A screening platform for optimizing the distance of baculovirus bidirectional promoters, a screening method and application thereof

Abstract

The application belongs to the technical field of bioengineering, and discloses a screening platform for optimizing the interval of baculovirus bidirectional promoters, a screening method and application. The application constructs a high-resolution screening platform with a reference gene integrated in a virus skeleton, and through two-stage screening, first, a transcription enhancement interval is determined by rough screening in the 98-245bp interval, and then, fine screening is performed in the transcription enhancement interval by 1bp steps, so that the optimal interval length of the p10 and polh promoters is determined as 153bp by normalizing the reference fluorescent signal to correct errors such as infection efficiency. The interval is applied to a recombinant adeno-associated virus (rAAV) packaging system, transcription interference between the bidirectional promoters is eliminated, the expression balance of Rep and Cap proteins is restored, the rAAV genome replication efficiency is improved by 3 times, the empty shell rate is reduced from 84.9% to 20.1%, and the transduction efficiency in mammalian cells can be maintained.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to a screening platform, screening method and application for optimizing the bidirectional promoter spacing of baculoviruses. Background Technology

[0002] Baculovirus expression vector systems (BEVS) are currently widely used eukaryotic expression systems. They can drive exogenous genes to achieve high-level expression using the very late promoters of baculoviruses, while also supporting post-translational modifications, accommodating large DNA insertions, and exhibiting good biocompatibility. For the need for multi-protein co-expression, back-to-back bidirectional promoter dual-expression systems based on the p10 and polh promoters have become the mainstream design in this field. Corresponding commercial vectors have been widely used in gene therapy vector production, vaccine development, and the expression of complex functional proteins. In the industrial production of recombinant adeno-associated virus (AAV), this system is used to simultaneously express Rep and Cap proteins to support the assembly and preparation of viral particles.

[0003] However, existing bidirectional promoter architectures have inherent technical flaws. When the two promoters are physically close together, the active transcription machinery on the promoters can cause spatial obstruction and transcriptional interference, leading to an imbalance in the transcriptional levels of the genes on both sides and making it impossible to achieve precise chemometric expression of the target protein. This flaw has caused a significant industrial bottleneck in the production of recombinant adeno-associated viruses. An imbalance in the expression ratio of Rep and Cap proteins can lead to a large number of Cap proteins assembling into empty capsids without therapeutic genomes. Under current processes, the rate of such empty capsids typically exceeds 80%, which not only dilutes the functional titer of the effective virus but may also trigger unnecessary host immune responses, while significantly increasing the cost and operational difficulty of downstream purification.

[0004] To address the aforementioned issues, existing technologies attempt to mitigate transcriptional interference by adjusting promoter spacing. However, most optimization schemes rely on traditional design-build-test cycles, allowing only a few discrete interval lengths to be preset for individual construction and testing. This results in extremely low overall throughput, failing to cover the continuous variation range of interval lengths and making it difficult to pinpoint the truly optimal spacing parameter. Furthermore, existing screening schemes lack effective internal reference correction mechanisms. Differences in vector copy number, fluctuations in cell infection efficiency, and batch-to-batch errors severely interfere with the accuracy of detection results, ultimately yielding only static optimization results for specific experimental systems. This lack of versatility fails to meet the precise optimization needs of different application scenarios. Therefore, there is an urgent need in this field for a universal, high-throughput, systematic screening scheme to achieve precise optimization of bidirectional promoter spacing and overcome the core technical bottlenecks of existing dual-expression systems. Summary of the Invention

[0005] This invention aims to solve the problem of target protein expression imbalance caused by transcriptional interference between promoters in existing bidirectional promoter dual-expression systems of baculoviruses. At the same time, it overcomes the shortcomings of existing optimization schemes, such as low throughput, lack of effective correction mechanisms, poor accuracy of detection results, and insufficient universality. This will alleviate the industrial bottleneck caused by the high empty shell rate in the industrial production of recombinant adeno-associated virus and provide a universal, high-throughput systematic solution for optimizing the spacing of bidirectional promoters.

[0006] In a first aspect, the present invention provides a screening platform for optimizing the bidirectional promoter spacing of baculoviruses, the platform comprising: a recombinant baculovirus backbone and a transfer vector carrying a bidirectional fluorescence assay unit;

[0007] The recombinant baculovirus backbone is a rod-particle backbone containing an internal reference fluorescent protein expression element; The transfer vector contains an expression cassette consisting of a p10 promoter and a polh promoter arranged back-to-back, with replaceable spacer slots between the transcription start sites of the p10 promoter and the polh promoter.

[0008] Furthermore, in the screening platform described above, the bidirectional fluorescence testing unit is site-specifically integrated into the polh locus of the rod granule backbone.

[0009] Furthermore, in the screening platform described above, in the bidirectional fluorescence testing unit, the p10 promoter drives mScarlet expression, and the polh promoter drives emiRFP670 expression.

[0010] Secondly, the present invention provides a high-resolution screening method for optimizing the bidirectional promoter spacing of baculoviruses using the above-mentioned screening platform, comprising the following steps: (1) Insert spacer sequences of different lengths into the spacer sequence slots of the transfer vector in the screening platform to construct a transfer vector variant library, and co-transfect the transfer vector variant library with the recombinant baculovirus backbone into host cells to prepare recombinant baculovirus; (2) First, a coarse screening is performed within the interval sequence length range of 98~245bp. After determining the transcription enhancement interval, a transfer vector variant library is constructed in the transcription enhancement interval with a step increment of 1bp for the second stage of fine screening. (3) Infect test cells with the recombinant baculovirus and detect the internal control fluorescence signal and the reporter gene fluorescence signal of the bidirectional fluorescence test unit in the cells; (4) The average fluorescence intensity of the reporter gene was normalized using the internal reference fluorescence signal; (5) Plot transcriptional activity curves based on the normalized data to determine the optimal spacing between bidirectional promoters.

[0011] Furthermore, in step (1) of the above high-resolution screening method, the host cell is an Sf9 insect cell.

[0012] Furthermore, in step (2) of the above high-resolution screening method, the step length of the coarse screening is 5~20bp.

[0013] Furthermore, in step (3) of the above high-resolution screening method, the fluorescence signal is detected by flow cytometry.

[0014] The aforementioned screening platform or high-resolution screening method can be applied to the production of recombinant adeno-associated virus (rAAV) and generate practical industrial value. Based on this: Thirdly, the present invention provides a recombinant adeno-associated virus expression system optimized based on the above-mentioned screening platform, which includes an inverted dual expression cassette driving the co-expression of Rep protein and Cap protein. The expression cassette is composed of a p10 promoter and a polh promoter arranged back to back. The length of the spacer sequence between the transcription start sites of the p10 promoter and the polh promoter is 153 bp.

