Recombinant oncolytic adenovirus based on PCV2 functional protein as well as preparation method and application of recombinant oncolytic adenovirus

Abstract

The invention provides a recombinant oncolytic adenovirus based on PCV2 functional protein as well as a preparation method and application of the recombinant oncolytic adenovirus, and belongs to the technical field of biological medicines. The genome of the recombinant oncolytic adenovirus based on the PCV2 functional protein provided by the invention comprises a PCV2 Cap gene and / or a PCV2 Rep gene. According to the invention, the Cap and / or Rep genes of PCV2 are / is taken as brand new therapeutic transgenosis, the recombinant oncolytic adenovirus is successfully constructed, and brand new candidate entities and action target bases different from traditional cell factors or tumor suppressor genes are provided for oncolytic virus therapy. According to the invention, the recombinant adenovirus is used as an efficient delivery vector, so that the transduction and expression efficiency of the PCV2 functional gene is greatly improved, and the limitations of low natural infection efficiency and weak replication ability of the PCV2 wild type virus are overcome. The recombinant oncolytic adenovirus, especially the recombinant Rep adenovirus, provided by the invention realizes a multi-mechanism synergistic efficient anti-tumor effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a recombinant oncolytic adenovirus based on the PCV2 functional protein, its preparation method, and its application. Background Technology

[0002] Oncolytic virus therapy is a novel treatment strategy that utilizes natural or genetically engineered viruses to selectively replicate and lyse tumor cells, while simultaneously stimulating systemic anti-tumor immunity. Currently, several viruses have been developed into oncolytic viruses, but they generally suffer from problems such as single-target targeting, limited oncolytic efficiency, susceptibility to neutralization by pre-existing immune responses, and the tendency for tumors to develop drug resistance. Therefore, the development of novel, highly efficient, and multi-mechanism-dependent oncolytic viruses is urgently needed.

[0003] Among numerous viral vectors, replication-deficient recombinant adenoviruses have become an ideal platform for expressing exogenous therapeutic genes due to their wide host range, high transgenic efficiency, large gene carrying capacity, and good safety profile. Currently, research on adenovirus-based oncolytic viruses largely focuses on carrying immunomodulatory factors (such as cytokines and chemokines) or tumor suppressor genes to enhance anti-tumor immunity or directly induce cancer cell death. However, existing strategies still face many challenges: First, many solid tumors exhibit high heterogeneity and immunosuppressive microenvironments, resulting in limited efficacy of single-mechanism therapies; second, tumor cells can develop strong chemotherapy resistance through pathways such as upregulating stress granule formation, greatly limiting the efficacy of combination therapies; furthermore, abnormal glycolytic metabolism (Warburg effect) and the powerful migration and invasion capabilities of tumor cells are key factors leading to tumor progression and metastasis, but existing oncolytic virus preparations still lack the ability to directly target these core metabolic and metastatic pathways.

[0004] Porcine circovirus type 2 (PCV2) is a traditional animal pathogen. Its encoded capsid protein (Cap) and replicase protein (Rep) play a central role in viral infection and replication. However, PCV2's limited natural infectivity, weak replication ability in tumor cells, and the potential unknown risks associated with its direct application as an animal-derived virus severely restrict its clinical translation. Current technologies lack a solution to utilize the unique characteristics of PCV2's Cap and Rep proteins to construct novel oncolytic viruses that simultaneously address multiple challenges such as targeting, drug resistance, metabolic abnormalities, and metastasis. Summary of the Invention

[0005] In view of this, the present invention addresses the problems existing in the current oncolytic virus technology, such as single targeting, easy development of drug resistance, difficulty in effectively inhibiting tumor metabolism and metastasis, as well as the low infection efficiency, unclear mechanism of action and potential safety risks when PCV2 wild-type virus is directly applied. The present invention aims to provide a novel recombinant oncolytic adenovirus that can effectively inhibit tumor growth and overcome chemotherapy resistance through synergistic action of multiple mechanisms.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a recombinant oncolytic adenovirus based on the PCV2 functional protein, wherein the genome of the recombinant oncolytic adenovirus includes the PCV2 Cap gene and / or the PCV2 Rep gene.

[0007] Preferably, the nucleotide sequence of the PCV2 Cap gene is shown in SEQ ID NO.1, and the nucleotide sequence of the PCV2 Rep gene is shown in SEQ ID NO.2.

