses significantly inhibited the proliferation of human ovarian cancer (A2780) and mouse melanoma (B16) cells and induced their apoptosis. In an in vivo nude mouse tumor-bearing model, both rAd5-GyH1-VP3 and rAd5-CAV-VP3 significantly inhibited tumor growth, with no significant difference in efficacy. This invention confirms that the rAd5-CAV-VP3 and rAd5-GyH1-VP3 recombinant adenoviruses have similar potent antitumor activities. The dual validation data in vivo and in vitro lay a solid foundation for the development of novel antitumor products with rAd5-CAV-VP3 and rAd5-GyH1-VP3 recombinant adenoviruses as active ingredients. Attached Figure Description
[0017] Figure 1 Complete genome maps of recombinant CAV-VP3 and GyH1-VP3 shuttle plasmids.
[0018] Figure 2 Transfection status of recombinant shuttle plasmid in 293T cells (400×).
[0019] Figure 3 Determination of rAd5-CAV-VP3 and rAd5-GyH1-VP3 titers (100×).
[0020] Figure 4 The figure shows the results of Western Blot detection of rAd5-CAV-VP3 and rAd5-GyH1-VP3 protein expression.
[0021] Figure 5 To assess the genetic stability of recombinant adenoviruses rAd5-CAV-VP3 and rAd5-GyH1-VP3.
[0022] Figure 6 The results of A2780 CCK8 infection with recombinant adenovirus rAd5-CAV-VP3 and rAd5-GyH1-VP3 are presented.
[0023] Figure 7 Results of A2780 Annexin-v / pe staining for rAd5-CAV-VP3 and rAd5-GyH1-VP3 infection (400×).
[0024] Figure 8 Results of B16 Annexin-v / pe staining for rAd5-CAV-VP3 and rAd5-GyH1-VP3 infection (400×).
[0025] Figure 9 Western blot analysis of protein expression of rAd5-CAV-VP3 and rAd5-GyH1-VP3 in A2780 and B16 cells.
[0026] Figure 10 Growth curves of subcutaneous melanoma in nude mice and tumor weight after 12 days of treatment.
[0027] Figure 11 For anatomical observation of the tumor and liver.
[0028] Figure 12 For pathological and histological observation of tumors and liver. Detailed Implementation
[0029] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.
[0030] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.
[0031] Example 1: Construction of pcADV-CAV-VP3 and pcADV-GyH1-VP3 shuttle plasmids.
[0032] Based on the CAV-VP3 gene sequence shown in SEQ ID NO:1 and the GyH1-VP3 gene sequence shown in SEQ ID NO:2, specific primers SEQ ID NO:5-SEQ ID NO:8 were designed for amplifying the CAV-VP3 and GyH1-VP3 target genes, respectively: CAV-VP3-F:ACGGAGCTCGAATTCGGATCCTTAGTGATGGTGGTGGTGATGCAGACGGA (SEQ IDNO: 5); CAV-VP3-R:TTAAGAAGGAGATATACATATGAATGCTCTACAAGA (SEQ ID NO: 6); GyH1-VP3-F:ACGGAGCTCGAATTCGGATCCTTAGTGATGGTGGTGGTGATGCAGACGGATA (SEQID NO:7); GyH1-VP3-R:TTAAGAAGGAGATATACATATGAATGCTCTACAAG (SEQ ID NO: 8).
[0033] PCR amplification and purification of the target gene fragments CAV-VP3 and GyH1-VP3.
[0034] Next, the pcADV-EF1-mNeonGreen-CMV vector was used... EcoRI and BamHI Enzyme digestion was performed, and In-FusionSnap Assembly Master Mix, linearized vector, and purified target fragment were added. Recombination was initiated by incubation at 50°C for 25 min. Subsequently, the ligation product was transformed into DH5α competent cells, and positive clones were screened after heat shock treatment. Finally, plasmids were extracted and processed... KpnI Single enzyme digestion and agarose gel electrophoresis further confirmed the successful construction of recombinant pcADV-CAV-VP3 and pcADV-GyH1-VP3 shuttle plasmids, such as... Figure 1 As shown.
[0035] Example 2: Packaging, amplification and purification of rAd5-CAV-VP3 and rAd5-GyH1-VP3 recombinant adenoviruses.