[0015] Furthermore, in the aforementioned recombinant adeno-associated virus expression system, the spacer sequence is used to relieve transcriptional interference between bidirectional promoters and restore the stoichiometric balance of Rep and Cap proteins.

[0016] Fourthly, the present invention provides a method for reducing the empty shell rate of recombinant adeno-associated virus in a baculovirus system: using the above-described recombinant adeno-associated virus expression system to produce recombinant adeno-associated virus particles in Sf9 insect cells.

[0017] Compared with the prior art, the present invention has the following significant advantages: Significantly improves screening accuracy, efficiency, and reliability: Achieves high-resolution interval scanning in 1bp increments, locks the transcription enhancement region of 133-161bp through a wide range of coarse screening of 98-245bp, and then precisely locates the optimal interval of 153bp through fine screening of 140-160bp, breaking through the resolution limitation of traditional 10-20bp coarse screening; the same vector internal reference normalization design eliminates systematic errors such as vector copy number and infection efficiency, improves the consistency and reproducibility of screening results, and the high-throughput cloning and detection strategy also significantly improves screening throughput and shortens experimental time.

[0018] Significantly improves rAAV production quality: effectively eliminates transcriptional interference between bidirectional promoters, achieving precise stoichiometric balance expression of two genes; in rAAV production, genome replication efficiency is increased by 3 times, empty capsid rate is reduced from 84.9% to 20.1%, Rep / Cap protein expression level is significantly improved, effective viral titer is increased, and the optimized rAAV also shows significantly enhanced in vitro transduction efficiency in various cell types such as HEK293T and HeLa.

[0019] Promising prospects for industrial application: It constructs a closed-loop process from screening to production validation, and laboratory screening results can be directly applied to industrial process scale-up, reducing intermediate steps and uncertainties; the accumulated high-resolution activity-spacing data can support the development of machine learning prediction models, and the screening platform and screening methods provide templates for the general optimization of baculovirus dual expression systems, expanding the industrial application of BEVS in gene therapy, vaccine development and other fields. Attached Figure Description

[0020] Figure 1 This diagram illustrates the grouping of Sf9 insect cells co-infected with three different baculovirus vectors to compare the effects of different promoter configurations on transgene expression. The left side shows the negative control construct (ITR+Cap), containing the Cap gene driven by the p10 promoter (vAc / p10-Cap) and the mCherry fluorescent reporter gene flanking the ITR (vAc / ITR-mCherry). The middle group is the segregation group (ITR+Cap+Rep), containing three independent baculoviruses (vAc / ITR-mCherry, vAc / p10-Cap, vAc / p10-Rep), driven by either the EF-1α or p10 promoter, respectively. The right side is the integration group (ITR+Cap-Rep), containing a back-to-back bidirectional promoter cassette (vAc / p10-Rep-polh-Cap), with the Rep and Cap genes driven by the p10 and polh promoters, respectively. All constructs are based on the BAC10:KO1629 baculovirus backbone and the polh site integration cassette.

[0021] Figure 2 Bar graphs were used to analyze the Rep-dependent genome replication efficiency in three constructs using quantitative real-time PCR (qPCR). The x-axis represents the three experimental groups (ITR+Cap control, ITR+Cap+Rep, and ITR+Cap-Rep), and the y-axis represents the relative copy number of the replicating genome targeting the ITR-flanking mCherry gene. Error bars represent mean ± SD (n=3); ns indicates no significant difference. This indicates that p < 0.001. The result indicates that p < 0.0001 is the result of one-way ANOVA combined with Tukey's multiple comparison test.

[0022] Figure 3 This is a quantitative analysis graph showing the relative mRNA expression levels of the Rep gene (left) and Cap gene (right) in the three constructs. The horizontal axis represents the experimental group (ITR+Cap control group, ITR+Cap+Rep, ITR+Cap-Rep), and the vertical axis represents the relative mRNA expression level. Error bars represent mean ± SD (n=3). Significance markers have the same meaning as above. Figure 2 .

[0023] Figure 4 SDS-PAGE electrophoresis images of Cap protein expression levels in different constructs. Lanes correspond to the different experimental groups mentioned above, and black arrows indicate the VP3 protein (Cap protein detection marker) band; molecular weight standards (kDa) are labeled on the right.

[0024] Figure 5 Western blotting results show the expression levels of Rep and Cap proteins in different constructs. Lanes correspond to different experimental groups; the left side labels the positions and molecular weight (kDa) of the Rep78, Rep52, and VP1 / 2 / 3 protein bands; β-tubulin is the loading control.

[0025] Figure 6 Transmission electron microscopy (TEM) images of rAAV particles generated from the ITR+Cap-Rep construct. Intact viral particles and empty capsid particles are visible in the images; the percentage of empty capsid particles in this field of view is 84.9 ± 3.0%; original magnification × 50,000, scale bar 100 nm.

[0026] Figure 7 This is a schematic diagram of the recombinant baculovirus structure for a dual-fluorescent reporter system, used to simulate a back-to-back promoter architecture. The core expression frame is “mScarlet←p10 promoter-spacer sequence-polh promoter→EGFP”, where the spacer sequence is the DNA fragment between the transcription start sites (TSS) of the p10 and polh promoters; mScarlet and EGFP are bidirectional fluorescent reporter genes.

[0027] Figure 8Bar graphs quantifying the mean fluorescence intensity (MFI) of p10-driven mScarlet and polh-driven EGFP at 3 dpi, 4 dpi, and 5 dpi post-infection for different interval lengths (98–245 bp). The x-axis represents the interval length (bp), and the y-axis represents the MFI value; different colored bars represent the fluorescence intensity of mScarlet and EGFP, respectively; error bars represent mean ± SD (n=3).

[0028] Figure 9 Scatter plots showing the correlation between mScarlet and EGFP fluorescence intensity at 3 dpi, 4 dpi, and 5 dpi. The x-axis represents the MFI value of mScarlet, and the y-axis represents the MFI value of EGFP; each point represents the detection result at one interval length; the Pearson correlation coefficient (r) and p-value are labeled in the plot to reflect the coupling of dual promoter expression.

[0029] Figure 10 This is a bar graph showing the MFI quantification of EGFP and mScarlet at 3 dpi within an interval length of 141–161 bp. The x-axis represents the interval length (bp), and the y-axis represents the MFI value; different colored bars represent the expression intensity of the two fluorescent proteins; error bars represent mean ± SD (n=3).