[0008] Preferably, the adenovirus vector is a replication-defective adenovirus vector, including human adenovirus type 5.

[0009] The present invention also provides a method for preparing the above-mentioned recombinant oncolytic adenovirus, comprising the following steps: integrating the PCV2 Cap gene and / or the PCV2 Rep gene into a shuttle vector to obtain a recombinant shuttle plasmid; co-transfecting the recombinant shuttle plasmid and the adenovirus backbone plasmid into cells, culturing and collecting the lysate supernatant to obtain the recombinant oncolytic adenovirus.

[0010] Preferably, the shuttle vector comprises pDC316, the adenovirus backbone plasmid comprises pBHGlox(delta)E1,3Cre, and the cells comprise HEK-293A cells.

[0011] The present invention also provides the use of the above-mentioned recombinant oncolytic adenovirus in the preparation of products for the treatment and / or prevention of tumors.

[0012] Preferably, the tumor includes lung cancer, liver cancer, and / or cervical cancer.

[0013] Preferably, the lung cancer includes non-small cell lung cancer.

[0014] This invention also provides the application of the above-mentioned recombinant oncolytic adenovirus in the preparation of products that reverse chemotherapy resistance.

[0015] The present invention also provides a drug for preventing or treating tumors or reversing chemotherapy resistance, wherein the active ingredient of the drug includes the above-mentioned recombinant oncolytic adenovirus.

[0016] The beneficial effects of this invention are: This invention utilizes the Cap and / or Rep genes of PCV2 as novel therapeutic transgenes to successfully construct recombinant oncolytic adenoviruses, providing a novel candidate entity and target for oncolytic virus therapy, distinct from traditional cytokines or tumor suppressor genes. This invention leverages recombinant adenoviruses as highly efficient delivery vectors, significantly improving the transduction and expression efficiency of PCV2 functional genes and overcoming the limitations of low natural infection efficiency and weak replication capacity of wild-type PCV2 viruses. The recombinant oncolytic adenoviruses provided by this invention, especially the recombinant Rep adenovirus, can synergistically attack tumors from multiple levels, including inducing apoptosis, reversing chemotherapy resistance, inhibiting metabolic reprogramming, and disrupting the tumor's main energy source and lactate accumulation, achieving a highly efficient anti-tumor effect through multi-mechanism synergy. In in vitro cell experiments (such as A549, HeLa, etc.) and in vivo xenograft animal models, the recombinant oncolytic adenoviruses provided by this invention have shown more significant tumor growth inhibition effects than traditional single-mechanism oncolytic viruses. This invention provides new candidate entities and target support for the development of novel oncolytic virus agents with multi-mechanism synergistic effects. Attached Figure Description

[0017] Figure 1 This is a diagram showing the enzyme digestion identification of the recombinant shuttle plasmid. Lane M is the DL-5000 Marker, lane 1 is the empty shuttle plasmid, lane 2 is the undigested recombinant PCV2 Cap shuttle plasmid, lane 3 is the double-digested recombinant PCV2 Cap shuttle plasmid, lane 4 is the undigested recombinant PCV2 Rep shuttle plasmid, and lane 5 is the double-digested recombinant PCV2 Rep shuttle plasmid. Figure 2 For identification of infectious clones of recombinant adenovirus, comet-shaped green fluorescence indicates successful viral release; Figure 3 The results of recombinant adenovirus protein expression identification; Figure 4 To assess the pathological effects of recombinant PCV2 Cap and / or Rep adenovirus infection on tumor cells, images were taken with adherent cells in sharp focus. Circular white bright spots in the images represent suspended dead cells or cell debris. In the adenovirus Rep group and the adenovirus-Cap+Rep synergistic group, due to the excessive number of dead cells and cell debris, the image could not focus on adherent cells. Figure 5 The effect of overexpression of PCV2 Cap and Rep proteins on the accumulation of P53 protein in A549 cells; Figure 6 The effects of PCV2 infection and overexpression of PCV2 Cap and Rep on key apoptosis signaling molecules in A549 cells; Figure 7The results show that PCV2 Cap and Rep proteins inhibit cisplatin-induced stress granule production by targeting G3BP1. Blue fluorescent signals represent DAPI-stained nuclei, green fluorescent signals represent G3BP1 protein, green bright spots represent stress granules, and red fluorescent signals represent Flag-tagged PCV2 Cap or PCV2 Rep proteins. Figure 8 To investigate the effects of overexpression of PCV2 Cap and / or Rep proteins on pyruvate kinase activity in human cells, This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001; Figure 9 For the effect of overexpression of PCV2 Cap and / or Rep proteins on ATP production in human cells, ns indicates no statistical significance. This indicates that p < 0.001; Figure 10 For the effect of overexpression of PCV2 Cap and / or Rep proteins on lactate production in human cells, ns indicates no statistical significance. This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001; Figure 11 Morphological illustration of xenograft tumors of human lung cancer cells (A549) from BALB / C nude mice treated with different adenoviruses; Figure 12 Growth curves of A549 xenograft tumors in BALB / C nude mice treated with different adenoviruses; Figure 13 A comparison of the weight of A549 xenograft tumors in BALB / C nude mice treated with different adenoviruses. Detailed Implementation