[0036] The recombinant shuttle plasmid prepared in Example 1 was co-transfected with the adenovirus backbone plasmid into HEK 293T cells to obtain high-quality recombinant adenovirus with high purity and titer. The specific steps are as follows: (1) Resuscitate and passage 293T cells. After the cells grow to the logarithmic growth phase, they are evenly seeded into 6-well plates. The transfection density of the cells is controlled at about 70%. The cells are placed in a 37-degree incubator and transfected the next day when the cells are in good growth condition.
[0037] (2) Discard the original culture medium and replace it with a new culture medium (1.5 mL of Opti-MEM medium) and put it back into the cell culture incubator to continue culturing.
[0038] (3) Dissolve 4 μg of the viral vector plasmid to be transfected (the ratio of backbone plasmid to shuttle plasmid is 1:1) in Opti-MEM medium, ensuring a total volume of 250 μL, and mix gently.
[0039] (4) Then, slowly add the dilution of the transfection reagent to the dilution of the plasmid while gently mixing, and then let it stand at room temperature for 20 minutes to form a stable transfection complex.
[0040] (5) Next, remove the cell culture plate from the incubator, add the DNA-transfection reagent complex prepared earlier into the culture plate and put it back into the incubator.
[0041] (6) After 6 hours, remove the culture medium, wash once with PBS, and then add 2 mL of fresh and complete culture medium for incubation.
[0042] (7) During the culture process, the culture medium should be changed every 3 days. From 2 to 15 days, a green fluorescence signal can be observed under a fluorescence microscope, such as... Figure 2 As shown. Viral plaques appeared on day 9. Once complete lesions were observed, the supernatant was collected for subsequent use.
[0043] The packaged recombinant adenovirus stock solution has a low titer, requiring large-scale amplification and purification of the virus to increase the viral titer. The specific steps are as follows: (1) Spread HEK293 cells evenly in small cell culture flasks. When the cells proliferate to 70%-80%, add 10µL of undiluted adenovirus to each culture flask to infect the cells. After complete cytopathic effect (2-3 days), add approximately 500µL of 10% Nonidet P 40 (NP40) to each culture flask to lyse the cells and release the virus.
[0044] (2) Collect cell lysates and collect the supernatant by centrifugation at 12,000 rpm for 10 min. Add 50 mL of virus precipitation solution (containing 20% PEG8000 and 2.5 M NaCl) to every 100 mL of supernatant and incubate at 4 °C for 1 h to precipitate virus particles.
[0045] (3) Centrifuge at 12000 rpm for 20 min and resuspend the virus precipitate in 10 mL of cesium chloride (CsCl) solution with a density of 1.10 g / mL. Centrifuge at 7000 rpm at 4℃ for 5 min and collect the virus.
[0046] (4) To perform density gradient centrifugation of the virus, first add 2.0 mL of cesium chloride (CsCl) solution with a density of 1.40 g / mL to a Beckman ultracentrifuge tube, then add 3.0 mL of cesium chloride (CsCl) solution with a density of 1.30 g / mL. Finally, slowly add 5 mL of the virus suspension to the centrifuge tube to form a density gradient. Centrifuge at 22,800 rpm for 2 h at 4 °C.
[0047] (5) Collect viral bands with a density of 1.30-1.40 g / mL and transfer them to a purified dialysis bag. Place the dialysis bag in dialysis buffer and dialyze overnight at 4°C, changing the dialysis buffer once during the process to ensure adequate dialysis.
[0048] (6) Collect the virus suspension after dialysis and store it at -80°C for subsequent experiments.
[0049] Example 3: Detection of titers and genetic stability of rAd5-CAV-VP3 and rAd5-GyH1-VP3 recombinant adenoviruses 1. Recombinant adenovirus titer detection (1) Remove the recombinant adenovirus sample from the -80°C freezer and quickly place it on ice to thaw. Gently shake to mix the virus sample, avoiding repeated freeze-thaw cycles.
[0050] (2) Preparation of HEK293T cells: Seed HEK293T cells into 6-well plates and culture until the cell density reaches 80%-90% confluence. Aspirate the culture medium and gently wash the cells once with PBS buffer. Perform 10-fold serial dilutions of the virus sample and select an appropriate dilution for inoculation. Inoculate each dilution in 3 replicates, adding 100 μL of virus dilution to each well, and set up negative control wells (add culture medium only). Place the inoculated cell plate in a 37℃, 5% CO2 incubator for 24 h for adsorption.
