棉铃虫c-Myc基因的原核表达纯化、抗体制备、时空表达谱测定与功能分析
作者:
基金项目:

国家自然科学基金(31401762,31672355);国家重点研发计划(2017YFD0200400);武汉市青年科技晨光计划项目(2017050304010320);遗传调控与整合生物学湖北省重点实验室课题(GRIB202210)


Prokaryotic expression, polyclonal antibody preparation, spatio-temporal expression profile and functional analysis of c-Myc of Helicoverpa armigera (Lepidoptera: Noctuidae)
Author:
  • SUO Qian

    SUO Qian

    College of Life Sciences, Central China Normal University, Wuhan 430079, Hubei, China;Institute of Entomology, Central China Normal University, Wuhan 430079, Hubei, China;Hubei Provincial Key Laboratory of Genetic Regulation and Integrative Biology, Central China Normal University, Wuhan 430079, Hubei, China
    在期刊界中查找
    在百度中查找
    在本站中查找
  • SUN Xiaoyan

    SUN Xiaoyan

    College of Life Sciences, Central China Normal University, Wuhan 430079, Hubei, China;Institute of Entomology, Central China Normal University, Wuhan 430079, Hubei, China;Hubei Provincial Key Laboratory of Genetic Regulation and Integrative Biology, Central China Normal University, Wuhan 430079, Hubei, China
    在期刊界中查找
    在百度中查找
    在本站中查找
  • ZHANG Ying

    ZHANG Ying

    College of Life Sciences, Central China Normal University, Wuhan 430079, Hubei, China;Institute of Entomology, Central China Normal University, Wuhan 430079, Hubei, China;Hubei Provincial Key Laboratory of Genetic Regulation and Integrative Biology, Central China Normal University, Wuhan 430079, Hubei, China
    在期刊界中查找
    在百度中查找
    在本站中查找
  • WANG Yujing

    WANG Yujing

    College of Life Sciences, Central China Normal University, Wuhan 430079, Hubei, China;Institute of Entomology, Central China Normal University, Wuhan 430079, Hubei, China;Hubei Provincial Key Laboratory of Genetic Regulation and Integrative Biology, Central China Normal University, Wuhan 430079, Hubei, China
    在期刊界中查找
    在百度中查找
    在本站中查找
  • LIU Kaiyu

    LIU Kaiyu

    College of Life Sciences, Central China Normal University, Wuhan 430079, Hubei, China;Institute of Entomology, Central China Normal University, Wuhan 430079, Hubei, China;Hubei Provincial Key Laboratory of Genetic Regulation and Integrative Biology, Central China Normal University, Wuhan 430079, Hubei, China
    在期刊界中查找
    在百度中查找
    在本站中查找
  • YANG Hong

    YANG Hong

    College of Life Sciences, Central China Normal University, Wuhan 430079, Hubei, China;Institute of Entomology, Central China Normal University, Wuhan 430079, Hubei, China;Hubei Provincial Key Laboratory of Genetic Regulation and Integrative Biology, Central China Normal University, Wuhan 430079, Hubei, China
    在期刊界中查找
    在百度中查找
    在本站中查找
  • HONG Huazhu

    HONG Huazhu

    College of Life Sciences, Central China Normal University, Wuhan 430079, Hubei, China;Institute of Entomology, Central China Normal University, Wuhan 430079, Hubei, China;Hubei Provincial Key Laboratory of Genetic Regulation and Integrative Biology, Central China Normal University, Wuhan 430079, Hubei, China
    在期刊界中查找
    在百度中查找
    在本站中查找
  • PENG Jianxin

    PENG Jianxin

    College of Life Sciences, Central China Normal University, Wuhan 430079, Hubei, China;Institute of Entomology, Central China Normal University, Wuhan 430079, Hubei, China;Hubei Provincial Key Laboratory of Genetic Regulation and Integrative Biology, Central China Normal University, Wuhan 430079, Hubei, China
    在期刊界中查找
    在百度中查找
    在本站中查找
  • PENG Rong

