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.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)

【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。

【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;

【DOI】

【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)

Download this article

    References

    Honig GR, Rabinovitz M, 1965.Actinomycin D:inhibition of protein synthesis unrelated to effect on template RNA synthesis.Science, 149 (3691) :1504-1506.

    Huang Q, Deveraux QL, Maeda S, Henning RS, Bruce DH, John CR, 2001.Cloning and characterization of an inhibitor of apoptosis protein (IAP) from Bombyx mori.Biochim.Biophys.Acta, 1499 (3) :191-198.

    Kamita SG, Majima K, Maeda S, 1993.Identification and characterization of the p35 gene of Bombyx mori nuclear polyhedrosis virus that prevents virus-induced apoptosis.J.Virol., 67 (1) :455-463.

    Kim EJ, Rhee WJ, Park TH, 2004.Inhibition of apoptosis by a Bombyx mori gene.Biotechnol.Prog., 20 (1) :324-329.

    Kumarswamy R, Chandna S, 2010.Inhibition of microRNA-14contributes to actinomycin-D-induced apoptosis in the Sf9 insect cell line.Cell Biol.Int., 34 (8) :851-857.

    Lindell TJ, 1976.Evidence for an extranucleolar mechanism of actinomycin D action.Nature, 5575:347-350.

    Livak KJ, Schmittgen TD, 2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CTmethod.Methods, 25 (4) :402-408.

    Mena JA, Kamen AA, 2011.Insect cell technology is a versatile and robust vaccine manufacturing platform.Expert Rev.Vacc., 10 (7) :1063-1081.

    Pan MH, Cai XJ, Liu M, Lv J, Tang H, Tan J, Lu C, 2010.Establishment and characterization of an ovarian cell line of the silkworm, Bombyx mori.Tissue Cell, 42 (1) :42-46.

    Pan MH, Chen M, Huang SJ, Yu ZS, Cheng CG, Lu C, 2009.Cloning and protein release of cytochrome C in the apoptosis cells of silkworm, Bombyx mori.Scientia Agricultura Sinica, 42 (7) :2546-2551.

    Pan MH, Deng WK, Chen M, Zhang JY, Du J, Lu C, 2008.Inhibition of BmE-SWU1 cell apoptosis by silkworm hemolymph.Acta Zoologica Sinica, 54 (5) :861-866.

    Perry RP, Kelley DE, 1970.Inhibition of RNA synthesis by actinomycin D:characteristic dose-response of different RNA species.J.Cell.Physiol., 76 (2) :127-139.

    Portt L, Norman G, Clapp C, 2011.Anti-apoptosis and cell survival:a review.Biochim.Biophys.Acta, 1813 (1) :238-259.

    Silke J, Meier P, 2013.Inhibitor of apoptosis (IAP) proteinsmodulators of cell death and inflammation.Cold Spring Harb.Perspect.Biol., 5 (2) :152-158.

    Simone F, Domagoj V, 2012.Targeting IAP proteins for therapeutic intervention in cancer.Nat.Rev.Drug Discov., 11 (2) :109-124.

    Song J, Yi HS, Hu YF, He Q, Liu TH, Pan CX, Lu C, Pan MH, 2014.Advances and phylogenetic analysis of inhibitors of apoptosis protein.Science of Sericulture, (1) :147-152.

    Summers MD, Smith GE, 1987.A manual of methods for baculovirus vectors and insect cell culture procedures.Bulletin B-Texas Agricultural Experiment Station, 40 (1555) :1-56.

    Sun YB, Li JH, Shi YH, 2006.Construction and expression of inset expression vector p IZT/V5-His harboring mI L-4.Journal of Jilin University (Medicine Edition) , 32 (2) :194-195, 198.

    Thornberry NA, Lazebnik Y, 1998.Caspases:enemies within.Science, 281 (5381) :1312-1316.

    Xia Q, Li S, Feng Q, 2014.Advances in silkworm studies accelerated by the genome sequencing of Bombyx mori.Annu.Rev.Entomol., 59 (1) :513-536.

    Xu Y, Zhao XH, 2010.IAP family and IAP-targeted cancer therapy.Chinese Bulletin of Life Sciences, (2) :161-168.

    Yi HS, 2014.Cloning and Functional Analysis of the BmI CE-2 Gene in Bombyx mori.Ph D Dissertation, Southwest University, Chongqing.

    Yi HS, Pan CX, Pan C, Song J, Hu YF, Wang L, Pan MH, Lu C, 2014.BmI CE-2 is a novel pro-apoptotic caspase involved in apoptosis in the silkworm, Bombyx mori.Biochem.Biophys.Res.Commun., 445 (1) :100-106.

This Article

ISSN:0454-6296

CN: 11-1832/Q

Vol 60, No. 01, Pages 18-23

January 2017

Downloads:4

Share
Article Outline

知识点

摘要

  • 1 材料与方法
  • 2 结果
  • 3 讨论
  • 参考文献