[0051] (3) Carefully remove the culture medium, and then slowly inject 0.5 mL of pre-cooled methanol along the side wall of the 6-well plate and fix it at -20℃ for 20 min.
[0052] (4) Wash the cells three times with PBS. Block the cells with PBS containing 1% BSA at 37°C for 1 hour.
[0053] (5) Add 0.2 mL of 1× anti-Hexon antibody solution to each well and incubate at 37°C for 1 h. Wash the cells three times with PBS.
[0054] (6) Add 0.2 mL of 1× horseradish peroxidase (HRP) labeled secondary antibody to each well and incubate at 37°C for 1 h. Then wash the cells three times with PBS.
[0055] (7) Add 0.2 mL of freshly prepared 1×DAB working solution to each well and incubate at room temperature for 5-10 min. Then discard the DAB and wash twice with PBS, adding 1 mL of PBS to each well each time.
[0056] (8) Observe the average number of positive cells under a microscope, selecting a specific gradient to ensure 5 to 50 positive cells per field of view, and randomly count in at least 5 such areas. In 10 -7 At the optimal dilution, the number of positive signals in the cell pores is most ideal, exhibiting 5 to 50 clear brown signals, such as... Figure 3 As shown, the titer of recombinant adenovirus was calculated. No dark brown signal was observed in the negative control cell wells, further validating the accuracy of the results. Finally, the titer of recombinant adenovirus was calculated using the formula: Viral titer (PFU / mL) = (Average number of positive cells / field of view) × (Number of fields of view / well) × Dilution factor / 0.1. The titer levels of recombinant adenovirus are as follows: rAd5-CAV-VP3 viral titer (PFU / mL) = (2 × 63 × 10) 7 ) / 0.1=1.58×10 10 (PFU / mL).
[0057] rAd5-GyH1-VP3 viral titer (PFU / mL) = (5 × 63 × 10) 7 ) / 0.1=3.95×10 10 (PFU / mL).
[0058] 2. Detection of genetic stability of recombinant adenovirus (1) Preparation of rabbit-derived CAV-VP3 and GyH1-VP3 polyclonal antibodies Using the genomes of chicken anemia virus (CAV) and goose circovirus (GyH1) strains preserved in our laboratory as templates, specific primers targeting their respective VP3 genes (CAV-VP3, GyH1-VP3) were designed and synthesized, and amplified by PCR. The purified PCR products were double-digested with NdeI and BamHI, and then ligated into the similarly treated pET-22b expression vector to construct recombinant plasmids pET-22b-CAV-VP3 and pET-22b-GyH1-VP3, respectively. The ligation products were transformed into *E. coli* DH5α competent cells, positive clones were screened, and samples were sent for sequencing verification.
[0059] The two correctly sequenced recombinant plasmids were transformed into *E. coli* BL21(DE3) expression strain. Single colonies were picked and cultured until the bacterial culture OD... 600 When the pH reached 0.6-0.8, different final concentrations of IPTG (isopropyl-β-D-thiogalactoside) were added, and the two recombinant bacteria were cultured and induced on a large scale under optimal induction conditions. After induction, the bacterial cells were collected by centrifugation, and inclusion bodies were collected after sonication. The inclusion bodies were dissolved in buffer containing 8M urea, and then the two recombinant proteins were purified separately by Ni-NTA affinity chromatography.
[0060] The purified protein was refolded using a gradient dialysis method to remove urea and restore protein activity. Finally, SDS-PAGE was used to verify the purity and molecular weight of the protein, and the BCA method was used to determine its concentration. Healthy New Zealand white rabbits were randomly divided into three groups: a CAV-VP3 immunization group, a GyH1-VP3 immunization group, and a PBS control group. The immunization groups received the corresponding purified recombinant protein at a dose of 0.5 mg / kg as the antigen. The initial immunization used Freund's complete adjuvant, followed by booster immunizations every two weeks using Freund's incomplete adjuvant. The control group received an equal volume of PBS. One week after the final immunization, serum was collected from the jugular vein of each group to collect antibodies.
