Construction method of human adenovirus type 4 protein v knockout mutant strain and application thereof

Abstract

The application discloses a construction method of a human adenovirus type 4 protein V knockout mutant strain and application thereof, and belongs to the technical field of biotechnology. The application precisely knocks out a protein V coding sequence in a human adenovirus type 4 full-genome infectious clone by means of a Red / ET homologous recombination system combined with a ccdB reverse screening technology, and obtains a recombination plasmid pBR322-Ad4-△V-EGFP which is clear in genetic background and highly homozygous in sequence. After the plasmid is transfected into HEK-293 cells, the human adenovirus type 4 protein V knockout mutant strain obtained exhibits a typical “replication-deficient” phenotype: although a particle containing a viral genome can be packaged, the physical titer (4.76×10 6 copies / μL) of the particle is about one order of magnitude lower than that of a wild type, and the progeny virus completely loses secondary infection ability. Transcriptome analysis reveals that the deletion mutation can specifically down-regulate a host proteasome pathway and activate a natural immune response. The mutant strain is high in safety and strong in immunogenicity, and can be used as an ideal attenuated vaccine carrier or a gene therapy tool.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for constructing a human adenovirus type 4 protein V knockout mutant and its application. Background Technology

[0002] Human adenoviruses (HAdVs) are a class of widespread pathogens, particularly prone to causing outbreaks of respiratory infections in enclosed environments (such as military camps and schools), posing a persistent threat to public health. Among them, HAdV-5, as the most maturely researched viral vector, plays a central role in vaccine development and gene therapy, and its vector system has been widely used in the development of vaccines for diseases such as COVID-19 and Ebola. However, HAdV-5 vectors have significant limitations in clinical application: the pre-existing neutralizing antibody positivity rate in the global population is as high as 70%-90%. This high level of pre-existing immunity significantly inhibits the transduction efficiency of viral vectors, reducing the immune protective effect of vaccines. For example, clinical trials have shown that after vaccination with a COVID-19 vaccine based on the HAdV-5 vector, the neutralizing antibody titer in individuals with pre-existing antibodies was more than 10 times lower than in those with negative antibodies. This deficiency severely limits the widespread adoption of HAdV-5 vectors in the general population, necessitating the development of novel adenovirus vectors with low pre-existing immunity.

[0003] To address these issues, researchers have begun to focus on rare serotypes of adenoviruses with low pre-existing immune levels. Human adenovirus type 4 (HAdV-4), the only member of the E genus, has a population antibody positivity rate of less than 20% and possesses unique respiratory tissue affinity, making it an ideal candidate for next-generation vaccine vectors. Epidemiological surveys show that while HADV-4 can easily cause outbreaks in specific situations (such as boot camps), its global prevalence is far lower than that of HADV-5, and its genome is highly stable and easily genetically modified. However, the pathogenic mechanism of HADV-4 is complex, and its direct use as a vector carries a potential risk of virulence reversion. Therefore, precise genetic modification is needed to balance its safety and immunogenicity, with the design of attenuation strategies being crucial.

[0004] Among adenovirus attenuation targets, core protein V has attracted much attention due to its multiple functions. Protein V is a key structural protein connecting the viral capsid and genomic DNA, playing a central role in the viral life cycle: on the one hand, it shields viral DNA from recognition by cytoplasmic sensors (such as cGAS), thereby suppressing the host's innate immune response; on the other hand, it acts as a "molecular switch" mediating the entry of the viral genome into the nucleus through the nuclear pore complex, initiating the infection process. Studies in HAdV-C5 (adenovirus C) have shown that knocking out protein V leads to a significant decrease in viral replication capacity, and the mutant strain still retains low levels of infectivity, suggesting its potential as an attenuation target. However, significant biological differences exist between different genera of adenoviruses: the core structural stability, genome packaging efficiency, and host interaction mechanisms of E-genus adenoviruses (such as HAdV-4) differ significantly from those of C-genus viruses. For example, the protein V coding region of HAdV-4 has low sequence conservation, and its interaction network with the host ubiquitin-proteasome system (UPS) may be genus-specific. Currently, there is a lack of research on the function of HAdV-4 protein V. The effects of its knockout on viral replication and host response are unclear. Directly applying the attenuation experience of HAdV-5 may lead to unforeseen biosafety risks.

[0005] In summary, current technologies lack specific attenuation techniques for HAdV-4, and especially lack systematic evaluation of the function of the core protein V. Developing a technology capable of precisely knocking out HAdV-4 protein V and clarifying its biological effects would have significant theoretical and applied value for advancing the development of novel adenovirus vectors. Summary of the Invention

[0006] Based on the technical problems existing in the prior art, the present invention provides a method for constructing a human adenovirus type 4 protein V knockout mutant and its application.

