中国生物工程杂志

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.