GI/GII型诺如病毒两联装甲RNA标准样品的研制

Preparation of Coupled Armored RNA Reference Material for Norovirus GI/GII

摘要:

针对目前缺乏适配多项检测标准、稳定、安全的诺如病毒RNA标准样品的问题，研制基于MS2噬菌体内含常见GI/GII型诺如病毒检测靶标两联装甲RNA标准参考样品。人工合成MS2噬菌体成熟酶基因、衣壳蛋白基因、包装位点及GI/GII型诺如病毒靶标基因，克隆于表达载体pET-28a(+)中，构建重组质粒pET-MS2-NoV。经大肠杆菌BL21诱导表达，先后利用PEG6000、酶处理和丙烯葡聚糖凝胶层析柱纯化表达产物。SDS-PAGE和透射电镜鉴定产物大小及结构，荧光定量PCR检测有无残留核酸。之后对纯化的病毒样颗粒（Virus-like particles，VLPs）开展定值、均匀性和短期稳定性研究。SDS-PAGE结果表明重组质粒在BL21中表达出了目的蛋白，大小在10~15 ku之间，与预期一致；纯化后的VLPs无杂蛋白和残留核酸；透射电镜下呈结构完整、大小均一的球状，直径约25 nm。纯化后AR-NoV中GI型和GII型靶标定值结果分别为(4.04±0.62)×107 copies/μL和(6.16±0.30)×107 copies/μL。单因素方差检验证实样品均一性良好，F

Abstract:

To provide a safe and stable reference material for Norovirus nucleic detection, the armored RNA containing target RNA of Norovirus GI and GII based on MS2 bacteriophage was developed in this work. DNA fragments including maturase coding gene, capsid protein coding gene and packing site of MS2 bacteriophage, target cDNA sequence of Norovirus GI and GII were synthesized artificially and then cloned into expression vector pET-28a(+) to construct recombinant plasmid pET-MS2-NoV. After expressed in E. coli BL21 cells by IPTG induction, the expression product was purified by PEG6000, enzyme digestion and molecular sieve chromatography. The purified product, also named AR-NoV, was identified by SDS-PAGE, transmission electron microscopy (TEM) and RT-PCR. The value, homogeneity and stability of the AR-NoV were evaluated. SDS-PAGE analysis showed that with the molecular weight of tar-get protein expressed in BL21 was 10~15 ku, which was consistent with the predicted value. There were no impure proteins and residual nucleic acids in AR-NoV after purification. The AR-NoV presented as spherical VLPs with uniform particle size (about 25 nm) and integrated structure under TEM. The values of GI and GII targets in AR-NoV were (4.04±0.62)×107 copies/μL and (6.16±0.30)×107 copies/μL, respectively. The good homogeneity of AR-NoV was confirmed by single-factor ANOVA test (F