Xu Peng-Wei, Guo Jian-Qiang, Yao Li-Hong, Chen Ai-Jun, Liu Xiao-Yu, Zeng Xian-Yin, Zhang Zhi-Qing
College of Life Science and Science, Sichuan Agricultural University, Yaan 625014, China.
Bing Du Xue Bao. 2012 May;28(3):231-6.
The M1 and HA genes of H1N1 influenza virus were amplified and then cloned into the pFastBac dual donor plasmid. The recombinant pFastBac Dual-M1-HA was identified by restriction enzyme digestion. After the pFastBacdual-M1-HA was transformed into the baculovirus shuttle plasmid (bacmid) in DH10Bac competent cells, the colonies were identified by antibiotics and blue-white selection. The rBac-mid-M1-HA was verified by PCR and transfected into S f9 cells to produce recombinant baculovirus (rBac-M1-HA). Gene insertion of rBac-M1-HA was verified and the expression of M1 and HA genes was analyzed by IFA and Western-blot, demonstrating M1 and HA were co-expressed successfully. This study provides the foundation for researching the formation mechanism of influenza VLP and developing new influenza vaccines.
扩增H1N1流感病毒的M1和HA基因，然后将其克隆到pFastBac双供体质粒中。通过限制性内切酶消化鉴定重组pFastBac Dual-M1-HA。将pFastBacdual-M1-HA转化到DH10Bac感受态细胞中的杆状病毒穿梭质粒（杆粒）后，通过抗生素和蓝白筛选鉴定菌落。通过PCR验证rBac-mid-M1-HA，并将其转染到S f9细胞中以产生重组杆状病毒（rBac-M1-HA）。验证rBac-M1-HA的基因插入，并通过IFA和Western-blot分析M1和HA基因的表达，表明M1和HA成功共表达。本研究为研究流感病毒样颗粒的形成机制和开发新型流感疫苗奠定了基础。