[0007] According to a first aspect of the technical solution of the present invention, the present invention first provides a method for constructing a human adenovirus type 4 protein V knockout mutant strain, comprising the following steps: (1) Using Red / ET homologous recombination technology, the ccdB-Kan selection frame was inserted into the protein V coding region of the HAdV-4 whole-genome infectious clone to obtain the intermediate plasmid pBR322-Ad4-ccdB; (2) The protein V knockout fragment ΔV was synthesized using overlap extension PCR technology; (3) The fragment △V was backfilled into the enzyme-digested linearized pBR322-Ad4-ccdB vector using seamless cloning technology to obtain the recombinant plasmid pBR322-Ad4-△V-EGFP.

[0008] In some embodiments, the targeting primers used in the Red / ET homologous recombination contain 50 bp sequences of upstream and downstream homologous arms of the protein V gene.

[0009] In some embodiments, the ccdB reverse screening system achieves positive clone screening through kanamycin resistance and the lethal effect of the ccdB gene on susceptible bacteria.

[0010] According to a second aspect of the technical solution of the present invention, the present invention also provides a human adenovirus type 4 protein V knockout mutant strain constructed by the above method.

[0011] According to a third aspect of the technical solution of the present invention, the present invention also provides an application of the above-mentioned mutant strain in the preparation of a live attenuated vaccine, wherein the mutant strain has replication defect characteristics and can induce high expression of inflammatory factors in host cells.

[0012] According to a fourth aspect of the technical solution of the present invention, the present invention also provides an application of the above-mentioned mutant strain in a gene therapy vector, wherein the mutant strain achieves controlled release of the viral genome through proteasome pathway interference.

[0013] According to a fifth aspect of the present invention, the present invention also provides a method for detecting the biological characteristics of a protein V knockout mutant, comprising: (1) Quantitative analysis of viral physical titer using qPCR; (2) Verify the infectivity of the progeny virus through a back-infection test; (3) The enrichment of differentially expressed genes and pathways in host cells was analyzed by transcriptome sequencing.

[0014] In some embodiments, the qPCR detection targets a conserved region of the viral fiber gene, and the primer sequences are shown in SEQ ID NO: 9-10.

[0015] According to a sixth aspect of the technical solution of the present invention, the present invention also provides a kit for constructing a protein V knockout mutant, comprising the above-mentioned recombinant plasmid pBR322-Ad4-△V-EGFP, ccdB-Kan targeting fragment primers, and seamless cloning reagent.

[0016] In some embodiments, the sequence of pBR322-Ad4-EGFP is shown in SEQ ID No. 11.

[0017] In some embodiments, the sequence of pR6K-ccdB-Kan is shown in SEQ ID No. 12.

[0018] In some embodiments, the sequence of pBR322-Ad4-ΔV-EGFP is shown in SEQ ID No. 13.

[0019] According to the seventh aspect of the technical solution of the present invention, the present invention provides an application of the above-mentioned mutant strain in screening anti-adenovirus drugs, and establishes a high-throughput drug evaluation model using the replication defect phenotype of the mutant strain.

[0020] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects: (1) Precise and efficient technology: By using Red / ET homologous recombination and ccdB reverse screening, the precise knockout of the protein V coding region was achieved. Without damaging other functional elements, Ad4-△V plasmids with clear genetic background and highly homozygous sequence were obtained, and the construction success rate was high.

[0021] (2) Outstanding safety: The constructed protein V knockout mutant strain exhibits a "replication defective" phenotype, showing obvious replication impairment in HEK-293 cells, with a significantly lower physical titer than the wild type, and the progeny virus completely loses its ability to infect again, eliminating the risk of environmental leakage and ensuring high safety.

[0022] (3) Enhanced immunogenicity: Mutant infection can specifically activate host cell innate immune response pathways, upregulate inflammatory factors such as CXCL8, IL6, and TNF, forming a "self-adjuvant" effect, which is suitable for vaccine development.

[0023] (4) The mechanism of action is clear: The molecular phenotypic characteristics of the knockout strain were revealed for the first time from the perspective of systems biology. It was found that the deletion of protein V can induce the downregulation of the host cell proteasome pathway and specifically activate the innate immune response, which provides important molecular evidence for elucidating its attenuation mechanism and assessing the safety of non-replicating adenovirus vectors.