[0030] Figure 11 This is a scatter plot showing the correlation between mScarlet and EGFP fluorescence intensities at 3 dpi. The x-axis represents the MFI value of mScarlet, and the y-axis represents the MFI value of EGFP. Pearson correlation coefficients (r) and p-values ​​are labeled to verify the synergistic regulatory effect of interval variation on the dual promoter.

[0031] Figure 12 This is a schematic diagram of a dual-fluorescence reporter system for high-resolution screening. The core expression box is “mScarlet←p10 promoter-spacer sequence-polh promoter→emiRFP670”; the construct is based on the BAC10:KO1629 / EGFP baculovirus backbone, which encodes a GFP biomarker as an internal control to reflect viral infection progress; the test unit and the internal control unit are integrated into the polh site of the same vector.

[0032] Figure 13 This is a bar graph showing the MFI quantification of mScarlet and emiRFP670 at 3 dpi within an interval length of 140–160 bp (1 bp increments). The x-axis represents the interval length (bp), and the y-axis represents the MFI value. Different colored bars represent the expression intensities of the two fluorescent proteins. Error bars represent mean ± SD (n=3), showing the periodic peaks of transcriptional activity.

[0033] Figure 14 The scatter plot shows the correlation between the fluorescence intensities of mScarlet and emiRFP670 at 3 dpi. The x-axis represents the MFI value of mScarlet, and the y-axis represents the MFI value of emiRFP670. Pearson correlation coefficients (r=0.8180) and p<0.0001 are labeled to confirm the expression coupling of the two reporter genes.

[0034] Figure 15 This is a schematic diagram of the experimental groups for traditional and optimized bidirectional promoter architectures in rAAV production. On the left is ITR+Cap-Rep-105, containing a standard back-to-back p10-Rep-polh-Cap cassette with a 105bp spacer (derived from the commercial pFastBac-Dual vector); on the right is ITR+Cap-Rep-153, containing an optimal 153bp spacer. Both groups are integrated into the polh site of the BAC10:KO1629 / EGFP baculovirus backbone and tandem with the mCherry reporter cassette flanking the ITR.

[0035] Figure 16 This is a quantitative RT-qPCR analysis of the relative mRNA expression levels of the Rep gene (left) and Cap gene (right) in the traditional and optimized groups. The horizontal axis represents the experimental group (ITR+Cap control group, ITR+Cap+Rep, ITR+Cap-Rep-105 traditional group, ITR+Cap-Rep-153 optimized group), and the vertical axis represents the relative mRNA expression level. Error bars represent mean ± SD (n=3); ns indicates no significant difference. This indicates that p < 0.01. The result indicates that p < 0.0001 is the result of one-way ANOVA combined with Tukey's multiple comparison test.

[0036] Figure 17 This is an SDS-PAGE electrophoresis image showing the expression levels of Cap protein in the conventional and optimized groups. Lanes correspond to the two experimental groups; black arrows indicate the VP3 protein band; molecular weight standards (kDa) are labeled on the right.

[0037] Figure 18 This image shows Western blotting results for Rep and Cap protein subtypes in the conventional and optimized groups. Lanes correspond to the two experimental samples; the left side labels the positions and molecular weight (kDa) of the Rep78, Rep52, and VP1 / 2 / 3 protein bands; β-tubulin serves as the loading control.

[0038] Figure 19 This is a qPCR-quantified bar chart of rAAV genome replication efficiency in the conventional and optimized groups. The horizontal axis represents the experimental group, and the vertical axis represents the relative DNA copy number of the mCherry gene targeting the ITR flanking region. Error bars represent mean ± SD (n=3). The result indicates that p < 0.0001, which is the result of a two-way ANOVA combined with Tukey's multiple comparison test.

[0039] Figure 20 TEM images of rAAV particles were generated for the optimization group (ITR+Cap-Rep-153). High-density, well-structured viral particles are visible in the images; the empty capsid percentage is indicated as 20.1±1.8%; original magnification ×50,000, scale bar 100 nm.

[0040] Figure 21 Fluorescence microscopy images of rAAV generated in the conventional and optimized groups during functional transduction in four mammalian cell types. The x-axis represents cell type (HEK293T, HeLa, AC16 human cardiomyocytes, SH-SY5Y neuroblastoma cells); the y-axis represents experimental group. Fluorescence intensity reflects the expression level of the mCherry transgene, indicating rAAV transduction efficiency; the multiplicity of infection (MOI) is 1 × 10⁻⁶. 5 Capsids / cells were photographed 48 hours after transduction. Detailed Implementation

[0041] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0042] Unless otherwise specified, the experimental and detection methods in the following embodiments are conventional methods; the reagents and materials mentioned are commercially available unless otherwise specified; and the index data are measured using conventional methods unless otherwise specified.

[0043] The experimental resource information involved in all embodiments of this patent is summarized as follows: I. Materials, Reagents and Equipment Cell lines and culture media: Sf9 or Hi5 insect cells (from a reliable supplier); Sf-900 III SFM or equivalent serum-free insect cell culture media; fetal bovine serum (added only if necessary).

[0044] The vector is a Bacmid:pQAV4 transfer vector (or an equivalent Bac-to-Bac compatible vector), BAC10:KO1629, or BAC10:KO1629 / EGFP (an example Bacmid with an EGFP internal reference).

[0045] Reporter genes: mScarlet, EGFP, emiRFP670 (or other validated fluorescent proteins); the reporter gene sequence should contain a stop codon and have a suitable polyA / transcription termination signal (such as SV40 polyA or viral polyA) at the 3' end.

[0046] Molecular biology reagents: high-fidelity DNA polymerase (e.g., Phusion), Gibson Assembly kit, restriction endonucleases, ligases, DNA purification kits, oligonucleotide synthesis services, bacterial strains (e.g., DH5α), and culture media.

[0047] Virus and titer assays: cell culture consumables, qPCR reagents and primers (for viral genome quantification), TCID50 or fluorescent titer assay reagents.

[0048] Flow cytometer: Equipped with at least three color detection channels, compatible with excitation / emission wavelengths: EGFP (488nm / ~530nm), mScarlet (561nm / ~610nm), emiRFP670 (633nm / ~670nm).

[0049] Software and data processing: FlowJo or equivalent streaming data analysis software, R / Python for data preparation and curve fitting (LOESS, ANOVA), GraphPad Prism for statistics and graphing.

[0050] Table 1: Relevant Primer Sequences

[0051] Example 1 Construction of a coarse screening platform for promoter-interval transcription enhancement regions mediated by a dual-fluorescence reporter system.

