Zhang Jin, Xue Xiaoning, Xu Hefei, Zhu Ke, Chen Xiaoguang, Zhang Juan, Zhang Qi, Lin Yuan
Bing Du Xue Bao. 2015 Nov;31(6):629-33.
To prepare virus-like particles containing FluA/B mRNA as RNA standard and control in Influenza RNA detection, the genes coding the coat protein and maturase of E. coli bacteriophage MS2 were amplified and cloned into D-pET32a vector. Then we inserted 6 histidines to MS2 coat protein by QuikChange Site-Directed Mutagenesis Kit to construct the universal expressing vector D-pET32a-CP-His. In addition, the partial gene fragments of FluA and FluB were cloned to the down-stream of expressing vector. The recombinant plasmid D-pET32a-CP-His-FluA/B was transformed to BL21 with induction by IPTG. The virus-like particles were purified by Ni+ chromatography. The virus-like particles can be detected by RT-PCR, but not PCR. They can be conserved stably for at least 3 months at both 4 degrees C and -20 degrees C. His-tagged virus-like particles are more stable and easier to purification. It can be used as RNA standard and control in Influenza virus RNA detection.
为制备含甲型/乙型流感病毒mRNA的病毒样颗粒作为流感病毒RNA检测中的RNA标准品和对照品，扩增了编码大肠杆菌噬菌体MS2外壳蛋白和成熟酶的基因，并将其克隆到D-pET32a载体中。然后通过快速定点突变试剂盒在MS2外壳蛋白中插入6个组氨酸，构建通用表达载体D-pET32a-CP-His。此外，将甲型流感病毒和乙型流感病毒的部分基因片段克隆到表达载体的下游。将重组质粒D-pET32a-CP-His-FluA/B转化至BL21感受态细胞，并用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达。通过镍离子亲和层析法纯化病毒样颗粒。该病毒样颗粒可通过逆转录-聚合酶链反应（RT-PCR）检测，但不能通过聚合酶链反应（PCR）检测。它们在4℃和-20℃下均可稳定保存至少3个月。带有组氨酸标签的病毒样颗粒更稳定且易于纯化。它可作为流感病毒RNA检测中的RNA标准品和对照品。
