SETD4蛋白在Bac-to-Bac杆状病毒系统的表达

doi:10.13418/j.issn.1001-165x.2015.04.011

中国临床解剖学杂志 ›› 2015, Vol. 33 ›› Issue (4): 426-429.doi: 10.13418/j.issn.1001-165x.2015.04.011

SETD4蛋白在Bac-to-Bac杆状病毒系统的表达

雷烨铭，　崔航，　钟玙沄，　王义乾，　黄穗，　赵舒祺，　蔡军伟，　姜勇，　刘靖华

广东省蛋白质组学重点实验室，南方医科大学基础医学院病理生理学教研室，广州 510515

收稿日期:2015-04-12 出版日期:2015-07-25 发布日期:2015-08-14
通讯作者: 姜勇，教授，博士生导师，Tel：020-61648231，E-mail: jiang48231@163.com；刘靖华，教授，博士生导师，Tel：（020）61648172-810，E-mail: liujhua@fimmu.com
作者简介:雷烨铭（1988-），女，湖南澧县人，在读硕士，主要从事脓毒症休克机制方向的研究，Tel：（020）61648172-136, E-mail：352139722@qq.com
基金资助:
国家自然科学基金（ 81072425, 81272149）

Expression of SETD4 in Bac-to-Bac baculovirus expression system

LEI Ye-ming, CUI Hang, ZHONG Yu-yun, WANG Yi-qian, HUANG Sui, ZHAO Shu-qi, CAI Jun-wei, JIANG Yong, LIU Jing-hua

Key laboratory for Functional Proteomics of Guangdong Province，Department of Pathophysiology，School of Basic Medical Science，Southern Medical University，Guangzhou 510515，China

Received:2015-04-12 Online:2015-07-25 Published:2015-08-14

摘要/Abstract

摘要：

目的　通过昆虫杆状病毒表达系统表达SETD4（SET domain-containing 4）蛋白，并纯化SETD4蛋白，为深入探讨SETD4的功能奠定基础。方法　提取小鼠正常肝组织的RNA，通过RT-PCR扩增SETD4基因，并克隆至pFastBac-HTB构建重组载体，再转座获得重组杆粒；通过脂质体介导将重组杆粒转染SF9细胞产生重组病毒，扩增病毒感染细胞并获得重组蛋白；利用Ni2+亲和柱来纯化蛋白，并通过Western Blot及考马斯亮蓝染色鉴定SETD4蛋白。结果　经双酶切鉴定及测序证实SETD4基因插入了供体质粒；经PCR鉴定证实SETD4基因插入了穿梭载体；经考马斯亮蓝染色证实纯化得到重组蛋白，用His-Tag抗体和SETD4特异性抗体在50 kD处可检测到目的条带。结论　成功利用昆虫杆状病毒表达系统够表达了SETD4，并纯化了SETD4。

关键词: SETD4蛋白, 　杆状病毒表达系统, 表达, 纯化

Abstract:

Objective To express SET domain-containing 4 (SETD4) protein through using baculovirus expression system and purify the expressed product to explore the functions of SETD4 protein and further understand the biological roles of SET family proteins. Methods The SETD4 gene was amplified by RT-PCR from mouse normal liver tissue. The gene was then inserted into pFastBac-HTB vector to form the recombinant donor plasmid which was further transformed into DH10Bac to construct the recombined bacmid. Next the bacmid was transfected to sf9 cells for package of the recombinant baculovirus particles. The recombinant SETD4 protein was expressed from the cells transduced by the recombinant baculovirus and was purified by NI-NTA resin. Purified protein was examined by coomassie brilliant blue staining and Western Blotting. Results The donor plasmid and recombined bacmid were successfully prepared and the recombinant baculovirus particles were produced from sf9 cells. The SETD4 protein was obtained and confirmed by brilliant blue staining and western blotting with a His-tag antibody and a specific SETD4 antibody. Conclusions The recombinant SETD4 protein was successfully expressed and prepared through baculovirus expression system.