    PENG Rong

    College of Life Sciences, Central China Normal University, Wuhan 430079, Hubei, China;Institute of Entomology, Central China Normal University, Wuhan 430079, Hubei, China;Hubei Provincial Key Laboratory of Genetic Regulation and Integrative Biology, Central China Normal University, Wuhan 430079, Hubei, China
    在期刊界中查找
    在百度中查找
    在本站中查找
  • 摘要
  • | |
  • 访问统计
  • |
  • 参考文献 [33]
  • |
  • 相似文献 [20]
  • | | |
  • 文章评论
    摘要:

    细胞致瘤基因(cellular-myelocytomatosis viral oncogene,c-Myc)编码的c-Myc蛋白通过Wnt/β-catenin信号通路调节相关基因的表达,近年来引起了广泛的研究关注。本研究旨在利用原核表达系统体外表达棉铃虫(Helicoverpa armigerac-MycHa-c-Myc)基因,制备多克隆抗体,明确Ha-c-Myc的时空表达模式,并探究Ha-c-Myc在调控棉铃虫胆固醇载体蛋白-2(sterol carrier protein-2,SCP-2)基因表达中的潜在作用。通过PCR扩增Ha-c-Myc基因并克隆至pET-32a (+)原核表达载体中,构建重组表达质粒pET-32a-Ha-c-Myc,转化大肠杆菌(Escherichia coli) BL21(DE3)细胞,使用IPTG诱导蛋白表达,并通过Ni2+-NTA柱纯化重组蛋白,用重组蛋白免疫新西兰兔制备抗Ha-c-Myc多克隆抗体。利用实时荧光定量PCR (real-time quantitative reverse transcription PCR,qRT-PCR)法检测棉铃虫不同发育时期(卵、幼虫、预蛹、蛹和成虫)和预蛹期不同组织(中肠、脂肪体、头部和表皮)中Ha-c-Myc基因的表达水平。设计合成Ha-c-Myc siRNA并转染棉铃虫Ha细胞,通过qRT-PCR分析Ha-c-Myc基因的RNA干扰后棉铃虫Ha-c-Myc基因和HaSCP-2基因mRNA表达的情况。结果表明,本实验构建了pET-32a-Ha-c-Myc重组质粒,在大肠杆菌中获得了高效表达的、大小约为65 kDa的可溶性Ha-c-Myc蛋白。经纯化得到纯度较高的蛋白,免疫新西兰兔制备了多克隆抗体,免疫印迹实验(Western blotting)表明抗体的特异性较好,酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)分析显示抗体具有较高的效价。qRT-PCR实验发现Ha-c-Myc基因在棉铃虫各发育阶段均有表达,在幼龄和大龄幼虫及预蛹中表达量较高,其中预蛹的表达量最高。组织表达结果显示,Ha-c-Myc基因在预蛹不同组织均有一定表达但存在差异,在中肠部位具有高表达,而在表皮和脂肪体表达水平较低。RNA干扰的结果显示,Ha-c-Myc表达的敲降对HaSCP-2基因的转录有较大影响,导致HaSCP-2基因的相对表达水平显著性降低了50%。上述研究表明,利用pET-32a (+)原核表达系统可以有效地表达可溶性Ha-c-Myc蛋白,并且制备高质量的抗Ha-c-Myc多克隆抗体。RNA干扰证明在中肠组织高表达的Ha-c-Myc能促进HaSCP-2基因的转录表达,在棉铃虫的脂质代谢中具有重要作用。本实验结果有助于深入研究Ha-c-Myc在棉铃虫中的潜在功能及其作用机理,为探究绿色农药的新型作用靶标提供实验数据。

