CN-122012623-A - Screening platform, screening method and application for optimizing baculovirus bidirectional promoter distance

Abstract

The invention belongs to the technical field of bioengineering, and discloses a screening platform, a screening method and application for optimizing the distance between baculovirus bidirectional promoters. The invention constructs a high resolution screening platform integrating reference genes into a virus skeleton, and by double-stage screening, a transcription enhancement region is firstly screened in a 98-245 bp region in a rough screening manner, then a 1bp step is used for fine screening in the transcription enhancement region, errors such as infection efficiency and the like are corrected by reference fluorescent signal normalization, and the optimal interval length between p10 and polh promoters is determined to be 153bp. The interval configuration is applied to a recombinant adeno-associated virus (rAAV) packaging system, can relieve transcription interference between bidirectional promoters, restore expression balance of Rep and Cap proteins, improve replication efficiency of rAAV genome by 3 times, reduce empty shell rate from 84.9% to 20.1%, and maintain transduction efficiency in mammalian cells.

Inventors

• NAN HAO
• LAN LAN
• QU YUCHEN
• CHEN XIYAN
• HAN YONGPING
• Song Wangcheng
• XU XIAODONG

Assignees

• 西北农林科技大学深圳研究院

Dates

Publication Date

20260512

Application Date

20260414

Claims (10)

1. 1. A screening platform for optimizing the distance between two-way promoters of baculovirus is characterized by comprising a recombinant baculovirus skeleton and a transfer vector carrying a two-way fluorescence test unit; The recombinant baculovirus skeleton is a bacmid skeleton containing an internal reference fluorescent protein expression element; The transfer vector comprises an expression cassette formed by arranging a p10 promoter and a polh promoter in a back-to-back mode, and an alternative interval sequence slot is arranged between transcription initiation sites of the p10 promoter and the polh promoter.
2. 2. The screening platform of claim 1, wherein the two-way fluorescence test unit is site-directed integrated at the polh locus of the bacmid scaffold.
3. 3. The screening platform of claim 1, wherein in the two-way fluorescence test unit, the p10 promoter drives mScarlet expression and the polh promoter drives emiRFP670 expression.
4. 4. A high resolution screening method for baculovirus bi-directional promoter spacing optimization using the screening platform of claim 1, comprising the steps of: (1) Inserting spacer sequences with different lengths into spacer sequence slots of a transfer vector of the screening platform, constructing a transfer vector variant library, and co-transfecting host cells with the transfer vector variant library and a recombinant baculovirus skeleton to prepare recombinant baculovirus; (2) First, carrying out first-stage coarse screening within the length range of an interval sequence of 98-245 bp, and after determining a transcription enhancement interval, constructing a transfer vector variant library in the transcription enhancement interval by taking 1bp as a stepping increment to carry out second-stage fine screening; (3) Infecting a test cell with the recombinant baculovirus, and detecting an internal reference fluorescent signal in the cell and a reporter gene fluorescent signal of a bidirectional fluorescent test unit; (4) Normalizing the average fluorescence intensity of the reporter gene by using the internal reference fluorescence signal; (5) And drawing a transcription activity curve according to the normalized data, and determining the optimal distance between the bidirectional promoters.
5. 5. The method of claim 4, wherein in step (1), the host cell is an Sf9 insect cell.
6. 6. The method of claim 4, wherein in step (2), the step length of the coarse screen is 5 to 20bp.
7. 7. The method of claim 4, wherein in step (3), the detection of the fluorescent signal is accomplished by flow cytometry.
8. 8. A recombinant adeno-associated virus expression system optimized based on the screening platform of claim 1, which is characterized by comprising a reverse double expression cassette for driving the co-expression of Rep protein and Cap protein, wherein the expression cassette consists of a p10 promoter and a polh promoter which are arranged back to back, and the length of a spacer sequence between transcription initiation sites of the p10 promoter and the polh promoter is 153bp.
9. 9. The expression system of claim 8, wherein the spacer sequence is used to relieve transcriptional interference between bi-directional promoters and restore stoichiometric balance of expression of Rep proteins and Cap proteins.
10. 10. A method of reducing the empty rate of recombinant adeno-associated virus in a baculovirus system, wherein recombinant adeno-associated virus particles are produced in Sf9 insect cells using the recombinant adeno-associated virus expression system of claim 8.