[0052] I. Experimental Methods (I) Construction of dual-fluorescent reporter vector To clarify the limitations of the traditional back-to-back starter architecture, three sets of components are first set up (such as...). Figure 1As shown): The left side represents the Rep-deficient group (ITR+Cap, containing only the Cap gene driven by the p10 promoter and the mCherry reporter gene flanking the ITR, negative control group, see...). Figure 1 The control group is marked in the middle; the segregation group (ITR+Cap+Rep, with Rep and Cap genes produced by independent promoters) is in the middle; and the integration group (ITR+Cap-Rep, with the p10 and phlh interval being the traditional 105 bp, and Rep and Cap genes co-expressed via a traditional promoter cassette) is on the right. All constructs are based on the BAC10:KO1629 baculovirus backbone. Based on this, a dual fluorescent reporter vector was constructed to screen for transcriptionally enhanced regions.

[0053] 1. Expression Frame Design: Constructing the expression frame structure of a dual-fluorescent reporter vector (e.g., Figure 7 As shown in the figure, the specific sequence is “mScarlet←p10 promoter-spacer sequence-polh promoter→EGFP”, where the spacer sequence is defined as the DNA fragment between the transcription start sites (TSS) of the two promoters. mScarlet and EGFP serve as transcriptional activity reporter genes for p10 and polh promoters, respectively, to achieve simultaneous detection of bidirectional transcription efficiency.

[0054] 2. Spacer sequence design: Using a step size of 14 bp, spacer sequences of different lengths covering 98~245 bp were designed. For each spacer sequence, 2~3 variants with different base compositions were designed to eliminate sequence-specific interference.

[0055] 3. Vector cloning: The vector was constructed using Gibson seamless cloning technology. The specific steps are as follows: (1) Using the pQAV vector as a backbone, after linearization, ensure that the backbone has 20~30nt linker sequences that are complementary to the spacer sequence at both ends; (2) The p10 promoter, polh promoter, mScarlet gene, EGFP gene and spacer sequences of various lengths were amplified by PCR. The amplification primers are shown in Table 1. The amplification products were recovered by a DNA purification kit. (3) Mix the vector and insert fragments at a molar ratio of 1:3, add Gibson Assembly reaction solution, and incubate at 50°C for 30 min to complete assembly; (4) The assembly product was transformed into DH5α competent Escherichia coli, and single clones were picked for colony PCR identification. Positive clones were sent for Sanger sequencing verification to ensure that the promoter sequence, spacer length and reporter gene sequence were correct. After the sequencing verification was qualified, the positive clones were amplified and cultured and plasmids were extracted to obtain dual fluorescent reporter vectors.

[0056] (II) Preparation of recombinant baculovirus 1. Preparation of P0 generation virus: Sf9 cells were prepared at a concentration of 1×10⁻⁶. 6 Cells / mL were seeded into 6-well plates and cultured for 24 h. Then, co-transfected with FuGENE HD transfection reagent at a ratio of 2 μg linearized BAC10:KO1629 backbone + 3 μg recombinant dual fluorescent reporter vector per well. After transfection, the cells were incubated in a cell culture incubator at 27 °C for 5 days. The supernatant was collected and centrifuged at 500 × g for 10 min to remove cell debris, and the P0 generation recombinant baculovirus stock solution was obtained.

[0057] 2. P1 generation virus amplification: Sf9 cells were seeded in 100 mm cell culture dishes and allowed to grow until the cell density reached 0.8 × 10⁻⁶ cells / mL. 6 At a concentration of cells / mL, P0 generation virus solution was added at a multiplicity of infection (MOI) of 0.05 and incubated at 27°C for 4 days. The supernatant was collected and purified by centrifugation at 1000×g for 15 min to obtain P1 generation recombinant baculovirus stock solution. The viral titer was determined by qPCR. The primer sequences were qPCR-gp41-F: 5'-CGTAGTGGTAGTAATCGCCGC-3' (SEQ ID NO:73) and qPCR-gp41-R: 5'-AGTCGAGTCGCGTCGCTTT-3' (SEQ ID NO:74).

[0058] (III) Cell infection and fluorescence detection 1. Infection treatment: Inoculate Sf9 cells at a rate of 1×10⁻⁶ 6 Cells were seeded at a density of 1 mL / well in 24-well plates. After the cells adhered, P1 generation virus solution corresponding to each length of the spacer sequence was added at an MOI of 3. Three biological replicates were set up for each spacer length, and uninfected cells were set up as a negative control.

[0059] 2. Culture and sampling: After infection, cells were placed in a 27°C incubator for static culture, and cell samples were collected at 3 days (dpi), 4 days (dpi), and 5 days (dpi) post-infection.

[0060] 3. Flow cytometry analysis: Cells were collected at each time point, washed twice with PBS, and the cell concentration was adjusted to 1×10⁻⁶. 6 cells / mL; mean fluorescence intensity (MFI) of mScarlet (excitation wavelength 561 nm, emission wavelength 610 nm) and EGFP (excitation wavelength 488 nm, emission wavelength 530 nm) was detected by flow cytometry, with at least 10,000 cell events collected for each sample; data preprocessing was performed using FlowJo software to remove cell debris and two-cell interference.

[0061] (iv) Data Statistical Analysis Data were processed using GraphPad Prism 8 software, and the results are expressed as mean ± standard deviation (mean ± SD). The differences in fluorescence intensity among groups with different interval lengths were analyzed by one-way ANOVA combined with Tukey's multiple comparison test, and the significance level was set at P < 0.05.

[0062] II. Test Results (I) Construction and Validation of Dual-Fluorescent Reporter Vector Sanger sequencing results showed that the p10 promoter, polh promoter, mScarlet gene, and EGFP gene sequences of all recombinant dual fluorescent reporter vectors were consistent with the design. The actual lengths of the nine spacer sequences were exactly the same as the preset lengths, and there were no base mutations, deletions, or insertions. The vectors were successfully constructed.

[0063] (ii) Detection of recombinant baculovirus titer qPCR results showed that the titers of each P1 generation recombinant baculovirus were all within 1×10⁻⁶. 8 ~5×10 8 There was no statistically significant difference in titers between groups at PFU / mL (P>0.05), which meets the requirements for subsequent unified MOI infection experiments. To clarify the limitations of traditional back-to-back promoter architectures, the Rep-dependent genome replication efficiency (e.g., ...) of three other constructs was simultaneously tested. Figure 2 As shown in the figure): the horizontal axis represents the three experimental groups, the vertical axis represents the relative copy number of the replicating genome targeting the mCherry gene, and the error bars represent mean ± SD (n=3). The result, p < 0.0001, indicates that the replication efficiency of the integration group (ITR+Cap-Rep) was significantly lower than that of the segregation group (ITR+Cap+Rep), confirming that the traditional promoter proximity inhibits Rep output, which provides a basis for subsequent screening of transcription enhancement regions.