[0024] (5) Wide range of applications: This replication-defective mutant can be used as a safe vaccine vector for the prevention of respiratory diseases (such as COVID-19 and influenza), or as a controllable gene therapy tool, and can also be used to establish a high-throughput antiviral drug screening model. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the construction strategy of the recombinant plasmid pBR322-Ad4-ΔV-EGFP. Note: Figure 1 The left side of the image shows the process of using the Red / ET recombination system to target and replace protein V with the ccdB-Kan resistance box; the right side shows the process of obtaining the knockout fragment ΔV using Overlap PCR; the ΔV fragment is then backfilled into the viral genome using Pac I / Spe I double digestion and seamless cloning technology to obtain the final recombinant plasmid.

[0026] Figures 2A-2E This is a graph showing the molecular biological identification results of the recombinant plasmid. Figure 2AColony PCR identification of the insertion status of the intermediate plasmid ccdB-Kan (M: DL5000; 1-5: positive clones, 2048 bp); Figure 2B PCR identification of overlap extension of the pV knockout fragment ΔV (M: DL5000; 1: ΔV fragment, 558 bp; 2: original fragment, 1584 bp). Figure 2C Electrophoresis image for BamH I restriction enzyme digestion identification of recombinant plasmid (M: DL15000; 1: original plasmid; 2: recombinant plasmid). Figure 2D The electrophoresis pattern of BamH I enzyme digestion simulated by SnapGene software; Figure 2E Sanger sequencing peak diagram for the knockout site.

[0027] Figure 3 This is a diagram showing the packaging and infection phenotype of the mutant strain in HEK-293 cells. Figure 3 Figure A shows the observation results of the Ad4-EGFP group during the adenovirus packaging stage (6 days after transfection); Figure 3 Figure B shows the observation results of the Ad4-ΔV-EGFP knockout strain during the adenovirus packaging stage (6 days after transfection). Figure 3 Figure C shows the results of the P0 generation progeny virus infection experiment (3 days post-infection) in the Ad4-EGFP group; Figure 3 Figure D shows the results of the P0 generation progeny virus infection experiment (10 days after infection) of the knockout strain Ad4-ΔV-EGFP group (the left side of each group is the fluorescence image, and the right side is the corresponding bright field image; magnification: 10×).

[0028] Figures 4A-4B This is a diagram showing the genome identification and titer analysis of the progeny virus of the mutant strain. Figure 4A PCR identification diagram of knockout regions in viral progeny genomes (M: DL5000; 1: Ad4-ΔV-EGFP P0 generation viral genome; 2: pBR322-Ad4-ΔV-EGFP plasmid control; 3: Ad4-EGFP P0 generation viral genome; 4: pBR322-Ad4-EGFP plasmid control). Figure 4B Molecular identification and titer analysis of the P0 generation viral genome. n=3; ***: indicates that the difference between the two groups is statistically significant (P<0.001).

[0029] Figures 5A-5E This is a map of the host cell transcriptome response characteristics. Figure 5A This is a volcano plot of differentially expressed genes in the Ad4-ΔV-EGFP group, with red representing upregulation and blue representing downregulation. Figure 5B Volcano plot of differentially expressed genes in the Ad4-EGFP group; Figure 5CThis is a differential protein-protein interaction (PPI) network, with node color representing log2FC value; the left side represents the Ad4-ΔV-EGFP group, and the right side represents the Ad4-EGFP group. Figure 5D Bubble diagram of KEGG pathway enrichment in the Ad4-ΔV-EGFP group; Figure 5E Bubble diagram of KEGG pathway enrichment in the Ad4-EGFP group. Detailed Implementation

[0030] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0031] This invention discloses a method for constructing a human adenovirus type 4 protein V knockout mutant and its application, belonging to the field of biotechnology. The method for constructing the human adenovirus type 4 protein V knockout mutant utilizes the Red / ET homologous recombination system combined with ccdB reverse selection technology to precisely knock out the protein V coding sequence in a whole-genome infectious clone of human adenovirus type 4, obtaining the recombinant plasmid pBR322-Ad4-△V-EGFP. After linearizing the plasmid, it is transfected into HEK-293 cells for virus packaging, resulting in a mutant strain with a viral particle physical titer of 4.76 × 10⁻⁶. 6 The human adenovirus type 4 protein V knockout mutant obtained in this invention has significantly reduced infectivity compared to the wild type, and the progeny viruses completely lose their infectivity. Transcriptome analysis shows that infection with this mutant strain can specifically downregulate the host proteasome pathway and activate the innate immune response. The mutant strain constructed in this invention has replication-defective characteristics, high safety, and strong immunogenicity, and can be used as an ideal attenuated vaccine vector or gene therapy tool.

[0032] The following description, in conjunction with the accompanying drawings and embodiments, illustrates the method for constructing the human adenovirus type 4 protein V knockout mutant strain disclosed in this invention and its application.

