Gholami Mohammad, Ravanshad Mehrdad, Baesi Kazem, Samiee Siamak M., Hosseini Rozbahani Negin, Mohraz Minoo
Department of Medical Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran.
Iran Biomed J. 2018 Nov;22(6):394-400. doi: 10.29252/.22.6.394. Epub 2018 May 19.
The human immunodeficiency virus type 1 (HIV-1) is an infectious viral agent that gradually extinguishes the immune system, resulting in acquired immune deficiency syndrome (AIDS). The aim of this study was to construct an RNA-positive control based on armored (AR) RNA technology, using HIV-1 RNA as a model.
The MS2 maturase, a coat protein gene (at positions 1765 to 1787) and HIV-1 pol gene were cloned into pET-32a plasmid. The prepared plasmid was transformed into Escherichia coli strain BL2 (DE3), and the expression of the construct was induced by 1 mM of isopropyl-L-thio-D-galactopyranoside (IPTG) at 37 °C for 16 h to obtain the fabricated AR RNA. The AR RNA was precipitated and purified using polyethylene glycol and Sephacryl S-200 chromatography.
The stability of AR RNA was evaluated by treatment with DNase I and RNase A and confirmed by transmission electron microscopy and gel agarose electrophoresis. Tenfold serial dilution of AR RNA from 101 to 105 was prepared. Real-time PCR assays had a range of detection between 101 and 105. In addition, R2 value was 0.998, and the slope of the standard curve was -3.33.
Prepared AR RNA, as a positive control, could be used as a basis for launching an in-house HIV-1 virus assay and other infectious agents. It can be readily available to laboratories and HIV research centers. The AR RNA is non-infectious and highly resistant to ribonuclease enzyme and can reduce the risk of infection in the clinical laboratory.
人类免疫缺陷病毒1型（HIV-1）是一种传染性病毒病原体，会逐渐破坏免疫系统，导致获得性免疫缺陷综合征（AIDS）。本研究的目的是以HIV-1 RNA为模型，构建基于装甲（AR）RNA技术的RNA阳性对照。
将MS2成熟酶、一个外壳蛋白基因（位于1765至1787位）和HIV-1 pol基因克隆到pET-32a质粒中。将制备好的质粒转化到大肠杆菌BL2（DE3）菌株中，在37℃用1 mM异丙基-L-硫代-D-半乳糖苷（IPTG）诱导构建体表达16小时，以获得制备的AR RNA。使用聚乙二醇和Sephacryl S-200层析对AR RNA进行沉淀和纯化。
通过用DNase I和RNase A处理评估AR RNA的稳定性，并通过透射电子显微镜和琼脂糖凝胶电泳进行确认。制备了从101到105的AR RNA十倍系列稀释液。实时PCR检测的检测范围在101到105之间。此外，R2值为0.998，标准曲线的斜率为-3.33。
制备的AR RNA作为阳性对照，可作为开展内部HIV-1病毒检测及其他感染因子检测的基础。实验室和HIV研究中心可以很容易地获得。AR RNA无传染性，对核糖核酸酶具有高度抗性，可降低临床实验室的感染风险。