    Abstract:

    c-Myc protein encoded by c-Myc (cellular-myelocytomatosis viral oncogene) gene regulates the related gene expression through the Wnt/β-catenin signaling pathway, and has received extensive attention in recent years. The purpose of this study was to express Helicoverpa armigera c-Myc gene (Ha-c-Myc) by using prokaryotic expression system, prepare the polyclonal antibody, examine the spatio-temporal expression profile of Ha-c-Myc, and investigate the possible function of Ha-c-Myc in regulating H. armigera sterol carrier protein-2 (SCP-2) gene expression. The Ha-c-Myc gene was amplified by PCR and cloned into a prokaryotic expression plasmid pET-32a(+). The recombinant plasmid pET-32a-Ha-c-Myc was transformed into Escherichia coli BL21. IPTG was used to induce the expression of the recombinant protein. Protein was purified by Ni2+-NTA column and used to immunize New Zealand rabbits for preparing the polyclonal antibody. The Ha-c-Myc expression levels in different developmental stages (egg, larva, prepupa, pupa, and adult) of H. armigera and different tissues (midgut, fat body, head, and epidermis) of the prepupa were determined by real-time quantitative reverse transcription PCR (qRT-PCR). Ha-c-Myc siRNA was synthesized and transfected into H. armigera Ha cells.The relative mRNA levels of Ha-c-Myc and HaSCP-2 in Ha cells were detected by qRT-PCR. Results showed that the pET-32a-Ha-c-Myc recombinant plasmid was constructed. The soluble Ha-c-Myc protein of about 65 kDa was expressed in E. coli. The polyclonal antibody was prepared. Western blotting analysis suggested that the antibody had high specificity. Enzyme linked immunosorbent assay (ELISA) showed that the titer of the antibody was high. Ha-c-Myc gene expressed at all developmental stages, with high levels in the early and late instars of larva, and the prepupal stage. Tissue expression profiles revealed that Ha-c-Myc expressed in various tissues of prepupa, with high expression level in the midgut, but low levels in the epidermis and fat body. RNAi results showed that the knockdown of Ha-c-Myc expression significantly affected transcription of HaSCP-2, leading to a 50% reduction in HaSCP-2 mRNA expression level. In conclusion, the Ha-c-Myc was expressed through a prokaryotic expression system, and the polyclonal anti-Ha-c-Myc antibody was obtained. Ha-c-Myc may promote the expression of HaSCP-2 and play an important role in the lipid metabolism of H. armigera. These results may facilitate further study on the potential role and function mechanism of Ha-c-Myc in H. armigera and provide experimental data for exploring new targets of green pesticides.