[0033] Example 1 1.1 Experimental Materials 1.1.1 Cell lines and strains Human embryonic kidney cells HEK-293 were preserved in our laboratory and passaged in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2 saturated humidity incubator. E. coli DH5α and DH10B chemocompetent cells were purchased from Beijing Qingke Biotechnology Co., Ltd. GB08red-gyr462 electrotransformation competent cells (possessing a Red / ET recombination system and responsive to...) were also purchased. ccdB (The lethal gene is insensitive) is preserved in this laboratory for homologous recombination modification of recombinant adenovirus plasmids.

[0034] 1.1.2 Plasmid Vectors The original plasmid used in this invention is pBR322-Ad4-EGFP (carrying a green fluorescent protein reporter gene, SEQ ID NO:11), which was constructed and preserved in our laboratory; the helper plasmid pR6K-ccdB-Kan (SEQ ID NO:12) is used to provide... ccdB Suicide genes and kanamycin resistance selection markers.

[0035] 1.2 Main Reagents Restriction endonucleases Spe I. Pac I. BamH I. Asis I. Purchased from NEB; 2×KeyPo Master Mix, 2×RapidTaq Master Mix, and 2×Taq Pro Universal SYBR qPCR Master Mix were purchased from Vazyme. DNA gel extraction kit and plasmid mini-preparation kit were purchased from UELandy; viral genome extraction was performed using the MiniBEST Viral RNA / DNA Extraction Kit Ver. 5.0, purchased from TaKaRa; seamless cloning kit was performed using the ClonExpress Ultra OneStep Cloning Kit, purchased from [unspecified source]. DMEM high-glucose medium and 0.25% Trypsin-EDTA digestion solution were purchased from EallBio; transfection reagents Lipofectamine 3000 and P3000 were purchased from Invitrogen.

[0036] 1.3 Primer Design and Synthesis Based on the pBR322-Ad4-EGFP sequence, targeting primers, overlap extension PCR primers, and identification primers were designed using SnapGene and Primer Premier 5.0 software. All primers were synthesized by Beijing Qingke Biotechnology Co., Ltd., and their sequences are detailed in Table 1.

[0037] Table 1. Primer sequences

[0038] 1.4 Construction of the recombinant plasmid pBR322-Ad4-ΔV-EGFP (SEQ ID No. 13) This invention employs a two-step Red / ET homologous recombination technique combined with seamless cloning technology for plasmid modification. The specific construction strategy is as follows: Figure 1 As shown.

[0039] 1.4.1 Construction of intermediate plasmid pBR322-Ad4-ccdB 500 ng of pBR322-Ad4-EGFP plasmid was transformed into GB08red-gyr462 competent cells using electroporation (electroporation parameters: 1.8 kV, 25 μF, 200 Ω). Positive clones were obtained by selection with ampicillin (100 μg / mL). After single colony amplification, when the OD600 of the bacterial culture reached 0.3-0.4, 10% L-arabinose was added, and Red recombinase expression was induced at 37℃ for 40 min. Subsequently, GB08red-gyr462-Ad4-EGFP electroporated competent cells were prepared.

[0040] Using pR6K-ccdB-Kan plasmid as a template, the ccdB-Kan targeting fragment was amplified by PCR. Primers carried 50 bp upstream and downstream homologous arm sequences of the protein V gene at both ends. After gel electroporation purification, 500 ng of the PCR product was electroporated into the induced competent cells. Homologous recombination mediated by the Red recombination system was used to replace the protein V coding region in the viral genome with the ccdB-Kan fragment. Selection was performed using kanamycin (50 μg / mL) resistance and... ccdB Positive clones were selected based on the lethal pressure of the gene on susceptible strains. The intermediate plasmid pBR322-Ad4-ccdB was obtained through colony PCR identification and sequencing verification.

[0041] 1.4.2 Obtaining the knockout fragment ΔV The fusion fragment (ΔV) without the protein V coding sequence was obtained using overlap extension PCR. First, using pBR322-Ad4-EGFP as a template, the upstream fragment A and downstream fragment B of protein V were amplified separately, with primers V-up-R and V-down-F containing a 40 bp complementary overlap sequence. Then, using the recovered mixture of fragments A and B as a template, a second round of amplification was performed using the outer primers V-up-F / V-down-R to obtain the full-length ΔV fragment. This fragment was cloned into the pTA vector and transformed into DH5α, and the sequence was confirmed to be completely correct by sequencing.