[0061] (2) Western blot identification of VP3 protein expression in 293T cells transfected with rAd5-CAV-VP3 and rAd5-GyH1-VP3 strains. 293T cells were cultured and infected with recombinant adenovirus. After 90% infection, the cells were observed under a fluorescence microscope. The original culture medium was discarded, and the cells were washed with PBS, digested with trypsin, and transferred to centrifuge tubes. The cells were centrifuged at 1000 rpm for 6 min, and the cell pellet was collected. 200 µL of RIPA lysis buffer and 2 µL of LPMSF protease inhibitor were added to the centrifuge tubes, and the cells were lysed at 4°C for 40 min. The cells were then centrifuged at 12000 rpm for 15 min, and 100 µL of supernatant was collected. 25 µL of loading buffer was added, and the cells were boiled at 100°C for 10 min. Western blot was performed using rabbit polyclonal antibodies CAV-VP3 and GyH1-VP3 prepared in our laboratory as the primary antibody, and HRP-labeled goat anti-rabbit IgG as the secondary antibody. Results are as follows: Figure 4 As shown, a specific band appeared at approximately 15 kDa, indicating that the VP3 protein in the recombinant adenovirus was effectively expressed.
[0062] (2) Detection of genetic stability of rAd5-CAV-VP3 and rAd5-GyH1-VP3 strains Both recombinant adenoviruses were used to infect 293T cells, and the cells were passaged 10 times consecutively. To verify the stability of the VP3 gene in the recombinant adenoviruses, the genomes of cells from passages 2, 4, 6, 8, and 10 were extracted and identified by PCR using CAV-VP3 and GyH1-VP3 specific primers. The gel electrophoresis results are shown below. Figure 5 As shown, the result that the target band matching the expected VP3 fragment size can be amplified in each generation indicates that the recombinant adenoviruses rAd5-CAV-VP3 and rAd5-GyH1-VP3 still have good genetic stability after 10 consecutive passages.
[0063] Example 4: Evaluation of the in vitro apoptosis effects of rAd5-CAV-VP3 and rAd5-GyH1-VP3 recombinant adenoviruses on A2780 and B16 tumor cells. The specific steps are as follows: (1) Cell resuscitation and culture B16 and A2780 cells were removed from liquid nitrogen and quickly placed in a preheated 37°C water bath, gently agitated to accelerate thawing. After thawing, the outer walls of the tubes were sterilized with alcohol, and the cell suspension was transferred to sterilized EP tubes on a laminar flow hood. Next, the cells were centrifuged at 1000 rpm for 6 minutes, the supernatant was discarded, 1 mL of culture medium was added to resuspend the cells, and then they were transferred to the appropriate culture flasks for further culture.
[0064] After culturing in a constant temperature incubator at 37℃ and 5% CO2 for 24 hours, observe the cell status and replace the culture medium as necessary to ensure normal cell growth. When the cell density reaches 80-90%, discard the culture medium in the culture flask and digest with 1 mL of trypsin for 30 seconds. Immediately afterwards, add 1 mL of culture medium to stop the digestion and gently mix the cells with a pipette. Finally, transfer a certain amount of cell suspension to a new culture flask and continue culturing.
[0065] (2) Inhibition of A2780 and B16 tumor cell proliferation by VP3 recombinant adenovirus (CCK-8) First, B16 and A2780 cells were counted, followed by 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates. After 24 hours of culture, the cells were infected with rAd5-CAV-VP3 and rAd5-GyH1-VP3 adenoviruses at MOIs of 50, 100, and 200, and treated for 1 hour each. Six replicates were performed for each group to ensure the accuracy of the results. After infection, 10 µL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C in the dark for 1 hour. Finally, the absorbance of each well was measured at 450 nm using a microplate reader to assess cell proliferation. The results are shown below. Figure 6 As shown, the proliferation of the two cell lines was detected using the CCK8 reagent 48 h post-infection. From the concentration gradient observations, regardless of the infection concentration (50 MOI, 100 MOI, or 200 MOI), the inhibitory effect of recombinant adenovirus on cells significantly increased with prolonged infection time; higher infection concentrations resulted in stronger inhibitory effects, exhibiting a significant concentration-time effect. Furthermore, at the maximum infection dose, there was no significant difference in cell proliferation inhibition between rAd5-CAV-VP3 and rAd5-GyH1-VP3 recombinant adenoviruses.