[0018] This invention provides a recombinant oncolytic adenovirus based on the PCV2 functional protein, wherein the genome of the recombinant oncolytic adenovirus includes the PCV2 Cap gene and / or the PCV2 Rep gene.

[0019] This invention is the first to propose that the Cap and Rep proteins of PCV2 can activate the p53-mediated apoptosis pathway, directly inducing programmed cell death in tumor cells; they can inhibit the formation of stress granules (SGs), thereby disrupting the self-protective mechanisms of tumor cells under chemotherapy or stress, effectively reversing chemotherapy resistance; simultaneously, they can inhibit glycolytic metabolism in tumor cells, suppressing energy supply and lactate accumulation, thus inhibiting tumor proliferation at the metabolic level. This invention overcomes the technical limitation of low infection efficiency of wild-type PCV2 virus by constructing a recombinant oncolytic adenovirus to achieve efficient delivery of the Cap and Rep genes. In in vitro cell experiments and in vivo animal models (such as the A549 xenograft model), the recombinant oncolytic adenovirus constructed in this invention showed significant tumor growth inhibition effects, with the recombinant Rep adenovirus showing particularly outstanding effects, completely lysing tumors.

[0020] The recombinant oncolytic adenovirus provided by this invention operably links the Cap gene and / or Rep gene to a heterologous promoter and can express the PCV2 Cap protein and / or Rep protein in host cells. In this invention, the preferred nucleotide sequence of the PCV2 Cap gene is shown in SEQ ID NO.1, and the preferred nucleotide sequence of the PCV2 Rep gene is shown in SEQ ID NO.2. In this invention, the adenovirus vector is preferably a replication-defective adenovirus vector, which preferably includes human adenovirus type 5 (Ad5). The E1 region genes of Ad5 (especially E1A and / or E1B genes) are absent or deleted, thereby ensuring that the virus cannot replicate in normal cells, but can specifically replicate in tumor cells with abnormal p53 function, achieving an oncolytic effect. This invention uses a replication-defective adenovirus backbone, avoiding the biosafety risks that may arise from the direct application of live PCV2 virus, and has greater potential for clinical translation. The recombinant oncolytic adenovirus provided by this invention can activate p53-mediated apoptosis, inhibit stress granule formation, inhibit tumor cell glycolysis, inhibit energy supply and lactate accumulation, and reverse tumor chemotherapy resistance.

[0021] The present invention also provides a method for preparing the above-mentioned recombinant oncolytic adenovirus, comprising the following steps: integrating the PCV2 Cap gene and / or the PCV2 Rep gene into a shuttle vector to obtain a recombinant shuttle plasmid; co-transfecting the recombinant shuttle plasmid and the adenovirus backbone plasmid into cells, culturing and collecting the lysate supernatant to obtain the recombinant oncolytic adenovirus.

[0022] In this invention, the shuttle vector preferably includes pDC316, the adenovirus backbone plasmid preferably includes pBHGlox(delta)E1,3Cre, and the cells preferably include HEK-293A cells. This invention does not have specific limitations on the specific sources of the shuttle vector, adenovirus backbone plasmid, and cells; commercially available products commonly used in the field are acceptable.

[0023] The present invention also provides the use of the above-mentioned recombinant oncolytic adenovirus in the preparation of products for the treatment and / or prevention of tumors.