[0042] 1.4.3 Screening and Identification of Recombinant Plasmids use Pac I and Spe I. The intermediate plasmid pBR322-Ad4-ccdB was digested with two restriction endonucleases to remove the ccdB-Kan fragment, and the linearized vector backbone was recovered. Using a seamless cloning kit, the linearized vector was ligated to the ΔV PCR fragment at a molar ratio of 1:4. The ligation product was transformed into DH10B competent cells, and after recovery, plated on ampicillin LB agar plates. Single colonies were randomly picked for preliminary screening using culture PCR. Subsequently, plasmids were extracted and used... BamHI. Enzyme digestion identification was performed, and the actual enzyme digestion band patterns were compared with the theoretical bands simulated by SnapGene software. Finally, Sanger sequencing was performed on the protein V knockout region and its flanking adapter sequences to ensure that there were no base mutations or frameshifts, and the correct recombinant plasmid pBR322-Ad4-ΔV-EGFP was finally obtained.

[0043] 1.5 Adenovirus Packaging and Cell Transfection 1.5.1 Plasmid linearization and purification To release the infectious adenovirus genome, restriction endonuclease is used. AsiS I. Linearization of recombinant plasmids pBR322-Ad4-ΔV-EGFP and pBR322-Ad4-EGFP. The reaction mixture was as follows: 4 μg plasmid DNA, 5 μL 10×CutSmart Buffer, 2 μL... AsiS I. Enzyme: Add ddH2O to 50 μL and digest in a 37℃ water bath for 4 h. The digestion product was purified using the classic phenol-chloroform-isoamyl alcohol (25:24:1) extraction method, followed by the addition of 2.5 volumes of anhydrous ethanol and 1 / 10 volume of sodium acetate (3 mol / L), and precipitated overnight at -80℃. After centrifugation at 12,000 rpm for 20 min, the precipitate was washed twice with 75% ethanol and finally dissolved in 25 μL of sterile ddH2O to determine the DNA concentration.

[0044] 1.5.2 HEK-293 cell transfection and virus packaging One day prior to the experiment, HEK-293 cells were seeded in T12.5 cell culture flasks. Transfection was performed when the cell confluence reached 70%–80%. Before transfection, the culture medium was replaced with DMEM containing 2% FBS. First, 4 μg of linearized genomic DNA was diluted with 9 μL of P3000 reagent in 125 μL of Opti-MEM; simultaneously, 9 μL of Lipofectamine 3000 was diluted in 125 μL of Opti-MEM. After incubation at room temperature for 5 min, the two solutions were mixed and incubated in the dark for 20 min to form a transfection complex. The mixture was slowly added to the cell culture flasks and incubated at 37°C in a 5% CO2 incubator for 6 h, after which the medium was replaced with DMEM containing 5% FBS.

[0045] 1.5.3 Virus Collection and Preparation of P0 Generation Virus Solution Following transfection, the expression of green fluorescent protein (EGFP) and cytopathic effect (CPE) in each group of cells were observed daily using an inverted fluorescence microscope. The initial appearance time of fluorescence and the evolution of fluorescence intensity were recorded. When cells exhibited obvious CPE (such as cell rounding, detachment, and grape-like aggregation) and the fluorescence intensity reached its peak, the cell culture flasks were subjected to repeated freeze-thaw cycles at -80℃ and 37℃ three times to ensure complete release of viral particles from the cells. The viral solution was collected, centrifuged at 12,000 rpm for 5 min at 4℃, and the supernatant was filtered through a 0.22 μm filter membrane for sterilization to obtain P0 generation viral solution, which was then aliquoted and stored at -80℃ for long-term storage.

[0046] 1.6 Preliminary identification of viral infectivity To verify the effect of protein V knockout on the infectivity of progeny viruses, the prepared Ad4-ΔV-EGFP and Ad4-EGFP P0 generation viral solutions were inoculated into newly formed HEK-293 cells (approximately 80% confluence). After the cells were replaced with DMEM medium containing 2% FBS, 50 μL of viral solution was inoculated into each flask. After 6 h, the medium was replaced with complete medium containing 5% FBS. Green fluorescence expression and CPE were observed and recorded for each group.

[0047] 1.7 Viral genome extraction and molecular identification 200 μL of P0 generation virus solution was used to extract viral genomic DNA using the MiniBEST Viral RNA / DNA Extraction Kit. PCR amplification and verification were performed using primers V_F and V_R. The PCR reaction system (20 μL) consisted of: 10 μL of 2×KeyPo MasterMix, 1 μL each of forward and reverse primers, 1 μL of viral DNA template, and 7 μL of ddH2O. Reaction conditions were: 95℃ for 3 min; 95℃ for 15 s, 58℃ for 5 s, 72℃ for 20 s, for a total of 35 cycles; a final extension at 72℃ for 5 min. The product was identified by 1% agarose gel electrophoresis.

