摘要
目的构建新型冠状病毒Omicron I1566V突变位点MS2噬菌体病毒样颗粒。方法选取并合成带有6×His标签的MS2噬菌体的衣壳蛋白(CP)、成熟蛋白(A蛋白)及Omicron I1566V突变基因序列,插入pACYCDuet-1质粒构建重组载体,通过原核系统诱导表达目的蛋白,纯化重组蛋白后利用透射电镜对蛋白质进行物理表征,最后通过反转录聚合酶链反应(RT-PCR)检测病毒样颗粒的热稳定性及耐核酸酶水解能力。结果成功构建包含有6×His标签的CP、A蛋白和Omicron I1566V突变基因序列的重组载体,经限制性内切酶BamHⅠ和KpnⅠ酶切鉴定和测序验证,结果均与预期相符。经诱导并纯化后,通过电镜观察到了大小均匀、直径为23~28 nm的病毒样颗粒,该病毒样颗粒经核酸酶消化后可在37℃条件下稳定储存20 d以上。结论该研究成功利用MS2噬菌体的CP和A蛋白构建了Omicron I1566V突变位点病毒样颗粒,为该突变位点的RT-PCR检测体系提供了可靠的质量保障。
Objective To construct MS2 phage virus-like particles with Omicron I1566V mutation site of SARS-CoV-2.Methods The capsid protein(CP),mature protein(A protein)and Omicron I1566V mutant gene sequences of MS2 phage with 6×His tag were selected and synthesized,and inserted into the pACYCDuet-1 plasmid to construct the recombinant vector.The recombinant protein was purified and characterized by transmission electron microscopy.Finally,the thermal stability and nuclease resistance of the virus-like particles were detected by reverse transcription polymerase chain reaction(RT-PCR).Results The recombinant vector containing CP and A protein with 6×His tag and Omicron I1566V mutant gene sequence was successfully constructed.The results were confirmed by restriction enzyme BamH I and Kpn I digestion and sequencing.After induction and purification,the virus-like particles with a diameter of 23-28 nm were observed by electron microscopy.The virus-like particles were digested by nuclease and stored stably at 37℃for more than 20 days.Conclusion In this study,the Omicron I1566V mutation site virus-like particles were successfully constructed using the CP and A protein of bacteriophage MS2,which provided reliable quality assurance for the RT-PCR detection system of Omicron I1566V mutation site.
作者
杨静远
李永鑫
史茜
刘春燕
梁梦洁
张新
YANG Jingyuan;LI Yongxin;SHI Qian;LIU Chunyan;LIANG Mengjie;ZHANG Xin(Medical College,Shihezi University,Shihezi,Xinjiang 832000,China;Department of Clinical Laboratory,the Second Affiliated Hospital of Medical College of Shihezi University/Xinjiang Production and Construction Corps Hospital,Urumqi,Xinjiang 830002,China)
出处
《检验医学与临床》
CAS
2024年第8期1030-1034,共5页
Laboratory Medicine and Clinic
基金
国家临床重点专科建设项目[兵财社(2023)16号]
兵团临床医学研究中心建设项目[兵科发(2023)12号]
兵团重点领域科技攻关计划项目(2022AB030)
新疆生产建设兵团医院院级科技计划项目(2022014)。