[0066] (3) Annexin-v / pe detection B16 and A2780 cells were revived, passaged, and plated. The cells were then infected with adenovirus, and a control group (Control group) was established that was not infected with rAd5-CAV-VP3 or rAd5-GyH1-VP3 viruses. Forty-eight hours after infection, the 6-well plates were removed, the culture medium in the wells was discarded, and the cells were washed with PBS. Next, 195 μL of Annexin-V / PE binding solution was added to each well, followed by 5 μL of PE for staining in the dark for 20 min. Afterward, the staining solution was discarded, and another 195 μL of Annexin-V / PE binding solution was added, followed by 5 μL of DAPI for staining in the dark for 10 min. Finally, the cells were observed under a fluorescence microscope, and apoptosis was analyzed. The results are as follows: Figure 7 , 8 As shown, both cell types exhibited significant apoptosis after infection with the recombinant virus, and viral aggregation in the nucleus exacerbated the accumulation of apoptin, thereby promoting apoptosis. Furthermore, under the same concentration of adenovirus infection, the rAd5-GyH1-VP3 group showed more pronounced apoptosis compared to the rAd5-CAV-VP3 group.
[0067] (4) Western Blot verification B16 and A2780 cells were resuscitated, passaged, and plated. Cells were then infected with recombinant adenovirus at a concentration of 100 MOI, and a control group (Control group) was established that was not infected with rAd5-CAV-VP3 or rAd5-GyH1-VP3 viruses. Forty-eight hours after infection, the 6-well plates were removed, the culture medium in the wells was discarded, and the cells were washed twice with PBS. Cells were then digested with 1 mL of trypsin for 30 seconds. Immediately afterwards, 1 mL of culture medium was added to stop the digestion, and the cells were gently reconstituted using a pipette. Cell lysis was successful, and proteins were extracted. SDS-PAGE gels were prepared, and Western blot experiments were performed. Results are as follows: Figure 9 As shown, recombinant adenoviruses rAd5-CAV-VP3 and rAd5-GyH1-VP3 still express VP3 in A2780 and B16 cells.
[0068] Example 5: Evaluation of the inhibitory effects of rAd5-CAV-VP3 and rAd5-GyH1-VP3 recombinant adenovirus on A2780 and B16 tumor cells in vivo. (1) Establishment of animal models B16 and A2780 cells were revived and cultured, and extensive cell expansion was performed using large-volume cell culture flasks. First, cells in good growth condition were selected and digested with trypsin. Then, the cell suspension was collected into 50 mL centrifuge tubes, and the supernatant was removed by centrifugation. The cells were washed twice with PBS, and the cell density was adjusted to 3 × 10⁶ cells / mL. 7 / mL. Next, 100μL of B16 and A2780 cell suspensions (containing 3×10⁶ cells per 100μL) were added. 6 (Number of cells) were inoculated subcutaneously into the right posterior side of male nude mice (6 weeks old, weighing 22±2 g) to observe tumor formation. When the tumor volume reached 0.5×0.5×0.5 cm³, the nude mice were randomly divided into four groups: saline injection group, adenovirus empty vector group (adenovirus vector obtained by co-transfecting pcADV-EF1-mNeonGreen-CMV vector and adenovirus backbone plasmid into HEK 293T cells), rAd5-CAV-VP3 group, and rAd5-GyH1-VP3 group, with 6 nude mice in each group.
[0069] Nude mice in the rAd5-CAV-VP3 group and the rAd5-GyH1-VP3 group were injected intratumorally with 6×10⁶ g of [unspecified substance]. 8 One plaque-forming unit (PFU) of rAd5-CAV-VP3 and rAd5-GyH1-VP3 was injected, with an injection volume of 100 μL. Simultaneously, the saline control group and the adenovirus empty vector group were also injected with the corresponding volume of the drug, 100 μL each. Injections were administered every 3 days for a total of 4 treatments.
[0070] (2) Measure changes in tumor size Nude mice successfully bearing tumors underwent weekly measurements starting from week 1, recording the long and short diameters of the tumor. These measurements continued for 2 weeks. A mean tumor growth curve was plotted with the measurement time (in weeks) on the x-axis and the average tumor volume (in mm³) on the y-axis. The results are shown below. Figure 10 As shown, in the experimental groups injected with recombinant adenovirus rAd5-CAV-VP3 and rAd5-GyH1-VP3, the growth rate of mouse melanoma was significantly lower than that of the blank control group and the adenovirus empty vector group. Furthermore, no significant difference was found between the rAd5-CAV-VP3 and rAd5-GyH1-VP3 groups. The tumors were weighed, and the results are as follows... Figure 10 As shown, the tumor weight in the experimental groups treated with recombinant adenovirus rAd5-CAV-VP3 and rAd5-GyH1-VP3 showed a significant decreasing trend compared with the blank control group and the adenovirus empty vector group. There was no significant difference in tumor weight between the recombinant adenovirus rAd5-CAV-VP3 group and the rAd5-GyH1-VP3 group.
