【摘要】 目的利用Bac-to-Bac杆状病毒表达系统表达鼠IL-4受体拮抗体(mIL-4RA)蛋白。方法PCR方法扩增小鼠IL-4C118截断型基因,定向克隆入转移质粒pFastBacHTB中,转化感受态DH10Bac细胞,在DH10Bac细胞内重组pFastBacHTB与杆粒发生转座。筛选阳性克隆,提取重组杆粒,转染sf9昆虫细胞株,获取完整重组杆状病毒。反复感染sf9细胞,扩增病毒同时表达目的蛋白;用ELISA方法进行蛋白鉴定并初步定量。结果经核苷酸序列测定及PCR方法,鉴定成功获得含mIL-4RA基因的重组杆粒;通过杆粒转染后sf9细胞所表现出来的细胞病变,推断成功转染并获得重组杆状病毒;最后ELISA方法初步定量sf9细胞培养上清中mIL-4RA蛋白表达量为(1.15±0.12)ng/mL。结论本研究利用Bac-to-Bac杆状病毒表达系统成功表达mIL-4RA蛋白,为进一步研究其生物学活性及功能奠定了实验基础,同时亦为其他蛋白质的真核表达提供了方法学的参考。更多还原

【Abstract】 Objective To express the mouse interleukin 4 receptor antagonist(mIL-4RA)protein with Bac-to-Bac baculovirus expression system.Methods mIL-4RA(C118 deletion)cDNA was obtained by polymerase chain reaction(PCR),and subcloned into the baculovirus transfer vector pFastBacHTB.Then the pFastBacHTB was transformed into competent DH10BacTM E.coli cells.The transposition was occurred between pFastBacHTB and bacmid and a recombinant bacmid was obtained.The positive clones were picked out and the recombinant bacmid was isolated,and then transfected into sf9 cells for producing recombinant baculovirus.The baculoviral stock was amplified and the mIL-4RA protein was expressed.Enzyme linked immunosorbent assay(ELISA)was used to identify and quantify the production of protein.Results The presence of mIL-4RA cDNA in the recombinant bacmid was verified by PCR and gene sequencing.The cytopathic effect(CPE)displayed by the infected sf9 cells assumed that the transfection was successful.The concentration of the mIL-4RA protein in the culture media determined by ELISA was(1.15±0.12)ng/mL.Conclusion The mIL-4RA protein is expressed successfully by Bac-to-Bac baculovirus expression system,which may lay foundation for further studying on biological activities and functions of the protein.Meanwhile,the research could provide technique support for eukaryotic expression of other proteins.更多还原