[0024] In this invention, the tumor preferably includes lung cancer, liver cancer, and / or cervical cancer. The lung cancer preferably includes non-small cell lung cancer. In this invention, the tumor preferably also includes tumors resistant to conventional chemotherapy. In this invention, the lung cancer preferably includes lung cancer A549 cells, and the cervical cancer preferably includes HeLa cells.

[0025] This invention also provides the application of the above-mentioned recombinant oncolytic adenovirus in the preparation of products for reversing chemotherapy resistance. In this invention, the type of product preferably includes a drug.

[0026] The present invention also provides a drug for preventing or treating tumors or reversing chemotherapy resistance, wherein the active ingredient of the drug includes the above-mentioned recombinant oncolytic adenovirus.

[0027] In this invention, the drug preferably further includes a pharmaceutically acceptable carrier or excipient; the dosage form of the drug preferably includes an injectable dosage form (intratumoral injection or intravenous injection) or a lyophilized dosage form. In this invention, the drug exerts its therapeutic effect through one or more mechanisms, including activating p53-mediated apoptosis, inhibiting stress granule formation to reverse chemotherapy resistance, and inhibiting tumor cell glycolysis and energy supply and lactate accumulation.

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Unless otherwise specified, the following embodiments are all conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] Example 1 Construction of recombinant shuttle plasmids and packaging, propagation, and identification of recombinant adenoviruses: Step 1: Construction and Identification of Recombinant Shuttle Plasmids The PCV2 Cap gene (nucleotide sequence shown in SEQ ID NO.1) and the Rep gene (nucleotide sequence shown in SEQ ID NO.2) were integrated into the NotⅠ and BamhⅠ restriction sites of the shuttle vector pDC316 via gene synthesis, yielding recombinant PCV2 Cap shuttle plasmid (SEQ ID NO.3) and recombinant PCV2 Rep shuttle plasmid (SEQ ID NO.4). Restriction digestion was then performed for identification. The empty vector plasmid, recombinant PCV2 Cap shuttle plasmid, and recombinant PCV2 Rep shuttle plasmid were digested overnight with restriction endonucleases NotⅠ and BamhⅠ. The results are as follows: Figure 1 As shown, the recombinant shuttle plasmids were successfully constructed and named pDC-316-cap and pDC-316-rep.

[0032] Step 2: Packaging and propagation of recombinant adenovirus 1. Plasmid co-transfection and initial virus packaging HEK-293A cells were cultured in 6-well plates until approximately 80% confluence was reached, at which point transfection was performed. First, two 1.5 mL centrifuge tubes were prepared: Tube A contained 200 μL of OPTI-MEM medium and 8 μL of Lipofectamine 2000 transfection reagent; Tube B contained an equal volume of 200 μL of OPTI-MEM medium, and the plasmids to be transfected (empty plasmid, recombinant shuttle plasmid (pDC-316-cap or pDC-316-rep)) and the adenovirus backbone plasmid pBHGlox(delta)E1,3Cre (purchased from Wuhan Zhijia Biotechnology Co., Ltd., product code P0883) were added at a 1:2 ratio (the ratio of the plasmid to be transfected to the adenovirus backbone plasmid is 1:2). The two tubes were mixed separately and allowed to stand for 5 minutes. Then, the plasmid mixture from tube B was combined with the transfection reagent mixture from tube A, gently mixed, and allowed to stand at room temperature for 20 minutes to form a DNA-liposome complex. During this process, the cells in the 6-well plate were replaced with fresh complete culture medium, and then the transfection complex was added dropwise to the wells and gently shaken.

[0033] The day after transfection, the culture medium was replaced with cell maintenance medium containing 2% serum, and this medium was changed regularly over the next four days. Approximately seven days after transfection, under a microscope, most cells exhibited the typical pathological effect of shrinkage and clumping. At this point, the cell culture was collected and subjected to three freeze-thaw cycles (first rapidly frozen in liquid nitrogen, then allowed to thaw naturally in an ice box, repeated three times). The culture was then centrifuged at 4°C and 16000 rpm for 20 minutes. The resulting supernatant was the successfully packaged first-generation recombinant oncolytic adenovirus solution.