[0064] (III) Determination of transcription enhancement regions Flow cytometry results showed that dual-fluorescent reporter vectors with different spacing lengths could drive the expression of mScarlet and EGFP in Sf9 cells, but the fluorescence intensity showed a significant non-linear change with the spacing length. 1. When the interval length is 98bp and 105bp, the MFI values ​​of mScarlet and EGFP are relatively low. Specifically, the mScarlet-MFI of the 98bp group at 3dpi is 152333±12915 and the EGFP-MFI is 397226±19562. The corresponding MFI values ​​of the 105bp group are 168907±2777 and 356865±5054, which are consistent with the expression level of the traditional commercial vector pFastBac-Dual with an interval length of 105bp.

[0065] Transcription level detection results of traditional 105bp spacer vectors are as follows Figure 3 As shown, the left side represents the relative mRNA expression levels of the Rep gene, and the right side represents the relative mRNA expression levels of the Cap gene. The p < 0.0001 indicates that the expression levels of Rep and Cap mRNA in this group (105 bp interval) were extremely low. However, the mRNA expression level of the vector in the 133-161 bp interval was significantly increased, further verifying the transcriptional enhancement effect of this interval.

[0066] 2. When the interval length is in the range of 133~161bp, the fluorescence intensity quantification results detected by flow cytometry are as follows: Figure 8 As shown, the horizontal axis represents the interval length from 98 to 245 bp, and the vertical axis represents the mean fluorescence intensity (MFI). Different colored bars represent the expression intensity of mScarlet and EGFP, respectively, and the error bars represent mean ± SD (n=3). It can be seen that the MFI values ​​of the two fluorescent proteins in the 133-161 bp interval are significantly higher than those in other interval lengths, and remain stable at the three time points of 3 dpi, 4 dpi, and 5 dpi, clearly indicating that this interval is a transcriptional enhancement window.

[0067] SDS-PAGE analysis showed (e.g.) Figure 4 As shown in the figure, the black arrow indicates the VP3 protein (Cap protein detection marker) band. The VP3 band of the traditional integrase is extremely weak, while the VP3 band of the vector in the 133~161bp range is significantly brighter, which directly reflects the difference in Cap protein expression level and is consistent with the fluorescence intensity detection results.

[0068] Western blotting further validated (e.g.) Figure 5 As shown in the figure, the left side indicates the location and molecular weight (kDa) of the Rep78, Rep52, and VP1 / 2 / 3 protein bands. β-tubulin is used as a loading control. The Rep78, Rep52, and Cap proteins in the traditional integration group are almost undetectable, while the target protein band of the vector in the 133~161bp range is clear, confirming that this range can effectively alleviate transcriptional interference.

[0069] 3. When the interval length exceeds 161bp (175bp, 200bp, 220bp, 245bp), the MFI values ​​of the two fluorescent proteins gradually decrease. Among them, the MFI value of the 245bp group is not significantly different from that of the 98bp group (P>0.05).

[0070] TEM observations showed that the proportion of empty capsids in the traditional integrase was as high as 84.9 ± 3.0% (e.g. Figure 6 As shown in the figure (scale bar is 100nm, original magnification × 50,000), when the vector in the 133~161bp range was subsequently used for rAAV production, the empty capsid rate was significantly reduced, indirectly confirming the transcriptional enhancement value of this range.

[0071] (iv) Correlation analysis of dual fluorescence expression Pearson correlation analysis was performed on mScarlet-MFI and EGFP-MFI at each time point (3 dpi, 4 dpi, 5 dpi). Figure 9 The x-axis represents the MFI value of mScarlet, and the y-axis represents the MFI value of EGFP. The results showed that the expression of the two fluorescent proteins was strongly positively correlated in all interval length groups, with correlation coefficients r greater than 0.86 (P < 0.0001), indicating that the p10 promoter and the polh promoter have intrinsic coupling characteristics in their back-to-back structure, and transcriptional interference affects the output of the two promoters proportionally in the form of system-level constraints.

[0072] Example 2 Construction of a 1bp resolution promoter spacing fine screening platform with internal reference normalization.

[0073] I. Experimental Methods (I) Construction of high-resolution dual-fluorescent reporter vector 1. Expression Frame Design: Construct a back-to-back bidirectional promoter dual-fluorescent reporter vector with internal parameters (see structural diagram). Figure 12 The core expression frame is “mScarlet←p10 promoter-spacer sequence-polh promoter→emiRFP670”. The vector backbone integrates the EGFP internal reference expression unit of BAC10:KO1629 / EGFP. A short insulating sequence is inserted between the test unit and the internal reference unit to avoid expression interference. The spacer sequence is still defined as the DNA fragment between the transcription start sites (TSS) of the two promoters.

[0074] 2.1bp Step-spacer Sequence Library Construction: Based on the 133-161bp transcriptional enhancement region determined in Example 1, focusing on the 140-160bp range, 31 spacer sequences of different lengths (140bp, 141bp, ..., 160bp) were designed in 1bp steps. Primer-sliding PCR was used to directly generate insert fragments of different lengths. Primer design ensured that the pairing region with the vector was ≥25nt to guarantee the specificity of PCR amplification. Two variants with different base compositions were designed for each length to eliminate sequence-specific interference.

[0075] 3. Vector Cloning and Validation: Using Gibson seamless cloning technology, the vector was assembled at a molar ratio of 1:3 to the insert fragment. After transformation into DH5α competent E. coli, single clones were selected for colony PCR identification. Positive clones were first verified for spacer length and sequence accuracy using Sanger sequencing, and then the integrity of the entire 140-160 bp spacer library was verified using an NGS sequencing platform to ensure that there were clones with the correct sequence at each length point.

[0076] (II) Preparation of recombinant baculovirus 1. Preparation of P0 generation virus: Recombinant dual fluorescent reporter vectors with 140~160bp intervals were co-transfected with the BAC10:KO1629 / EGFP linearized baculovirus backbone into Sf9 cells. The transfection conditions were the same as in Example 1 (2 μg backbone + 3 μg vector per well, FuGENE HD transfection reagent mediated). After incubation at 27℃ for 5 days, the supernatant was collected by centrifugation at 500×g for 10 min to obtain the P0 generation recombinant baculovirus stock solution.

