Qin Li-Ting, Wang Xi-Jun, Hu Sen, Li Zhi-Zhong, Chen Wei-Ye, Ge Jin-Ying, Liu Si-Dang, Bu Zhi-Gao
Key Laboratory of National Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agriculture Science, Harbin 150001, China.
Sheng Wu Gong Cheng Xue Bao. 2007 May;23(3):386-91.
The full-length porcine interferon gamma(PoIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into honor plasmid pFastBac 1 of Bac-To-Bac Baculovirus Expression System. These recombinant plasmids, pFastBac -PoIFN-gamma, were transformed into DH(10Bac) host bacteria to get recombinant shuttle plasmids, rBacmid-PoIFN-gamma. Recombinant baculovirus, rBac-PoIFN-gamma, was generated for expressing PoIFN-gamma, by transfecting rBacmid-PoIFN-gamma with Cellfectin Reagent into sf9 insect cells. The expression of PoIFN-gamma in insect cells was confirmed by Western Blot, indirect immunofluorescence assay and indirect ELISA. The antiviral activity assay shows that PoIFN-gamma expressed by the rBac-PoIFN-gamma can efficiently inhibit the replication of the recombinant Vesicular Stomatitis Virus expressing green fluorescence protein in PK-15 cells. The antiviral activity of PoIFN-gamma can be specifically blocked by anti-PoIFN-gamma mouse serum. The antiviral titer of culture supernatant of insect cells infected by rBac-PoIFN-gamma is 2 x 10(4) IU/mL. The results demonstrat that the rBac-PoIFN-gamma can express rPoIFN-gamma efficiently and rPoIFN-gamma has high antiviral activity.
将包含分泌信号肽编码区的全长猪干扰素γ（PoIFN-γ）cDNA 重新克隆到杆状病毒表达系统的pFastBac 1 载体质粒中。这些重组质粒pFastBac -PoIFN-γ 被转化到DH(10Bac)宿主菌中，以获得重组穿梭质粒rBacmid-PoIFN-γ。通过使用Cellfectin试剂将rBacmid-PoIFN-γ 转染到sf9昆虫细胞中，产生用于表达PoIFN-γ 的重组杆状病毒rBac-PoIFN-γ。通过蛋白质免疫印迹、间接免疫荧光测定和间接酶联免疫吸附测定法证实了PoIFN-γ 在昆虫细胞中的表达。抗病毒活性测定表明，rBac-PoIFN-γ 表达的PoIFN-γ 能够有效抑制表达绿色荧光蛋白的重组水疱性口炎病毒在PK-15细胞中的复制。PoIFN-γ 的抗病毒活性可被抗PoIFN-γ 小鼠血清特异性阻断。rBac-PoIFN-γ 感染的昆虫细胞培养上清液的抗病毒效价为2×10(4) IU/mL。结果表明，rBac-PoIFN-γ 能够高效表达rPoIFN-γ，且rPoIFN-γ 具有高抗病毒活性。
