昆虫学报 ›› 2017, Vol. 60 ›› Issue (1): 18-23.doi: 10.16380/j.kcxb.2017.01.003

• 研究论文 • 上一篇    下一篇

家蚕凋亡蛋白抑制因子(BmIAP)基因真核表达与功能分析

易华山1,2, 马鲜平1, 潘敏慧2,*, 鲁成2,*   

  1. (1. 西南大学荣昌校区动物医学系, 重庆 402460; 2. 家蚕基因组生物学国家重点实验室, 重庆 400715)
  • 出版日期:2017-01-20 发布日期:2017-01-20

Eukaryotic expression and function analysis of Bombyx mori inhibitor of apoptosis protein (BmIAP) gene

YI Hua-Shan1,2, MA Xian-Ping1, PAN Min-Hui2,*, LU Cheng2,*   

  1. (1. Department of Animal Medicine, Rongchang Campus of Southwest University, Rongchang, Chongqing 402460, China; 2. State Key Laboratory of Silkworm Genome Biology, Chongqing 400715, China)
  • Online:2017-01-20 Published:2017-01-20

摘要: 【目的】家蚕Bombyx mori凋亡蛋白抑制因子(BmIAP)是在家蚕中发现的一个凋亡蛋白抑制因子(IAP)。本研究旨在验证家蚕BmIAP蛋白在家蚕细胞内的功能特征,以进一步研究BmIAP在家蚕细胞凋亡中的作用。【方法】对构建的瞬时表达载体PIZ/V5-BmIAP-dsRed,应用脂质体转染家蚕BmN-SWU1细胞;应用实时荧光定量PCR和Western blot分析BmIAP mRNA及蛋白表达水平;通过150 ng/mL放线菌素D诱导家蚕BmN-SWU1细胞12, 18和24 h,应用免疫荧光及实时荧光定量PCR方法,分析BmIAP瞬时表达与细胞凋亡的关系。【结果】构建的瞬时表达载体PIZ/V5-BmIAP-dsRed能在家蚕BmN-SWU1细胞表达BmIAP蛋白,其BmIAP mRNA表达水平上调近45倍,融合蛋白约为60 kD;BmIAP基因瞬时表达72 h后,BmIAP能显著抑制放线菌素D诱导12 h时的家蚕BmN-SWU1细胞凋亡。【结论】BmIAP蛋白在家蚕细胞中能抑制家蚕细胞凋亡,是家蚕的一种凋亡蛋白抑制因子。

关键词: 家蚕, 细胞凋亡, 凋亡蛋白抑制因子, 真核表达, 瞬时表达

Abstract: 【Aim】 Bombyx mori inhibitor of apoptosis protein (BmIAP) has been found in the silkworm, B.mori, and characterized as a protein of the IAP (inhibitor of apoptosis protein) family. This study aims to verify its function as a suppressor of apoptosis in B.mori cells, so as to further study the roles of BmIAP in cell apoptosis. 【Methods】 The transient expression vector named PIZ/V5-BmIAP-dsRed was transfected in the BmN-SWU1 cells of B.mori by liposome, and the mRNA and protein expression levels of BmIAP gene were evaluated by real-time quantitative PCR and Western blot analysis, respectively. The BmN-SWU1 cells were induced by 150 ng/mL actinomycin D for 12, 18 and 24 h, and the relationship between BmIAP protein and cell apoptosis was assayed by immunofluorescence and real-time quantitative PCR methods. 【Results】 The PIZ/V5-BmIAP-dsRed vector effectively expressed BmIAP fusion protein of about 60 kD, and the mRNA expression level of BmIAP was up-regulated 45-fold in BmN-SWU1 cells of B.mori. After BmIAP was transiently expressed for 72 h using PIZ/V5-BmIAP-dsRed vector in the BmN-SWU1 cells, BmIAP significantly inhibited the apoptosis in the BmN-SWU1 cells at 12 h after induction by actinomycin D. 【Conclusion】 BmIAP can inhibit apoptosis in B.mori cells and is one of the inhibitor of apoptosis proteins in B.mori.

Key words: Bombyx mori; apoptosis; inhibitor of apoptosis protein(IAP); eukaryotic expression, transient expression