含五种RNA病毒核酸假病毒的制备及特性研究  

Preparation and characterization of pseudovirus containing five kinds of RNA virus

作　　者：程巧珍 陈春 应婷 伍义行 叶子弘 廖学俊 胡华军 CHENG Qiaozhen;CHEN Chun;YING Ting;WU Yihang;YE Zihong;LIAO Xuejun;HU Huajun(Zhejiang Provincial Key Laboratory of Biometrology,Inspection and Quarantine,College of Life Sciences,China Jiliang University,Hangzhou 310018,China)

机构地区：[1]中国计量大学生命科学学院浙江省生物计量与检验检疫技术重点实验室,浙江杭州310018

出　　处：《中国计量大学学报》2022年第2期281-287,共7页Journal of China University of Metrology

基　　金：国家重点研发计划课题(No.2018YFA0108403)。

摘　　要：目的:制备一种应用于细胞制剂中丙型肝炎病毒(HCV)、人类免疫缺陷病毒1型和2型(HIV-1/2)、人类T细胞白血病病毒1型和2型(HTLV-1/2)五种RNA病毒核酸检测的阳性质控品,即装载上述五种病毒核酸的假病毒5V(5V)。方法:将上述病毒的部分基因片段连接成一条基因,与MS2噬菌体部分基因共同插入原核表达载体中构建pACYCDuet-1-MS2/5Virus重组质粒,转化到E.coli BL21(DE3)感受态中表达并鉴定;表达产物纯化浓缩,电镜分析5V形态;对5V进行DNase I、RNase A抗性及保存期的稳定性试验。结果:重组基因有效表达,5V纯度高无杂带;5V成功包装目的基因序列,具有明显的病毒结构特征;5V能抵抗高浓度的DNase I和RNase A的降解,在-20℃稳定保存6个月。结论:制备的5V稳定,达到了作为细胞制剂中RNA病毒核酸检测阳性质控品的基本要求。Aims: This paper aims to prepare a positive quality control product for nucleic acid detection of hepatitis C virus, human immunodeficiency virus type 1 and 2, human T-cell leukemia virus type 1 and 2 in cytomedicine, namely, preudovirus loaded with the nucleic acids of the above five kinds of virus. Methods: The gene fragments of the above viruses were connected into a single gene and inserted into prokaryotic expression vector together with part of the MS2 phage gene, to construct pACYCDuet-1-MS2/5 virus recombinant plasmid. The plasmid was transformed into E.coli BL21(DE3) for expression and evaluation. The expression product was purified and concentrated;and the morphological identification of 5V was analgsed by scanning electron microscopy. The stability of 5V was evaluated by challenging with DNase I and RNase A and was evaluated for long-term storage. Results: The recombinant gene was effectively expressed and the 5V purity was high. 5V had successfully packaged the target gene sequence and had obvious virus-like structural features. Stability evaluation showed that the 5V exhibited strong resistance to degradation by DNase I and RNase A and long-lasting protection of coated RNA for at least six months when stored at-20 ℃. Conclusions: The prepared 5V is stable and meets the basic requirements to be used as a quality control material for RNA virus nucleic acid detection.

关 键 词：丙型肝炎病毒 人类免疫缺陷病毒 人类T细胞白血病病毒 假病毒 MS2噬菌体 

分 类 号：R373[医药卫生—病原生物学]