基于Rep蛋白定向进化与高通量筛选提高rAAV产量

doi:10.13560/j.cnki.biotech.bull.1985.2026-0320

摘要/Abstract

摘要：

目的对Rep蛋白编码基因进行随机突变，构建突变体文库并建立高通量筛选平台，以获得可提高rAAV产量的Rep蛋白突变体，并解析其作用机制。方法采用易错PCR构建Rep蛋白突变文库。利用带挡板24孔深孔板培养HEK293细胞并进行三质粒转染，以rAAV基因组滴度为指标，将产量接近或高于对照组80%的培养孔定义为候选突变组。对候选组中Rep蛋白突变体进行摇瓶复验，获得Rep蛋白有益突变体。通过分析rep基因和cap基因转录、Rep蛋白和Cap蛋白表达、rAAV转导效率，并基于AlphaFold3预测Rep78蛋白结构以及突变位点定位，解析突变体rAAV产量提高机制。结果基于高通量筛选与摇瓶验证获得2个Rep蛋白有益突变体P39L与Q202H，其rAAV产量分别提高至对照组的2.0倍与1.8倍。对Rep蛋白的P39位点与Q202位点进行饱和突变未进一步提高rAAV产量。进一步分析表明，Rep蛋白突变体P39L和Q202H未显著影响Rep蛋白与Cap蛋白的表达量及rAAV的转导效率。结构定位结果表明，P39位于起源结构域（origin-binding domain，OBD）区域，Q202位于OBD与同源异型结构域（homeodomain，HD）的过渡区域且靠近OBD。结论建立了Rep蛋白定向进化与高通量筛选技术，获得了可将rAAV产量分别提高至对照组的2.02倍和1.87倍的Rep蛋白有益突变体P39L和Q202H。OBD及其邻近区域是影响rAAV产量的重要敏感区域。

Abstract:

Objective This study aimed to perform random mutagenesis of the Rep-coding gene, construct a mutant library, and establish a high-throughput screening platform for the identification of Rep variants capable of enhancing rAAV production, and then elucidate their mechanism of action. Methods A Rep mutant library was constructed by error-prone PCR. HEK293 cells cultured in baffled 24-well deep-well plates were subjected to triple-plasmid transfection. Using rAAV genome titer as the screening criterion, culture wells with yields close to or exceeding 80% of the control group were selected as candidate mutant groups. Rep mutants from candidate groups were further validated in shake-flask cultures to obtain beneficial Rep variants. The underlying mechanisms of the enhanced rAAV production were investigated by analyzing rep/cap gene transcription, Rep/Cap protein expression, and rAAV transduction efficiency, as well as by predicting the Rep78 structure using AlphaFold3 and locating the mutation sites. Results Through high-throughput screening and shake-flask verification, we identified two beneficial Rep protein mutants, P39L and Q202H, which enhanced rAAV yield to 2.02-fold and 1.87-fold that of the control, respectively. Saturation mutagenesis at positions P39 and Q202 of the Rep protein did not further improve rAAV yield. P39L and Q202H did not significantly affect Rep/Cap expression levels or rAAV transduction efficiency. Structural localization indicated that P39 is located in the origin‑binding domain (OBD), while Q202 is situated in the OBD-HD (homeodomain) transition region, in proximity to the OBD. Conclusion This study established a directed evolution and high-throughput screening platform for Rep protein and identified two beneficial Rep mutants, P39L and Q202H, which enhanced rAAV yield to 2.02-fold and 1.87-fold that of the control, respectively. The results further indicate that OBD and its nearby regions are important sensitive areas affecting rAAV yield.

Key words: recombinant adeno-associated virus, Rep protein, random mutagenesis, high-throughput screening, viral yield

宋紫川, 任和, 莫婉婷, 聂简琪, 白仲虎. 基于Rep蛋白定向进化与高通量筛选提高rAAV产量[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0320.

SONG Zi-chuan, REN He, MO Wan-ting, NIE Jian-qi, BAI Zhong-hu. Enhancing rAAV Production through Directed Evolution and High-Throughput Screening of Rep Protein[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0320.

图/表 5

图1 Rep蛋白突变体高通量筛选与功能分析流程图

Fig. 1 Schematic workflow of high-throughput screening and functional analysis of Rep protein mutants

图2 Rep蛋白突变体高通量筛选及摇瓶验证A，B：候选突变组的rAAV产量；C：候选突变组中单点突变体摇瓶复验结果；D：候选突变组中多点组合突变体摇瓶验证结果。*表示P<0.05，**表示P<0.01，***表示P<0.001，ns表示差异无统计学意义，下同

Fig. 2 High-throughput screening and shake-flask validation of Rep protein mutantsA, B: rAAV yields of candidate mutant groups. C: Shake-flask validation results of single-point mutants from candidate mutant groups. D: Shake-flask validation results of multi-point combination mutants from candidate mutant groups. *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively; ns indicates no significant difference. The same below

图3 P39和Q202位点饱和突变体rAAV产量分析A：P39位点饱和突变体rAAV产量；B：Q202位点饱和突变体rAAV产量

Fig. 3 rAAV yields of saturation mutants at P39 and Q202 sitesA: rAAV yields of saturation mutants at the P39 site. B: rAAV yields of saturation mutants at the Q202 site

图4 P39L和Q202H突变体对rep/cap基因转录、蛋白表达及rAAV转导效率的影响A：转染后24 h和72 h的rep基因转录水平；B：转染后24 h和72 h的Rep78/68基因转录水平；C：转染后24 h和72 h的cap基因转录水平；D：Western blot检测转染72 h后Rep78/68、Rep52/40、VP1/2/3蛋白表达情况；E：转染后48 h流式细胞术检测EGFP阳性细胞比例；F：转染后48 h EGFP表达的代表性荧光显微照片，比例尺100 μm

Fig. 4 Effects of P39L and Q202H mutations on rep/cap gene transcription, protein expression and rAAV transduction efficiencyA: rep gene transcription levels at 24 h and 72 h post-transfection. B: Rep78/68 gene transcription levels at 24 h and 72 h post-transfection. C: cap gene transcription levels at 24 h and 72 h post-transfection. D: Western blot analysis of Rep78/68, Rep52/40, and VP1/2/3 protein expression at 72 h post-transfection. E: Percentage of EGFP-positive cells detected by flow cytometry at 48 h post‑infection. F: Representative fluorescence micrographs showing EGFP expression at 48 h post-transduction. Scale bars, 100 μm

图5 基于AlphaFold3预测的Rep78蛋白结构及P39、Q202位点定位A：Rep78蛋白预测结构及主要功能区域标注；B：P39位点在OBD区域中的空间位置；C：Q202位点在OBD-HD过渡区域中的空间位置

Fig. 5 AlphaFold3‑predicted structure of Rep78 protein and the localization of P39 and Q202A: Predicted structure of Rep78 protein with annotation of major functional regions. B: Spatial localization of P39 in the OBD region. C: Spatial localization of Q202 in the OBD-HD transition region

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