Key words: SETD4 protein, Baculovirus expression system, Expression, Purification

雷烨铭,崔航,钟玙沄,王义乾,黄穗,赵舒祺,蔡军伟,姜勇,刘靖华. SETD4蛋白在Bac-to-Bac杆状病毒系统的表达[J]. 中国临床解剖学杂志, 2015, 33(4): 426-429.

参考文献

[1] Stephen S. Care planning for independence[J]. Provider, 2000, 26(9):2-3.
[2] 薛红，田方. SET基因家族研究进展[J]. 医学分子生物学杂志,2005，(03):190-193.
[3] Carr SM, La Thangue NB. Cell cycle control by a methylation-phosphorylation switch[J]. Cell Cycle, 2011, 10(5):733-734.
[4] Ali A, Veeranki SN, Tyagi S. A SET-domain-independent role of WRAD complex in cell-cycle regulatory function of mixed lineage leukemia[J]. Nucleic Acids Res, 2014,42(12):7611-7624.
[5] Kim DW, Kim KB, Kim JY, et al. Characterization of a novel histone H3K36 methyltransferase setd3 in zebrafish[J]. Biosci Biotechnol Biochem,2011,75(2):289-294.
[6] Tyagi S, Chabes AL, Wysocka J, et al. E2F activation of S phase promoters via association with HCF-1 and the MLL family of histone H3K4 methyltransferases[J]. Mol Cell,2007,27(1):107-119.
[7] Chang Y, Levy D, Horton JR, et al. Structural basis of SETD6-mediated regulation of the NF-kB network via methyl-lysine signaling[J]. Nucleic Acids Res, 2011, 39(15):6380-6389.
[8] p38信号通路对SETD4在细胞中的表达和定位的影响[J]. 中国病理生理杂志, 2012, 28(5):878-883.
[9] Faria JA, Correa NC, de Andrade C, et al. SET domain-containing Protein 4 (SETD4) is a newly identified cytosolic and nuclear lysine methyltransferase involved in breast cancer cell proliferation[J]. J Cancer Sci Ther,2013,5(2):58-65.
[10] Li G, Wang Y, Pan Q, et al. RNAi screening with shRNAs against histone methylation-related genes reveals determinants of sorafenib sensitivity in hepatocellular carcinoma cells[J]. Int J Clin Exp Pathol , 2014,7(3):1085-1092.
[11] Gros F. Control of gene expression in prokaryotic systems[J]. FEBS Lett,1974,40(0):S19-S27.
[12] Vel'Kov VV. Gene amplification in prokaryote and eukaryote systems[J]. Genetika,1982,18(4):529-543.
[13] Jarvis DL. Baculovirus-insect cell expression systems[J]. Methods Enzymol, 2009,463:191-222.
[14] Jarvis DL. Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production[J]. Virology, 2003, 310(1):1-7.
[15] Xiao S, White JF, Betenbaugh MJ, et al. Transient and stable expression of the neurotensin receptor NTS1: a comparison of the baculovirus-insect cell and the T-REx-293 expression systems[J]. PLoS One, 2013, 8(5):e63679.
[16] Luckow VA. Baculovirus systems for the expression of human gene products[J]. Curr Opin Biotechnol, 1993, 4(5):564-572.
[17] Luckow VA, Lee SC, Barry GF, et al. Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli[J]. J Virol, 1993, 67(8):4566-4579.
[18] Verburg JG, Rangwala SH, Samac DA, et al. Examination of the role of tyrosine-174 in the catalytic mechanism of the Arabidopsis thaliana chitinase: comparison of variant chitinases generated by site-directed mutagenesis and expressed in insect cells using baculovirus vectors[J]. Arch Biochem Biophys,1993,300(1):223-230.

SETD4蛋白在Bac-to-Bac杆状病毒系统的表达

Expression of SETD4 in Bac-to-Bac baculovirus expression system

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