[0048] 1.8 Real-time quantitative PCR (qPCR) determination of viral titer The plasmid pBR322-Ad4-EGFP containing the target fragment was serially diluted 10-fold (10... 1 -10 8Standards were constructed, and a standard curve was established. Primers VqPCR_F and VqPCR_R were designed targeting the conserved fiber regions of the virus. The qPCR reaction system (20 μL) consisted of: 10 μL of 2×Taq Pro Universal SYBR qPCR Master Mix, 1 μL each of forward and reverse primers, 1 μL of DNA template, and 7 μL of ddH2O. Reaction conditions were: 95℃ for 30 s; 95℃ for 10 s, 58℃ for 30 s, for a total of 40 cycles, with fluorescence signals collected during the extension phase. The genome copy number (copies / μL) of the P0 generation virus was calculated based on the Ct value and the standard curve.

[0049] 2 Results 2.1 Construction and Molecular Identification of Recombinant Plasmid pBR322-Ad4-ΔV-EGFP according to Figure 1 The construction strategy shown in this invention first utilizes Red / ET homologous recombination technology to... ccdB-Kan An expression cassette was introduced into the viral genome to replace the coding region of protein V. Colony PCR identification results showed ( Figure 2A The positive clones obtained by kanamycin screening all amplified a clear specific band at approximately 2048 bp, confirming that the intermediate plasmid pBR322-Ad4-ccdB had been successfully constructed.

[0050] Subsequently, the protein V knockout fragment ΔV was prepared using overlap PCR technology. The amplified product ΔV band was located at 558 bp ( Figure 2B The amplified fragment size was exactly as expected; while the control group, using the Ad4-EGFPP genome without protein V knockout as a template, had an amplified fragment size of 1584 bp. The electrophoretic bands proved that the protein V coding region (1026 bp) had been successfully removed.

[0051] Furthermore, the ΔV fragment is backfilled into the system using seamless cloning technology. Pac I and Spe I. In the linearized vector backbone after double enzyme digestion. To verify the molecular characteristics of the final recombinant plasmid pBR322-Ad4-ΔV-EGFP, the following was used: BamH I. Enzyme digestion and identification were performed. The experimental results showed that ( Figure 2C The actual distribution of enzyme digestion bands of the recombinant plasmid and the electrophoresis diagram simulated by SnapGene software ( Figure 2D The complete correspondence proves that no accidental recombination of the plasmid backbone has occurred.

[0052] Finally, Sanger sequencing analysis was performed on the protein V knockout region and its flanking adapter sequences. Sequencing peak diagram ( Figure 2EThe results showed that the ΔV fragment was precisely coupled to the vector backbone, the sequence characteristics on both sides of the knockout site were completely consistent with the design scheme, and no base mutations or frameshifts were found. The above molecular biological evidence collectively confirms that this invention successfully constructed a whole-genome infectious cloning plasmid of HAdV-4 with protein V knockout.

[0053] 2.2 Effects of protein V knockout on HAdV-4 packaging efficiency and progeny viral infectivity 2.2.1 Protein V knockout significantly delays the viral packaging process. The linearized Ad4-ΔV-EGFP recombinant plasmid genome and the Ad4-EGFP genome were transfected into HEK-293 cells to initiate the viral packaging process. On day 6 post-transfection, fluorescence microscopy revealed... Figure 3 Ad4-EGFP cells exhibited severe cytopathic effects (CPE), with numerous cells becoming rounded, detaching, and exhibiting typical "grape-like" aggregations. High-density green fluorescence signals were visible across the entire field of view, indicating that the virus was replicating and packaging efficiently. Figure 3 A).

[0054] In contrast, the cell morphology of the knockout Ad4-ΔV-EGFP group remained largely normal at the same time point, and no obvious CPE phenomenon was observed; fluorescence observation showed that only a single cell expressed fluorescence ( Figure 3 B). This phenomenon indicates that the knockout of protein V significantly inhibits the assembly and release of progeny virus particles, resulting in a substantial decrease in packaging efficiency.

[0055] 2.2.2 Protein V knockout leads to loss of functional infectivity in progeny viruses To further verify the infectivity of the P0 generation virus solution generated during packaging, a back-pass infection experiment was conducted. The results showed that on day 3 after infection of HEK-293 cells with Ad4-EGFP P0 generation virus, diffuse strong green fluorescence and significant CPE were observed, indicating that the progeny virus had successfully completed cross-cell infection and amplification. Figure 3 C).