[0034] 2. Virus passage and fluorescence verification 500 μL of first-generation recombinant oncolytic adenovirus solution was used to infect confluent HEK-293A cells, and the cells were incubated for 1 hour to allow for virus adsorption. The viral solution was then removed and replaced with maintenance medium containing 2% serum. Approximately 24 hours after infection, green fluorescent protein expression was observed under a fluorescence microscope; by 48 hours, the fluorescence distribution exhibited a unique "comet-like" pattern. Figure 2 As shown, this initially demonstrates the successful packaging and proliferation of the recombinant adenovirus. Over time, the fluorescence intensity and range gradually increased. Approximately 7 days later, when about 80% of the cells showed cytopathic effects, the viral fluid was harvested again and frozen at -80°C; this is the second-generation virus.

[0035] 3. Large-scale viral amplification To obtain sufficient virus for subsequent research, the harvested virus solution was further used to infect HEK-293A cells in T75 cell culture flasks. This method was used to amplify and harvest large quantities of virus solution from different passages.

[0036] Step 3: Identification of recombinant oncolytic adenovirus protein expression To further verify the expression of Cap and Rep proteins in the constructed recombinant oncolytic adenovirus PCV2, the following experiment was designed: 1. Sample preparation The experimental group (infected with recombinant oncolytic adenovirus) and the negative control group (infected with empty vector adenovirus) were set up. Cells were collected, total protein was extracted using lysis buffer, and protein concentration was accurately determined. The protein sample was mixed with loading buffer and boiled for denaturation before electrophoresis.

[0037] 2. SDS-PAGE electrophoresis Add equal amounts of protein sample and pre-stained protein marker to the sample wells. Perform gel electrophoresis: initially use low pressure, then switch to high pressure after the sample enters the separating gel until the bromophenol blue indicator reaches the bottom of the gel, causing the proteins to separate according to their molecular weight.

[0038] 3. Transfer membrane The protein bands separated in the gel were transferred to a PVDF membrane using a wet transfer method.

[0039] 4. Enclosed Immerse the PVDF membrane in a 5% skim milk solution and block it at room temperature for 1-2 hours to block the non-specific binding sites of the antibody.

[0040] 5. Primary antibody incubation The sealed membrane was incubated overnight at 4°C with a specific primary antibody targeting PCV2 Cap or Rep protein to allow the primary antibody to fully bind to the target protein.

[0041] 6. Secondary antibody incubation The primary antibody was recovered, and the membrane was washed three times with TBST buffer to remove unbound primary antibody. The membrane was then incubated with horseradish peroxidase-labeled secondary antibody at room temperature for 1–2 hours.

[0042] 7. Chemiluminescence detection The secondary antibody was recovered, and the membrane was thoroughly washed again with TBST. ECL chemiluminescent substrate was then uniformly dropped onto the membrane, and exposure and image acquisition were performed using a gel imaging system.

[0043] 8. Results Analysis The final result is as follows Figure 3 As shown, specific Cap and Rep protein expression was observed in the experimental group; however, no such specific bands were observed in the negative control group. This result confirms that the recombinant oncolytic adenovirus successfully expressed the Cap and Rep target proteins of PCV2.

[0044] Example 2 Recombinant oncolytic adenovirus infects human tumor cells, causing pathological effects. To verify the oncolytic effects of the recombinant oncolytic PCV2 Cap adenovirus and the recombinant oncolytic PCV2 Rep adenovirus obtained in Example 1, the following cytopathic effect experiment was designed: 1. Cell Culture and Grouping: Human non-small cell lung cancer A549 cells and cervical cancer HeLa cells were selected as in vitro models. Cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator until the logarithmic growth phase. The experiment was set up with 5 groups: a blank control group (cell maintenance medium without virus), an empty vector virus control group (inoculated with an empty vector adenovirus without the target gene), a Cap adenovirus infection group, a Rep adenovirus infection group, and a Cap+Rep adenovirus infection group (referring to infection with a mixture of Cap and Rep adenovirus).

[0045] 2. Viral Infection and Observation: When cell confluence reaches 70%–80%, discard the old culture medium and gently wash the cells twice with PBS. At an infection dose of MOI=1, dilute the recombinant adenovirus and empty adenovirus with serum-free culture medium and add them to the corresponding experimental group and empty virus control group cells, respectively. The blank control group is replaced with an equal volume of maintenance culture medium. Return the cells to the incubator and allow the virus to adsorb for 4–6 hours. Then discard the virus solution and replace with fresh complete culture medium for continued culturing.

