【摘要】 目的:利用昆虫细胞表达着床丝氨酸蛋白酶-2(implantation serine proteinase-2,ISP2)。方法:将ISP2cDNA克隆入核型多角体病毒(AcNPV)非融合蛋白基因表达载体pVL1392,获得的重组表达载体pVL1392/ISP2,与BaculGoldTM线性杆状病毒DNA共转染昆虫细胞Sf9,经多次扩增后获得高滴度的重组病毒AcNPV-ISP2。将此重组病毒感染昆虫细胞,收集转染细胞培养上清液。结果:用SDS-PAGE考马斯亮蓝染色和Westernblot测得培养上清液中有表达蛋白,分子量为40kD左右。结论:用杆状病毒系统表达的ISP2能分泌到细胞外,有利于产物的分离纯化,为进一步研究ISP2在着床中的生物功能奠定了基础。更多还原

【Abstract】 Objective: To investigate the expression of implantation serine proteinase-2 (ISP2) using insect cells. Methods: We constructed an expression vector pVL1392/ISP2, using ISP2 cDNA cloned into unfused protein nuclear polyhedrosis virus (AcNPV) expression vector pVL1392. The insect cells (Sf9) were cotransfected with the expression vector and BaculoGold?linearized baculovirus DNA, and recombinant viruses AcNPV-ISP2 were collected after several times amplification. The insect cells (Sf9) were infected with the recombinant viruses AcNPV-ISP2 and the supernatant was collected and analysed using Coomassie brilliant blue stained SDS-PAGE and Western blot. Results: SDS-PAGE and Western blot results suggested that the expressed protein with a molecular weight of about 40 kD existed in the conditioned media of Sf9 cells infected by AcNPV-ISP2. Conclusion: The ISP2 expressed in Sf9 cells infected by AcNPV-ISP2 was secreted into media, which contributed the purification of the expressed product. Our study prepared basic necessary data for further investigating the biological function of ISP2 in implantation.更多还原