[0056] However, in the Ad4-ΔV-EGFP P0 generation infection group, cells maintained a healthy morphology throughout day 3 and subsequent continuous observations, showing no signs of CPE and no detectable green fluorescence expression. Even when the observation period was extended to day 10, this group still showed no viral replication signals. Figure 3 D). The above results strongly demonstrate that protein V is a structural protein essential for HADV-4 to complete its full life cycle, and its knockout results in the complete loss of functional infectivity of the progeny viral particles.

[0057] 2.3 Molecular identification and titer analysis of progeny viral genomes 2.3.1 PCR Validation of the P0 generation virus genome knockout region Agarose gel electrophoresis results showed ( Figure 4A The band amplified using the Ad4-ΔV-EGFP P0 generation viral genome as a template was approximately 1107 bp, which was completely consistent with the amplification results of the recombinant plasmid template (pBR322-Ad4-ΔV-EGFP). In contrast, the bands amplified from the Ad4-EGFP viral genome and the corresponding original plasmid control group were both 2133 bp. This significant difference in band size (knockout of approximately 1026 bp) confirms that the protein V coding region was successfully knocked out in the progeny viral genome produced during packaging, and that the virus's genetic characteristics remained stable during packaging.

[0058] 2.3.2 Protein V knockout led to a significant decrease in progeny viral titers. The titer of P0 generation virus solution was determined using qPCR absolute quantification. First, a standard curve was constructed using serially diluted standard plasmids, and the resulting linear regression equation was y = 3.009 x + 35.107, coefficient of determination R 2 = 0.993.

[0059] Quantitative results show ( Figure 4B The titer of the Ad4-EGFP P0 generation virus was 3.47 × 10⁻⁶. 7 The titer of Ad4-ΔV-EGFP P0 generation virus was only 5.87 × 10⁻⁶ copies / μL, while the titer of Ad4-ΔV-EGFP P0 generation virus was only 5.87 × 10⁻⁶ copies / μL. 6 The number of copies / μL decreased by nearly an order of magnitude compared to the former. Combined with the aforementioned fluorescence observation results, this quantitative data further confirms that although the knockout of protein V did not completely block viral genome replication, it greatly limited the packaging efficiency of mature viral particles, resulting in a significant reduction in progeny virus yield.

[0060] Example 2: Transcriptome response analysis of host cells to HAdV-4 infection Transcriptome sequencing and preliminary bioinformatics analysis: (1) Sample preparation and sequencing On day 6 after transfection and packaging, cells from the HEK-293 group, Ad4-EGFP group, and Ad4-ΔV-EGFP group were collected. The culture medium was aspirated, and the cells were washed twice with pre-chilled PBS. Then, 1 mL of Trizol reagent was added to each group of cells, and after complete lysis, the cells were transferred to RNase-free centrifuge tubes and sent to Beijing Qingke Biotechnology Co., Ltd. on dry ice for subsequent sequencing.

[0061] (2) Data Analysis Raw sequencing data were aligned to the human reference genome (GRCh38), and differentially expressed genes (DEGs) were screened based on a fold change of ≥ 2. Subsequently, relevant bioinformatics tools were used to perform clustering heatmaps, GO enrichment analysis, and KEGG signaling pathway analysis.

[0062] To elucidate the molecular mechanism by which protein V knockout leads to decreased HAdV-4 packaging efficiency and loss of infectivity in progeny viruses from a systems biology perspective, this invention performed transcriptome sequencing analysis on host cells from various groups. Figure 5A -E). Volcano map analysis shows ( Figure 5A (B) Compared to the 2989 differentially expressed genes (DEGs) induced by the Ad4-EGFP group, the knockout strain Ad4-ΔV-EGFP group ( Figure 5A This induced more drastic changes in the host transcriptional profile, with a total of 9045 DEGs (4648 upregulated and 4397 downregulated), suggesting that protein V knockout impairs the stability of the viral core structure, making it more susceptible to premature exposure of viral DNA and inducing large-scale cellular stress. Further analysis using protein-protein interaction (PPI) networks revealed... Figure 5C The knockout group (left) induced a highly concentrated pro-inflammatory cytokine interaction network centered on CXCL8, IL6, and TNF, with significantly higher inflammatory response activation intensity than the Ad4-EGFP group, confirming the key role of protein V in shielding viral DNA recognition and suppressing host innate immunity. At the functional enrichment level ( Figure 5D ,E), knockout group ( Figure 5D The virus exhibits unique pathway response characteristics: on the one hand, proteasome-related pathways show a significant downregulation trend, which may interfere with the host ubiquitin-proteasome system-mediated uncoating process, causing progeny viruses to lose their functional infectivity due to their inability to initiate nucleus entry; on the other hand, compared with the Ad4-EGFP group ( Figure 5E The knockout strain exhibits a severe hijacking of host metabolism and cell cycle pathways. The expression of cell cycle-related genes in the knockout group is relatively stable, showing transcriptional characteristics that are more similar to those of normal cells. This decline in the ability to take over host cells explains, at the molecular level, the biological phenotype of the knockout strain with low packaging efficiency and slow toxicity.

