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

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.