家蚕凋亡蛋白抑制因子 (BmIAP) 基因真核表达与功能分析
(2.家蚕基因组生物学国家重点实验室, 重庆 400715)
【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Bm N-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/m L放线菌素D诱导家蚕Bm N-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Bm N-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 k D;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Bm N-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。
【基金资助】 西南大学博士基金 (SWU114062) ; 中央高校基本业务费专项资金 (XDJK2015C032) ;
Eukaryotic expression and function analysis of Bombyx mori inhibitor of apoptosis protein (BmIAP) gene
(2.State Key Laboratory of Silkworm Genome Biology, Rongchang, Chongqing, China 400715)
【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 by 45 folds 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.
【Keywords】 Bombyx mori; apoptosis; inhibitor of apoptosis protein (IAP) ; eukaryotic expression; transient expression;
【Funds】 Doctoral Scientific Fund of Southwest University (SWU114062); Fundamental Research Funds for the Central Universities (XDJK2015C032);
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