[0046] 3. Monitoring and recording of cytopathic effects: The results are as follows Figure 4As shown, 48 hours after infection, compared with the control group and empty vector adenovirus, infection with recombinant oncolytic PCV2 Cap adenovirus and recombinant oncolytic PCV2 Rep adenovirus resulted in a large number of cells becoming rounded and detaching. The cytopathic effect was more pronounced under the synergistic effect of recombinant oncolytic PCV2 Cap and recombinant oncolytic PCV2 Rep adenovirus. Compared with PCV2 infection of A549 cells and HeLa cells, PCV2 only showed a significant cytopathic effect after 5 passages of virus-carrying virus, indicating that the recombinant oncolytic adenovirus has a stronger oncolytic effect than empty vector adenovirus and PCV2 virus.

[0047] Example 3 Overexpression of PCV2 Cap and PCV2 Rep in A549 cells promotes apoptosis pathway activation by inducing p53 accumulation. To investigate the oncolytic mechanism of PCV2 Cap / Rep proteins, Cap or Rep proteins were first overexpressed in A549 cells. The overexpression method involved transfection with pECMV-3×FLAG-N-PCV2 CAP or pECMV-3×FLAG-N-PCV2 Rep plasmids, with the PCV2 Cap or Rep sequence inserted between EcoR1 and Not1. The recombinant plasmids were prepared by Wuhan Tianyi Huayu Gene Technology Co., Ltd. through gene synthesis. The effects on the key tumor suppressor protein p53 were then detected using Western blotting. Results are as follows: Figure 5 As shown, compared with the control group with empty virus, the p53 protein level in the experimental group with Cap or Rep overexpression was significantly increased, indicating that Cap and Rep proteins can promote the accumulation of p53 in cells.

[0048] It is known that p53 initiates the apoptosis process primarily through both transcription-dependent and non-transcription-dependent pathways. To verify whether p53 accumulation successfully activates downstream apoptosis signaling pathways, key protein markers in multiple apoptosis pathways were further examined. Western blot analysis results (see...) Figure 6 The results showed that in cells overexpressing Cap, Rep, or simultaneously overexpressing Cap and Rep (simultaneous overexpression of Cap and Rep refers to simultaneous transfection with pECMV-3×FLAG-N-PCV2 CAP and pECMV-3×FLAG-N-PCV2 Rep plasmids), the expression level of the pro-apoptotic protein BAX was significantly upregulated, directly demonstrating the activation of p53's transcriptional regulatory function. Simultaneously, varying degrees of activation and cleavage were observed in both Caspase 9, a key executor of the mitochondrial pathway, and Caspase 8, a key executor of the death receptor pathway. Ultimately, these two pathways converged on the activation of the apoptosis-executing protein Caspase 3.

[0049] In summary, overexpression of PCV2 Cap and / or Rep proteins promotes the accumulation of p53 protein; the accumulated p53 then activates caspase 3 through both transcription-dependent pathways (upregulation of BAX expression, activation of caspase 9) and non-transcription-dependent pathways (activation of caspase 8), thereby initiating the apoptosis program. This mechanism provides a solid molecular biological basis for explaining the oncolytic effect of recombinant adenoviruses in effectively killing cancer cells.

[0050] Example 4 PCV2 Cap and Rep proteins target G3BP1 to inhibit cisplatin-induced stress granule production. When cells are subjected to external stress or internal stress, such as oxidative stress, heat shock, viral infection, and drug treatment, it can lead to disorders in intracellular protein synthesis and metabolism, which in turn promotes the formation of cellular stress granules. Stress granules help cells suspend the synthesis of non-essential proteins by aggregating translated but arrested mRNA and related proteins, and prioritize the allocation of resources to cope with stress.

[0051] This mechanism is highly detrimental to drug treatment of tumors, severely impacting the killing effect of chemotherapy drugs on tumor cells. It helps tumor cells escape death under chemotherapy, targeted therapy, or microenvironmental stress by dynamically regulating intracellular translational arrest and signaling pathway reprogramming.

[0052] To verify whether the hijacking effect of PCV2 Cap and Rep proteins on G3BP1 would inhibit the production of stress granules, PCV2 Cap or Rep proteins were overexpressed in A549 cells (overexpression method as in Example 3), and cisplatin (DDP) was used as a stress granule inducer to observe the formation of stress granules.