[0071] (3) Gross necropsy and histopathological observation On days 6, 9, and 12 of the experiment, nude mice in each group were euthanized. Subcutaneous melanomas and livers were then removed and photographed for documentation. Some tissue samples were stored in liquid nitrogen, while others were fixed in 10% neutral formalin solution. Paraffin sections were prepared and stained with hematoxylin and eosin (HE) to allow observation of pathological changes in each tissue under an optical microscope.
[0072] After necropsy and observation, the results were as follows: Figure 11 In the rAd5-CAV-VP3 and rAd5-GyH1-VP3 recombinant adenovirus groups at 6d, 9d, and 12d, tumor growth was significantly slower than in the control and empty vector groups, and atypia was also significantly lower. The liver and kidneys were reddish-brown with no obvious lesions.
[0073] Pathological histological observation revealed the following results: Figure 12 As shown, the tissue morphology of the groups injected with rAd5-CAV-VP3 and rAd5-GyH1-VP3 remained intact, with no obvious atypia. In contrast, the tissue morphology of the control group and the empty group was significantly damaged, with unclear structure and more widespread necrosis.
[0074] The above description is merely a preferred embodiment of this application and is not intended to limit the application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications made within the spirit and principles of this application are not permitted. Equivalent substitutions and improvements should all be included within the scope of protection of this application.
Claims
1. A recombinant adenovirus for inhibiting tumor cell growth and / or inducing tumor cell apoptosis, characterized in that, The recombinant adenovirus is either rAd5-CAV-VP3 recombinant adenovirus or rAd5-GyH1-VP3 recombinant adenovirus; The rAd5-CAV-VP3 recombinant adenovirus was obtained by cloning the chicken anemia virus VP3 gene into a shuttle plasmid vector, then co-transfecting it with an adenovirus backbone vector into 293T cells for packaging and recombination. The rAd5-GyH1-VP3 recombinant adenovirus was obtained by cloning the human type I VP3 gene of circular virus into a shuttle plasmid vector, then co-transfecting it with an adenovirus backbone vector into 293T cells for packaging and recombination.
2. The recombinant adenovirus according to claim 1, characterized in that, The nucleotide sequence of the chicken anemia virus VP3 gene is shown in SEQ ID NO:1, and the nucleotide sequence of the human type I VP3 gene of the circle virus is shown in SEQ ID NO:
2.
3. The recombinant adenovirus according to claim 2, characterized in that, The amino acid sequence of the protein encoded by the VP3 gene of the chicken anemia virus is shown in SEQ ID NO:3, and the amino acid sequence of the protein encoded by the human type I VP3 gene of the circle virus is shown in SEQ ID NO:
4.
4. The recombinant adenovirus according to claim 1, characterized in that, The shuttle plasmid vector is pcADV-EF1-mNeonGreen-CMV-MCS, and the adenovirus backbone vector is human replication-defective adenovirus type 5.
5. The recombinant adenovirus according to claim 1, characterized in that, The viral titer of the rAd5-CAV-VP3 recombinant adenovirus was 1.58 × 10⁻⁶. 10 The viral titer of the rAd5-GyH1-VP3 recombinant adenovirus was 3.95 × 10⁻⁶ PFU / mL. 10 PFU / mL.
6. The use of the recombinant adenovirus according to claim 1 in either (1) or (2) below: (1) Prepare products for inhibiting the growth of tumor cells; (2) Prepare products for inducing tumor cell apoptosis.
7. The application according to claim 6, characterized in that, The tumor cells include: human ovarian cancer cells or mouse melanoma cells.
8. The application according to claim 6, characterized in that, The product includes a vaccine with the recombinant adenovirus of claim 1 as the active ingredient or a drug with the recombinant adenovirus of claim 1 as the active ingredient.