[0063] In summary, this invention first provides a method for the precise deletion of the protein V gene in human adenovirus type 4 (HAdV-4): using Red / ET homologous recombination technology, the coding sequence for protein V is precisely knocked out in infectious clones of the entire HADV-4 genome. This method, by introducing the ccdB suicide gene for reverse screening, ensures the acquisition of highly pure ΔV mutant plasmids without damaging other functional regions of the genome.

[0064] This invention also discovered that the absence of protein V leads to a "replication-deficient" phenotype in HADV-4: the HADV-4ΔV mutant strain constructed using this technique exhibits significant replication impairment in conventional HEK-293 cells. Experiments confirmed that although this mutant strain can package viral particles containing the genome, its physical titer is reduced by about an order of magnitude compared to the wild type, and the progeny viruses completely lose their ability to infect new cells in the second generation.

[0065] Finally, this invention utilizes transcriptome sequencing technology to systematically elucidate the remodeling characteristics of host cellular physiological pathways caused by the deletion of Protein V type 4 human adenovirus. Analysis confirmed that this gene deletion can induce downregulation of the host proteasome pathway and specific activation of the innate immune response pathway. This discovery clarifies the molecular phenotypic trend of this recombinant strain from a systems biology perspective, providing crucial data and molecular criteria for assessing the biosafety of non-replicating adenovirus vectors and host interaction mechanisms. This invention not only deepens our understanding of the pathogenic mechanism of HAdV-4 but also provides new strategic considerations for the prevention and control of outbreaks of adenovirus infection.

[0066] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles 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 method for constructing a human adenovirus type 4 protein V knockout mutant strain, characterized in that, It includes the following steps: (1) Using Red / ET homologous recombination technology, the ccdB-Kan selection frame was inserted into the protein V coding region of the HAdV-4 whole-genome infectious clone to obtain the intermediate plasmid pBR322-Ad4-ccdB; (2) The protein V knockout fragment ΔV was synthesized using overlap extension PCR technology; (3) The fragment △V was backfilled into the enzyme-digested linearized pBR322-Ad4-ccdB vector using seamless cloning technology to obtain the recombinant plasmid pBR322-Ad4-△V-EGFP.

2. The construction method according to claim 1, characterized in that, The targeting primers used in the Red / ET homologous recombination contain 50 bp sequences of upstream and downstream homologous arms of the protein V gene.

3. The construction method according to claim 1, characterized in that, The ccdB reverse screening system achieves positive clone screening through kanamycin resistance and the lethal effect of the ccdB gene on sensitive bacteria.

4. A human adenovirus type 4 protein V knockout mutant strain constructed according to the construction method of any one of claims 1-3.

5. The application of the human adenovirus type 4 protein V knockout mutant strain constructed according to claim 4 in the preparation of an attenuated live vaccine, characterized in that, The human adenovirus type 4 protein V knockout mutant strain has replication-defective characteristics and can induce high expression of inflammatory factors in host cells.

6. The application of the human adenovirus type 4 protein V knockout mutant strain constructed according to claim 4 in a gene therapy vector, characterized in that, The mutant strain achieves controlled release of the viral genome by interfering with the proteasome pathway.

7. A method for detecting the biological characteristics of a protein V knockout mutant constructed by the construction method according to any one of claims 1-3, characterized in that, include: (1) Quantitative analysis of viral physical titer using qPCR; (2) Verify the infectivity of the progeny virus through a back-infection test; (3) The enrichment of differentially expressed genes and pathways in host cells was analyzed by transcriptome sequencing.

8. The method according to claim 7, characterized in that, The qPCR detection targets the conserved region of the viral fiber gene, and the primer sequences are shown in SEQ ID NO:9-10.

9. A kit for constructing protein V knockout mutant strains, characterized in that, It contains the recombinant plasmid pBR322-Ad4-△V-EGFP, ccdB-Kan targeting fragment primers, and seamless cloning reagent as described in claim 1.

10. The application of the human adenovirus type 4 protein V knockout mutant strain constructed according to claim 4 in screening anti-adenovirus drugs, characterized in that, A high-throughput drug evaluation model was established using the replication-deficient phenotype of this mutant strain.

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