[0053] The results are as follows Figure 7 As shown, under normal conditions, G3BP1 is uniformly distributed in the cytoplasm. Upon the addition of DDP, G3BP1 rapidly forms stress granules within the cytoplasm. Overexpression of PCV2 Cap or Rep proteins significantly reduced DDP-induced stress granules. This indicates that PCV2 Cap and Rep proteins inhibit cisplatin-induced stress granule production by targeting G3BP1, thereby suppressing tumor drug resistance.

[0054] Example 5 Overexpression of PCV2 Cap and Rep proteins inhibits tumor cell glycolysis, energy supply, and lactate accumulation. Based on the applicant's previous research, which found that PCV2 Cap and Rep proteins interact with PKM2, a key metabolic enzyme in human tumor cells, it was hypothesized that this interaction might be a crucial step in the PCV2 oncolytic mechanism. To verify this hypothesis, a series of experiments were conducted to investigate the impact of this interaction on tumor cell energy metabolism.

[0055] First, to verify the direct effect of Cap and / or Rep proteins on PKM2 enzyme activity, normal human lung epithelial cells BEAS-2B and three human tumor cell lines (lung cancer A549, cervical cancer HeLa, and liver cancer HepG2) were selected and transfected with pECMV-3×FLAG-N-PCV2 CAP and / or pECMV-3×FLAG-N-PCV2 Rep plasmids, respectively. Forty-eight hours after transfection, cell lysates were collected from each group, and the enzyme activity was accurately measured spectrophotometrically using a pyruvate kinase activity assay kit. The results are as follows: Figure 8 As shown, overexpression of Cap and / or Rep proteins significantly inhibited pyruvate kinase activity in all test cells compared to the control group (Flag) transfected with an empty vector. This indicates that the interaction between PCV2 Cap and / or Rep and PKM2 can indeed "hijack" and inhibit the catalytic function of PKM2.

[0056] Next, the functional consequences of enzyme activity inhibition were explored in depth. In the glycolysis pathway, PKM2 is responsible for catalyzing the conversion of phosphoenolpyruvate to pyruvate and the production of ATP, a key step in the Warburg effect in tumor cells. To assess whether Cap and / or Rep proteins disrupt the energy supply to tumor cells by inhibiting PKM2, the following assays were performed: Intracellular ATP level detection: The ATP content in cells directly infected with PCV2 virus and transfected with Cap and / or Rep plasmids was detected using an ATP luminescence assay kit.

[0057] Detection of lactic acid content in glycolysis end products: Collect cell culture supernatant and use a lactic acid content detection kit to quantitatively analyze the amount of lactic acid produced.

[0058] Experimental results are as follows Figure 9 and Figure 10 As shown, both PCV2 virus infection and Cap and / or Rep protein overexpression significantly inhibited ATP production in tumor cells and lactate accumulation in the culture medium. However, in normal BEAS-2B cells, neither ATP nor lactate levels showed significant changes.

[0059] In summary, this invention reveals a novel oncolytic mechanism of PCV2. The Cap and Rep proteins of PCV2 can directly inhibit the pyruvate kinase activity of PKM2 protein through interaction. This effectively chokes the tumor cells' aerobic glycolysis, blocking the energy and raw material supply for their rapid proliferation, thereby selectively downregulating the glycolytic flux of tumor cells and inhibiting the production of ATP and lactate. Since normal BEAS-2B cells do not rely on glycolysis as their primary energy source, this process has minimal impact on them, which to some extent explains the selective killing effect of PCV2 and its recombinant virus on tumor cells.

[0060] Example 6 Validation of the oncolytic efficacy of recombinant PCV2 Cap and / or Rep adenovirus in A549 cell xenograft tumor BALB / c nude mouse model. To verify the oncolytic effect of recombinant adenovirus in vivo, a BALB / c nude mouse xenograft model of human lung cancer cells (A549) was constructed. The tumor was established when the subcutaneous tumor volume in the mice grew to approximately 100 mm². 3 At that time, they were randomly divided into the following four groups for intervention and treatment: PBS control group (blank group); empty adenovirus group (adenovirus group); recombinant oncolytic PCV2 Cap adenovirus obtained in Example 1 group (adenovirus Cap group); recombinant oncolytic PCV2 Rep adenovirus obtained in Example 1 group (adenovirus Rep group); and combined recombinant oncolytic PCV2 Cap adenovirus obtained in Example 1 and recombinant oncolytic PCV2 Rep adenovirus obtained in Example 1 group (adenovirus Cap+Rep group).