[0077] 2. P1 generation virus amplification and titer standardization: P1 generation virus was amplified according to the method in Example 1. The titers of all P1 generation viruses were determined by qPCR (targeting the baculovirus gp41 gene, primer sequences as in Example 1), ensuring that the titers of all variants were within 1×10⁻⁶. 8 ~5×10 8 The PFU / mL range was uniformly diluted to the same concentration according to the titer for subsequent unified MOI infection.

[0078] (III) Cell infection and multifluorescence detection 1. Infection treatment: Inoculate Sf9 cells at a rate of 1×10⁻⁶ 6 Cells were seeded at a density of 1 mL / well in 24-well plates. After the cells adhered, diluted P1 generation virus solution was added at an MOI of 3. Three biological replicates were set up for each interval length. Uninfected cells were set up as a negative control. The infection conditions were the same as in Example 1.

[0079] 2. Culture and sampling: Cells were cultured statically at 27°C and collected at 3 dpi post-infection (which was verified by preliminary experiments as the peak time point of late promoter activity).

[0080] 3. Flow cytometry analysis: Collect infected cells, wash twice with PBS buffer, and adjust the cell concentration to 1×10⁻⁶. 6 cells / mL; three fluorescence signals were simultaneously detected by flow cytometry: mScarlet (excitation wavelength 561 nm, emission wavelength 610 nm), emiRFP670 (excitation wavelength 633 nm, emission wavelength 670 nm), and EGFP internal control (excitation wavelength 488 nm, emission wavelength 530 nm). At least 10,000 cellular events were collected from each sample, and gating was performed using FlowJo software (FSC / SSC to exclude debris, and FSC-A vs FSC-H dual-parameter gating to exclude double cells), and the mean fluorescence intensity (MFI) of each fluorescence channel was recorded.

[0081] (iv) Internal reference normalization and data processing 1. Normalization Calculation: To eliminate systematic errors such as virus infection efficiency and cell growth status, the detection signal of each sample is normalized using internal references. The calculation formula is as follows: Normalized mScarlet fluorescence intensity (Ratio1) = MFI mScarlet / MFI EGFP ; Normalized emiRFP670 fluorescence intensity (Ratio2) = MFI emiRFP670 / MFI EGFP .

[0082] 2. Statistical Analysis: GraphPad Prism 8 software was used, and the results are expressed as "normalized Ratio ± standard deviation (mean ± SD)". One-way ANOVA combined with Tukey's multiple comparison test was used to analyze the differences in normalized Ratio values ​​among groups with different interval lengths, and the significance level was set at P < 0.05. At the same time, Pearson correlation analysis was performed to evaluate the association between the expression levels of the two reporter genes mScarlet and emiRFP670.

[0083] II. Test Results (I) Construction and Validation of High-Resolution Vector Library Sanger sequencing results showed that the 31 vectors with 1bp step intervals in the range of 140~160bp had interval lengths that were completely consistent with the preset lengths, with no base deletions, insertions or mutations. Library integrity verification using the NGS platform showed that the accuracy of vector sequences at all length points was ≥99%, with no omissions or erroneous clones, and the 1bp step interval sequence library was successfully constructed.

[0084] (ii) Improvement of data stability by internal reference normalization Unnormalized data showed that the original MFI of mScarlet and emiRFP670 vectors with different interval lengths fluctuated to some extent, and some differences between groups were difficult to distinguish due to experimental errors. After normalization with EGFP internal control, the data dispersion was significantly reduced (the coefficient of variation decreased from 12.3% to 3.7%), which effectively eliminated the systematic errors caused by uneven viral infection efficiency and cell state fluctuations, and ensured that the detected expression differences were only caused by changes in interval length.

[0085] (III) Determination of the optimal spacing at 1bp resolution Analysis of the normalized ratio values ​​showed that the expression of mScarlet and emiRFP670 exhibited synchronous nonlinear fluctuations within the 140–160 bp interval. 1. When the interval length is 140~146bp, the normalized Ratio value gradually increases; obvious periodic transcriptional activity peaks appear in the 147~153bp interval, where Ratio1 (normalized mScarlet) reaches its maximum value (3.73±0.05) and Ratio2 (normalized emiRFP670) also reaches its peak value (4.70±0.08) at 153bp, which are significantly higher than other interval length groups (P<0.001); when the interval length is 154~160bp, the normalized Ratio value gradually decreases.

[0086] Fluorescence quantification results in the fine region of 141~161bp are as follows: Figure 10 As shown, the horizontal axis represents the interval length, and the vertical axis represents the MFI value. Different colored bars represent the expression intensity of EGFP and mScarlet, respectively, and the error bars represent mean ± SD (n=3). It can be seen that the MFI of the two fluorescent proteins exhibit synchronous nonlinear fluctuations, which locks in the key range for subsequent 1bp step-by-step fine screening and avoids blind screening.

[0087] 2.140~160bp 1bp step-screening fluorescence quantification results are as follows Figure 13As shown, the horizontal axis represents the interval length, and the vertical axis represents the MFI value. Different colored bars represent the expression intensity of mScarlet and emiRFP670, respectively. Error bars represent mean ± SD (n=3). The results show that transcriptional activity has obvious periodic peaks. At 153 bp, the normalized ratio values ​​of both reporter genes reached their peak, which was significantly higher than other length groups (p<0.001), clearly indicating that 153 bp is the optimal interval.

[0088] (iv) Correlation analysis of dual reporter gene expression To verify the expression coupling of dual promoters within the subdivided region, Pearson correlation analysis was performed on the fluorescence intensity in the 141–161 bp region (e.g., Figure 11 As shown in the figure, the horizontal axis represents the MFI value of mScarlet, and the vertical axis represents the MFI value of EGFP. The Pearson correlation coefficient is r=0.9004 and p=0.0002, which confirms that the interval variation has a synergistic consistency in the regulation of dual promoters, further supporting the scientific validity of the dual reporter gene system for transcriptional activity detection.

[0089] Pearson correlation analysis showed that among all spacer sequence variants with 1bp-1bp steps, mScarlet and emiRFP670 maintained a strong positive correlation in normalized ratio, with a correlation coefficient r = 0.8180 (P < 0.0001). Figure 14 As shown in the figure, the results are consistent with those of Example 1, indicating that the p10 promoter and the polh promoter are still intrinsically coupled transcriptional functional units in the back-to-back structure, and the interval length has a synergistic consistency in the regulation of the two promoters.

[0090] Example 3 Application of high-resolution screening platform in rAAV production (reducing empty shell rate).

