作为自我扩增mRNA疫苗载体的复制缺陷塞姆利基森林病毒衍生颗粒的包装、纯化和滴定

Q2 Biochemistry, Genetics and Molecular Biology
Nastaran Sadat Savar, Thomas Vallet, Arash Arashkia, Kenneth Lundstrom, Marco Vignuzzi, Hamid Mahmoudzadeh Niknam
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引用次数: 0

摘要

背景:自扩增mRNA是新一代疫苗平台,在感染性疾病和癌症的疗效和开发速度方面具有潜在优势。主要目的是提出优化和快速的SFV-PD SAM制备、包装和效价测定方法。这些方案是为疫苗研究生产和收获高产量vrp包装的SAM提供的。方法:将pSFV-PD-EGFP质粒线性化,进行体外转录。先将不同浓度的SFV-PD SAM转染到HEK-293和BHK-21细胞系,荧光显微镜观察不同时间点EGFP的表达情况。通过将SFV-PD SAM和pSFV-Helper2 RNA共转染到BHK-21细胞中,实现了复制子颗粒包装。采用截止时间为100 kDa的超滤法浓缩VRPs。RT-qPCR检测复制子颗粒滴度。结果:体外转录的SAM编码EGFP在HEK-293和BHK-21细胞系中成功转染表达。与HEK-293细胞相比,BHK-21细胞中EGFP的表达水平更高,显示出更稳定的蛋白过表达和VRP包装。采用超滤技术,可以快速获得高产量的SFV-PD-EGFP颗粒,并且只有少量的复制子颗粒损失。RT-qPCR可准确、快速测定复制缺陷颗粒滴度。结论:通过优化的SAM转染、VRP包装和浓缩方法,可以生产和纯化高产量的SFV-PD VRP。RT-qPCR被证明是一种准确、快速的测定复制缺陷VRPs滴度的方法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Packaging, Purification, and Titration of Replication-Deficient Semliki Forest Virus-Derived Particles as a Self-Amplifying mRNA Vaccine Vector

Background: Self-amplifying mRNA is the next-generation vaccine platform with the potential advantages in efficacy and speed of development against infectious diseases and cancer. The main aim was to present optimized and rapid methods for Semliki Forest virus (SFV)-PD self-amplifying mRNA (SAM) preparation, its packaging, and titer determination. These protocols are provided for producing and harvesting the high yields of virus replicon particle (VRP)-packaged SAM for vaccine studies.

Methods: pSFV-PD-EGFP plasmid was linearized and subjected to in vitro transcription. Different concentrations of SFV-PD SAM were first transfected into human embryonic kidney 293 cells (HEK-293) and baby hamster kidney cell line 21 (BHK-21) cell lines, and EGFP expression at different time points was evaluated by fluorescent microscopy. Replicon particle packaging was achieved by co-transfection of SFV-PD SAM and pSFV-Helper2 RNA into BHK-21 cells. The VRPs were concentrated using ultrafiltration with 100 kDa cut-off. The titers of replicon particles were determined by reverse transcription quantitative real-time PCR (RT-qPCR).

Results: In vitro transcribed SAM encoding EGFP was successfully transfected and expressed in HEK-293 and BHK-21 cell lines. Higher levels of EGFP expression was observed in BHK-21 compared to HEK-293 cells showing more stable protein overexpression and VRP packaging. Using ultrafiltration, the high yields of purified SFV-PD-EGFP particles were rapidly obtained with only minor loss of replicon particles. Accurate and rapid titer determination of replication-deficient particles was achieved by RT-qPCR.

Conclusion: Using optimized methods for SAM transfection, VRP packaging, and concentration, high yields of SFV-PD VRPs could be produced and purified. The RT-qPCR demonstrated to be an accurate and rapid method for titer determination of replication deficient VRPs.

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来源期刊
Iranian Biomedical Journal
Iranian Biomedical Journal Biochemistry, Genetics and Molecular Biology-Biochemistry, Genetics and Molecular Biology (all)
CiteScore
3.20
自引率
0.00%
发文量
42
审稿时长
8 weeks
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