基于MS2噬菌体病毒样颗粒的RT-PCR检测新型冠状病毒(SARS-CoV-2)质控品制备*

目的: 制备热稳定性好、耐RNase攻击及可全程监控操作的核酸检测新型冠状病毒阳性质控品。方法: 分别扩增MS2噬菌体外壳蛋白CP(含PAC位点)基因以及成熟酶蛋白A基因序列(含核糖体结合位点),先后插入质粒pET28a多克隆位点不同位置,构建通用重组载体pET28a/CP-A。合成包含ORF1ab基因、N基因和E基因三个靶标的特定核酸序列,插入到重组载体pET28a/CP-A中PAC位点的下游,构建包含靶序列的重组载体pET28a/CP-A/S。通过原核表达系统表达目的蛋白,采用硫酸铵和凝胶过滤层析进行纯化,利用透射电镜和动态光散射对蛋白质进行物理表征。全能核酸酶消化形成的盔甲RNA,通过RT-PCR检测其残余核酸和热稳定性。结果: 成功构建包含MS2噬菌体外壳蛋白CP基因、成熟酶蛋白A基因和外源靶核酸的重组载体,目的蛋白在25℃、IPTG 0.3mmol /L、诱导14h时以可溶性形式得到高效表达,纯化后,得到了大小均一、直径为23~28nm的病毒样颗粒,经核酸酶消化后RT-PCR检测,颗粒溶液中几乎无核酸残余且形成了包封靶基因的盔甲RNA。加速破坏试验表明该盔甲RNA无菌过滤后可在37℃稳定保持10天。结论: 在体外,利用MS2噬菌体外壳蛋白和成熟酶蛋白自组装包封外源靶序列制备的盔甲RNA,其热稳定性好,可全程监控整个检测过程,可作为核酸检测SARS-CoV-2的定性或定量质控品。

Preparation of Quality Control Materials for RT-PCR Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Based on MS2 Phage Virus-like Particles

Objective: Preparation of positive quality control products with good thermal stability, resistance to RNase attack and full monitoring for RT-PCR detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: The MS2 phage coat protein CP (including the PAC site) gene sequence and the mature enzyme protein A gene sequence (including the ribosome binding site) were amplified and inserted into the plasmid pET28a to construct the universal recombinant vector pET28a/CP-A. Synthesize a specific nucleic acid sequence containing ORF1ab gene, N gene and E gene of SARS-Cov-2, and insert it into the downstream of the PAC site, which is named pET28a/CP-A.The recombinant protein is expressed through the prokaryotic expression system, and purified by ammonium sulfate and gel filtration chromatography. The purified protein is physically characterized by electron microscopy and dynamic light scattering. The formed armor RNA is digested with omnipotent nuclease, and its thermal stability is verified by fluorescent RT-PCR. Results: A recombinant vector containing the MS2 bacteriophage coat protein gene, the mature enzyme protein gene and exogenous nucleic acid was successfully constructed. VLPs was efficiently expressed in the form of soluble protein at 25℃ and IPTG at 0.3mmol /L for 14h. After purification, The VLPs were observed under transmission electron microscopy in uniform shape and size,with a diameter of about 23-28nm. The VLPs were digested with Benzonase nuclease, and detected by RT-PCR, which confirmed that they formed armor RNA that encapsulated the target gene. The armor RNA can exist stably at 37℃ for 10-15 days under sterile conditions. Conclusion: In vitro, the armor RNA encapsulating foreign target sequence prepared by self-assembly of MS2 phage coat protein and mature enzyme protein has good thermal stability and can monitor the entire detection process. It can be used as a qualitative or quantitative quality control product for the detection of SARS-CoV-2 by RT-PCR.