Description

Screening platform, screening method and application for optimizing baculovirus bidirectional promoter distance Technical Field The invention belongs to the technical field of bioengineering, and relates to a screening platform, a screening method and application for optimizing baculovirus bidirectional promoter intervals. Background The baculovirus expression vector system (baculovirus expression vector system, BEVS) is a eukaryotic expression system which is widely applied at present, can realize high-level expression by means of the extremely late promoter driving exogenous genes of baculovirus, supports posttranslational modification, can accommodate large-fragment DNA insertion, and has good biological safety. Aiming at the requirement of multi-protein co-expression, a back-to-back bi-directional promoter double expression system constructed based on p10 and polh promoters becomes the mainstream design in the field, and corresponding commercial vectors have been widely applied to gene therapy vector production, vaccine research and development and expression scenes of complex functional proteins, wherein in the industrial production of recombinant adeno-associated viruses, the system is used for synchronously expressing Rep and Cap proteins so as to support the assembly preparation of virus particles. However, the existing bidirectional promoter architecture has inherent technical defects, when two promoters are physically close to each other, an active transcription machine on the promoters can generate space obstruction and transcription interference, so that the transcription levels of genes at two sides are unbalanced, and the accurate stoichiometric expression of target proteins cannot be realized. The defect causes a remarkable industrial bottleneck in the production of recombinant adeno-associated viruses, the unbalanced expression proportion of Rep and Cap proteins can lead a large amount of Cap proteins to be assembled into empty capsids without therapeutic genome, the empty capsids usually exceed 80 percent in the prior art, the functional titer of effective viruses can be diluted, unnecessary host immune response can be initiated, and the cost and the operation difficulty of downstream purification links are greatly improved. Aiming at the problems, the prior art tries to relieve transcription interference by adjusting the distance between promoters, but related optimization schemes depend on traditional design-construction-test cycles, only a few discrete interval lengths can be preset for construction and test one by one, the overall flux is extremely low, the continuous variation range of the interval lengths cannot be covered, the real optimal distance parameters are difficult to locate, meanwhile, the existing screening schemes lack effective internal reference correction mechanisms, carrier copy number differences, cell infection efficiency fluctuation and errors among batches can seriously interfere with the accuracy of detection results, and finally only static optimization results under a specific experiment system can be obtained, so that the universality is poor, the accurate optimization requirements under different application scenes cannot be met, and a general and high-flux systematic screening scheme is needed in the field so as to realize the accurate optimization of the distance between bidirectional promoters, and the core technical bottleneck of the existing double-expression system is broken through. Disclosure of Invention The invention aims to solve the problem of unbalanced target protein expression caused by transcription interference among promoters in the existing baculovirus bi-directional promoter double expression system, and simultaneously overcomes the defects of low flux, lack of an effective correction mechanism, poor accuracy of detection results and insufficient generality of the existing optimization scheme, thereby relieving the industrial bottleneck brought by high-altitude rate in the industrial production of recombinant adeno-associated viruses and providing a general and high-flux systematic solution for the interval optimization of bi-directional promoters. In a first aspect, the invention provides a screening platform for optimizing baculovirus bi-directional promoter spacing, the platform comprising a recombinant baculovirus backbone and a transfer vector carrying a bi-directional fluorescence test unit; The recombinant baculovirus skeleton is a bacmid skeleton containing an internal reference fluorescent protein expression element; The transfer vector comprises an expression cassette formed by arranging a p10 promoter and a polh promoter in a back-to-back mode, and an alternative interval sequence slot is arranged between transcription initiation sites of the p10 promoter and the polh promoter. Further, in the above screening platform, the two-way fluorescence test unit is site-directed integrated at the polh locus of the baculoskeletal framework