[0091] I. Experimental Methods (I) Construction of rAAV packaging carrier 1. Vector Design: Two sets of rAAV packaging vectors were constructed, both with the core expression frame "Rep←p10 promoter-spacer sequence-polh promoter→Cap", and tandem with the mCherry reporter gene cassette flanking the ITR. All constructs were integrated into the polh site of the BAC10:KO1629 / EGFP baculovirus backbone. Figure 15 ).

[0092] 1) Optimized group (ITR+Cap-Rep-153): The spacer sequence length is 153bp, which is the optimal spacing determined in Example 2. The sequence was verified by Sanger sequencing.

[0093] 2) Traditional group (ITR+Cap-Rep-105): The interval sequence length is 105bp, which is the traditional commercial vector pFastBac-Dual, and serves as the existing industry benchmark.

[0094] 2. Vector Cloning and Validation: The Rep and Cap genes were amplified from the pAAV2 / 2 plasmid and inserted flanking the p10 and polh promoters, respectively, using Gibson seamless cloning technology. Simultaneously, spacer sequences of corresponding lengths and ITR-mCherry cassettes were inserted. In the cloning reaction, the molar ratio of vector to insert fragment was 1:3, and assembly was completed by incubation at 50℃ for 30 min. The assembly products were transformed into DH5α competent *E. coli*. Positive clones were selected and identified by colony PCR. Sanger sequencing confirmed that the promoter sequence, spacer length, Rep gene, Cap gene, and ITR sequence were free of base mutations, deletions, or insertions, and the sequence accuracy met the design requirements.

[0095] (II) Preparation of recombinant baculovirus and production of rAAV 1. Preparation of recombinant baculovirus: The optimized and conventional vectors were co-transfected into Sf9 cells with the BAC10:KO1629 / EGFP linearized backbone (2 μg backbone + 3 μg vector per well in a 6-well plate). Transfection was mediated by FuGENE HD transfection reagent. After incubation at 27°C for 5 days, the supernatant was collected by centrifugation at 500×g for 10 min to obtain P0 generation recombinant baculovirus. Sf9 cells in 100 mm cell culture dishes were infected with the P0 generation recombinant baculovirus at a multiplicity of infection (MOI) of 0.05. After incubation at 27°C for 4 days, P1 generation virus was amplified. The titer was determined by qPCR (targeting the baculovirus gp41 gene), ensuring that the titers of both groups were within 1×10⁻⁶. 8 ~5×10 8 Within the PFU / mL range.

[0096] 2. rAAV production and purification: Sf9 cells were produced at a concentration of 1×10⁻⁶ cells / mL. 6Cells were seeded at a density of 100 mm cells / mL in 100 mm cell culture dishes and co-infected with recombinant baculovirus expressing ITR, Cap, and Rep at an MOI ratio of 3. Cells were harvested after 5 dpi at 27°C. Cells were lysed under three freeze-thaw cycles (lysis buffer: 500 mM NaCl, 2 mM MgCl2, 50 mM Tris-HCl, pH 8.0). 50 U / mL benzo[a]nuclease was added to the lysis buffer, and the cells were digested at 37°C for 30 min. The supernatant was collected by centrifugation at 12000 × g for 30 min, filtered through a 0.22 μm filter, and purified by affinity chromatography using POROS CaptureSelect AAVX resin (Thermo Fisher Scientific, catalog number A36739). The purified rAAV was concentrated through a 100 kDa Amicon filter (Millipore, catalog number UFC8100), sterilized again through a 0.22 μm filter, and the purification efficiency was assessed by SDS-PAGE.

[0097] (III) rAAV performance testing 1. Transcriptional level detection (RT-qPCR): Sf9 cells were collected 5 days post-infection, total RNA was extracted and reverse transcribed into cDNA. Using cDNA as a template, the relative mRNA expression levels of Rep and Cap genes were detected by RT-qPCR. β-actin was used as an internal reference gene. - Ct The relative mRNA expression level was calculated using a method with three biological replicates per group.

[0098] 2. Protein level detection (SDS-PAGE and Western blotting): Sf9 cells were collected 5 dpi after infection, and SDS loading buffer (composition: 50 mM Tris-HCl, 2% SDS, 10% glycerol, 1.5 mM bromophenol blue, 100 mM DTT) was added. The cells were denatured at 95℃ for 5 min and centrifuged at 12,000×g for 1 min. Equal volumes of samples were subjected to 10% SDS-PAGE electrophoresis, then transferred to a PVDF membrane (Millipore, catalog number IPVH00010), blocked with 5% skim milk powder TBST buffer for 1 h, and incubated overnight at 4°C with primary antibodies (anti-Rep antibody 1:300 dilution, anti-VP1 / 2 / 3 antibody 1:300 dilution). After washing 3 times with TBST, secondary antibody (1:5000 dilution) was added and incubated at room temperature for 1 h. Finally, the samples were developed with Immobilon Western Chemiluminescent HRP Substrate (Millipore, catalog number WBKLS0100) and imaged using the ChemiDocXRS+ system. β-tubulin was used as a loading control.

[0099] 3. Genome replication efficiency assay (qPCR): Total DNA was extracted from Sf9 cells 5 days post-infection. Using the mCherry gene flanking the ITR as the target and the baculovirus gp41 gene as the reference gene, rAAV genome replication efficiency was quantified by qPCR. Primer sequences are as follows: qPCR-mCh-F: 5'-AGTTCATGTACGGCTCCAAG-3' (SEQ ID NO:75), qPCR-mCh-R: 5'-TTGTAGATGAACTCGCCGTC-3' (SEQ ID NO:76).

[0100] qPCR-gp41-F: 5'-CGTAGTGGTAGTAATCGCCGC-3' (SEQ ID NO: 73); qPCR-gp41-R: 5'-AGTCGAGTCGCGTCGCTTT-3' (SEQ ID NO:74). Three biological replicates were set up for each group.

[0101] 4. Empty Capsule Ratio Detection (TEM): The purified and concentrated rAAV sample (concentration approximately 10) was subjected to TEM. 13Capsids / mL (dissolved in PBS buffer) were added dropwise to a 200-mesh carbon-coated copper grid, incubated for 2 min, excess liquid was aspirated, and the grid was stained with 2% phosphotungstic acid negative staining solution for 2 min. The grid was washed three times with ultrapure water and air-dried at room temperature. The cells were observed and imaged using a transmission electron microscope (accelerating voltage 80 kV, magnification 50000×). Four independent fields of view were randomly selected, and at least 2000 virus particles were quantified to calculate the proportion of empty capsids.

[0102] 5. In vitro transduction efficiency assay: HEK293T, HeLa, AC16, and SH-SY5Y cells were seeded at 50% confluence in 96-well plates. Purified rAAV sample was added to each well, with a multiplicity of infection (MOI) of 1 × 10⁻⁶. 5 Capsids / cells. 48 hours post-transduction, mCherry fluorescence expression was observed and recorded using fluorescence microscopy to assess transduction efficiency.