[0061] Each group received either the corresponding virus or PBS via intratumoral injection every other day, for a total of two injections. During treatment, the major and minor axes of the tumor were measured regularly using calipers to calculate tumor volume and plot growth curves. Simultaneously, the mice's weight and activity level were closely monitored to assess treatment safety. Finally, the mice were euthanized, the tumors were harvested, and weighed.

[0062] The results are as follows Figure 11 , 12 and 13 shown by Figure 11 It can be seen that both recombinant oncolytic PCV2 Cap adenovirus and recombinant oncolytic Rep adenovirus exhibited stronger oncolytic effects than empty vector adenovirus, and the recombinant PCV2 Rep adenovirus group even achieved 100% tumor clearance, demonstrating strong therapeutic potential. Figure 12It can be seen that the recombinant oncolytic PCV2 Cap adenovirus and the recombinant oncolytic PCV2 Rep adenovirus completely inhibited tumor growth from the start of inoculation. In the recombinant oncolytic PCV2 Rep adenovirus group, some tumors even underwent complete ablation within one week of inoculation. Figure 13 It can be seen that both the recombinant oncolytic PCV2Cap adenovirus group and the recombinant oncolytic PCV2 Rep adenovirus group significantly reduced tumor weight, with the recombinant oncolytic PCV2 Rep adenovirus group showing a particularly significant reduction.

[0063] However, when recombinant oncolytic PCV2 Cap is used in combination with recombinant oncolytic PCV2 Rep adenovirus, the final oncolytic effect is weaker than when recombinant oncolytic PCV2 Rep adenovirus is used alone. This may be because, in the tumor microenvironment, when the two viruses co-infect the same cell population, they compete for limited cellular resources (such as transcription / translation machinery), which to some extent limits the amplification of the more efficient recombinant oncolytic Rep adenovirus, ultimately preventing its oncolytic efficacy from reaching its optimal level.

[0064] In summary, this invention confirms that recombinant oncolytic PCV2 Rep adenovirus has extremely strong antitumor activity in vivo, but its combined application strategy with recombinant oncolytic PCV2 Cap adenovirus may have antagonistic effects, which provides important experimental evidence for future optimization of virus administration strategies.

[0065] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant oncolytic adenovirus based on PCV2 functional proteins, characterized in that, The genome of the recombinant oncolytic adenovirus comprises a PCV2 Cap gene and / or a PCV2 Rep gene.

2. The recombinant oncolytic adenovirus according to claim 1, characterized in that, The nucleotide sequence of the PCV2 Cap gene is shown as SEQ ID NO. 1, and the nucleotide sequence of the PCV2 Rep gene is shown as SEQ ID NO.

2.

3. The recombinant oncolytic adenovirus of claim 1, wherein, The adenovirus vector is a replication-defective adenovirus vector, and the replication-defective adenovirus vector comprises a human adenovirus type 5.

4. The method of producing the recombinant oncolytic adenovirus according to any one of claims 1 to 3, wherein the adenovirus is a serotype 5 adenovirus. The method comprises the following steps: integrating the PCV2 Cap gene and / or the PCV2 Rep gene into a shuttle vector to obtain a recombinant shuttle plasmid; co-transfecting the recombinant shuttle plasmid and an adenovirus backbone plasmid into a cell, culturing and collecting a lysate supernatant, and obtaining the recombinant oncolytic adenovirus.

5. The production method according to claim 4, characterized by, The shuttle vector comprises pDC316, the adenovirus backbone plasmid comprises pBHGlox(delta)E1,3Cre, and the cell comprises HEK-293A cells.

6. Use of the recombinant oncolytic adenovirus according to any one of claims 1-3 in the preparation of a product for treating and / or preventing tumors.

7. Use according to claim 6, characterized in that, The tumors comprise lung cancer, liver cancer and / or cervical cancer.

8. Use according to claim 7, characterized in that, The lung cancer comprises non-small cell lung cancer.

9. Use of the recombinant oncolytic adenovirus according to any one of claims 1-3 in the preparation of a product for reversing chemotherapy resistance.

10. A medicament for preventing a tumor or reversing resistance to chemotherapy, comprising the compound of claim 1. The active ingredient of the drug comprises the recombinant oncolytic adenovirus according to any one of claims 1-3.

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