[0103] (iv) Statistical Analysis Data processing was performed using GraphPad Prism 8 software, and results are expressed as mean ± standard deviation (mean ± SD). Differences between two groups were analyzed using independent samples t-tests, and differences among multiple groups were analyzed using one-way ANOVA combined with Tukey's multiple comparison test. The significance level was set at P < 0.05.

[0104] II. Test Results (I) Construction and Validation of rAAV Packaging Vector Sanger sequencing results showed that the p10 promoter, polh promoter, Rep gene, Cap gene, spacer sequence, and ITR-mCherry cassette sequence of the optimized group (ITR+Cap-Rep-153) and the conventional group (ITR+Cap-Rep-105) were consistent with the design, with spacer lengths of 153bp and 105bp, respectively. There were no base mutations, deletions, or insertions, and the vector was successfully constructed.

[0105] (ii) Increased transcription and protein expression levels 1. Transcription level: RT-qPCR results showed ( Figure 16 The expression levels of Rep and Cap gene mRNA in the traditional group (105bp) were extremely low, while the relative expression level of Rep mRNA in the optimized group (153bp) was significantly increased, reaching a level comparable to that of the isolated group (ITR+Cap+Rep) (P<0.0001). The relative expression level of Cap mRNA was also significantly higher than that in the traditional group (P<0.01), indicating that the 153bp interval effectively relieved the transcriptional interference of the bidirectional promoter.

[0106] 2. Protein levels: SDS-PAGE analysis showed ( Figure 17 The optimized group (153 bp) showed a clear VP3 protein band (indicated by the black arrow), while the conventional group (105 bp) showed almost no corresponding band. Western blotting results further confirmed this. Figure 18 The optimized group can efficiently express Rep protein isoforms (Rep78, Rep52) and Cap protein isoforms (VP1, VP2, VP3), and the band brightness is significantly higher than that of the traditional group. The Rep and Cap protein isoforms in the traditional group are almost undetectable, indicating that the 153bp interval achieves efficient synergistic expression of Rep and Cap proteins.

[0107] (iii) Improved genome replication efficiency qPCR test results showed ( Figure 19 The optimized group (153bp) showed significantly higher rAAV genome replication efficiency than the conventional group (105bp), with a relative DNA copy number approximately three times that of the conventional group (P<0.0001). This confirms that the 153bp interval effectively improved the rAAV genome replication efficiency by restoring the normal expression level of Rep protein.

[0108] (iv) The empty shell rate is significantly reduced. TEM observation results showed ( Figure 20 In the traditional group (105bp spacer), the proportion of empty capsids in the rAAV particles was as high as 84.9±3.0%, which is consistent with the empty capsid rate of the traditional back-to-back promoter architecture. In contrast, the rAAV particles in the optimized group (153bp spacer) had a complete structure and the proportion of empty capsids was only 20.1±1.8%, which was significantly lower than that of the traditional group (P<0.0001). This indicates that the 153bp spacer effectively inhibited the generation and accumulation of empty capsids by balancing the expression ratio of Rep protein and Cap protein.

[0109] (v) Maintaining in vitro transduction efficiency Fluorescence microscopy observation results show that ( Figure 21 In four mammalian cell lines—HEK293T, HeLa, AC16 (human cardiomyocytes), and SH-SY5Y (neuroblastoma)—the optimized group (153 bp) produced rAAVs that mediated stronger mCherry fluorescence expression, with significantly higher transduction efficiency than the conventional group (105 bp). This result is attributed to the reduced proportion of empty capsids in the optimized group, leading to a corresponding increase in the proportion of functional rAAV particles containing therapeutic genomes, thereby improving productive transduction efficiency.

[0110] The above embodiments can well illustrate the technical solution of the present invention, but they are only describing preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, all kinds of changes and improvements made by those skilled in the art to the technical solution of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A screening platform for optimizing the bidirectional promoter spacing of baculoviruses, characterized in that, include: Recombinant baculovirus backbone and transfer vector carrying a two-way fluorescence assay unit; The recombinant baculovirus backbone is a rod-particle backbone containing an internal reference fluorescent protein expression element; The transfer vector contains an expression cassette consisting of a p10 promoter and a polh promoter arranged back-to-back, with replaceable spacer slots provided between the transcription start sites of the p10 promoter and the polh promoter. The bidirectional fluorescence assay unit is site-specifically integrated into the polh locus of the rod granule skeleton, wherein the p10 promoter drives mScarlet expression and the polh promoter drives emiRFP670 expression. A short insulating sequence is inserted between the assay unit and the internal control unit to avoid expression interference.

2. A high-resolution screening method for optimizing bidirectional promoter spacing of baculoviruses using the screening platform described in claim 1, characterized in that, Includes the following steps: (1) Insert spacer sequences of different lengths into the spacer sequence slots of the transfer vector in the screening platform to construct a transfer vector variant library, and co-transfect the transfer vector variant library with the recombinant baculovirus backbone into Sf9 insect host cells to prepare recombinant baculovirus; (2) First, a coarse screening is performed in the interval sequence length range of 98~245bp. The step length of the coarse screening is 5~20bp. After determining the transcription enhancement interval, a transfer vector variant library is constructed in the transcription enhancement interval with a step increment of 1bp for the second stage of fine screening. (3) Infect test cells with the recombinant baculovirus and detect the internal control fluorescence signal and the reporter gene fluorescence signal of the bidirectional fluorescence test unit by flow cytometry; (4) The average fluorescence intensity of the reporter gene was normalized using the internal reference fluorescence signal; (5) Plot transcriptional activity curves based on the normalized data to determine the optimal spacing between bidirectional promoters.

3. A recombinant adeno-associated virus expression system optimized based on the screening platform described in claim 1, characterized in that, The expression cassette contains a reverse dual expression cassette that drives the co-expression of Rep and Cap proteins. The cassette consists of a p10 promoter and a polh promoter arranged back-to-back. The spacer sequence between the transcription start sites of the p10 promoter and the polh promoter is 153 bp in length.

4. The expression system according to claim 3, characterized in that, The spacer sequence is used to remove transcriptional interference between bidirectional promoters and restore the stoichiometric balance of Rep and Cap proteins.

5. A method for reducing the empty shell rate of recombinant adeno-associated virus in a baculovirus system, characterized in that, Recombinant adeno-associated virus particles were produced in Sf9 insect cells using the recombinant adeno-associated virus expression system of claim 3.

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