    参考文献
    [1] STONE J, de LANGE T, RAMSAY G, JAKOBOVITS E, BISHOP JM, VARMUS H, LEE W. Definition of regions in human c-myc that are involved in transformation and nuclear localization[J]. Molecular and Cellular Biology, 1987, 7(5):1697-1709.
    [2] ZHOU JN, ZHANG B, WANG HY, WANG DX, ZHANG MM, ZHANG M, WANG XK, FAN SY, XU YC, ZENG Q, JIA YL, XI JF, NAN X, HE LJ, ZHOU XB, LI S, ZHONG W, YUE W, PEI XT. A functional screening identifies a new organic selenium compound targeting cancer stem cells:role of c-Myc transcription activity inhibition in liver cancer[J]. Advanced Science, 2022, 9(22):2201166.
    [3] DANG CV. MYC on the path to cancer[J]. Cell, 2012, 149(1):22-35.
    [4] PATEL JH, LOBODA AP, SHOWE MK, SHOWE LC, MCMAHON SB. Analysis of genomic targets reveals complex functions of MYC[J]. Nature Reviews Cancer, 2004, 4(7):562-568.
    [5] KATO GJ, DANG CV. Function of the c-Myc oncoprotein[J]. The FASEB Journal, 1992, 6(12):3027-3122.
    [6] BLACKWELL TK, KRETZNER L, BLACKWOOD EM, EISENMAN RN, WEINTRAUB H. Sequence-specific DNA binding by the c-Myc protein[J]. Science, 1990, 250(4984):1149-1151.
    [7] DANG CV, BARRETT J, VILLA-GARCIA M, RESAR LM, KATO GJ, FEARON ER. Intracellular leucine zipper interactions suggest c-Myc hetero-oligomerization[J]. Molecular and Cellular Biology, 1991, 11(2):954-962.
    [8] AMATI B, BROOKS MW, LEVY N, LITTLEWOOD TD, EVAN GI, LAND H. Oncogenic activity of the c-Myc protein requires dimerization with Max[J]. Cell, 1993, 72(2):233-245.
    [9] AMATI B, DALTON S, BROOKS MW, LITTLEWOOD TD, EVAN GI, LAND H. Transcriptional activation by the human c-Myc oncoprotein in yeast requires interaction with Max[J]. Nature, 1992, 359(6394):423-426.
    [10] NIKIFOROV MA, KOTENKO I, PETRENKO O, BEAVIS A, VALENICK L, LEMISCHKA I, COLE MD. Complementation of Myc-dependent cell proliferation by cDNA expression library screening[J]. Oncogene, 2000, 19(42):4828-4831.
    [11] HOFFMAN B, AMANULLAH A, SHAFARENKO M, LIEBERMANN DA. The pro-oncogene c-myc in hematopoietic development and leukemogenesis[J]. Oncogene, 2002, 21(21):3414-3421.
    [12] 陈龙, 陈洁平, 林晨. 重组反义c-myc腺病毒诱导HL-60细胞分化的作用[J]. 第三军医大学学报, 2004, 26(17):1553-1556. CHEN L, CHEN JP, LIN C. Effects of recombinant antisense c-myc adenovirus in induction of differentiation of HL-60 cells[J]. Acta Academiae Medicinae Militaris Tertiae, 2004, 26(17):1553-1556(in Chinese).
    [13] JÄRVINEN K, HOTTI A, SANTOS L, NUMMELA P, HÖLTTÄ E. Caspase-8, c-FLIP, and caspase-9 in c-Myc-induced apoptosis of fibroblasts[J]. Experimental Cell Research, 2011, 317(18):2602-2615.
    [14] IACCARINO I, HANCOCK D, EVAN G, DOWNWARD J. c-Myc induces cytochrome c release in Rat1 fibroblasts by increasing outer mitochondrial membrane permeability in a bid-dependent manner[J]. Cell Death & Differentiation, 2003, 10(5):599-608.
    [15] MEYER N, PENN LZ. Reflecting on 25 years with MYC[J]. Nature Reviews Cancer, 2008, 8(12):976-990.
    [16] AMATI B, ALEVIZOPOULOS K, VLACH J. Myc and the cell cycle[J]. Frontiers in Bioscience-Landmark, 1998, 3(4):250-268.
    [17] LUTZ W, LEON J, EILERS M. Contributions of Myc to tumorigenesis[J]. Biochimica Biophysica Acta, 2002, 1602(1):61-71.
    [18] CLAASSEN GF, HANN SR. A role for transcriptional repression of p21CIP1 by c-Myc in overcoming transforming growth factor β-induced cell-cycle arrest[J]. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(17):9498-9503.
    [19] LÜSCHER B, LARSSON LG. The world according to MYC[J]. EMBO Reports, 2007, 8(12):1110-1114.
    [20] GALLANT P, SHIIO Y, CHENG PF, PARKHURST SM, EISENMAN RN. Myc and Max homologs in Drosophila[J]. Science, 1996, 274(5292):1523-1527.
    [21] de la COVA C, JOHNSTON LA. Myc in model organisms:a view from the flyroom[J]. Seminars in Cancer Biology, 2006, 16(4):303-312.
    [22] GALLANT P. Myc function in Drosophila[J]. Cold Spring Harbor Perspectives in Medicine, 2013, 3(10):a014324.
    [23] CHEN W, XU WH. Wnt/β-catenin signaling regulates Helicoverpa armigera pupal development by up-regulating c-Myc and AP-4[J]. Insect Biochemistry and Molecular Biology, 2014, 53:44-53.
    [24] KREBS KC, LAN Q. Isolation and expression of a sterol carrier protein-2 gene from the yellow fever mosquito, Aedes aegypti[J]. Insect Molecular Biology, 2003, 12(1):51-60.
    [25] DYER DH, LOVELL S, THODEN JB, HOLDEN HM, RAYMENT I, LAN Q. The structural determination of an insect sterol carrier protein-2 with a ligand-bound C16 fatty acid at 1.35-Å resolution[J]. The Journal of Biological Chemistry, 2003, 278(40):39085-39091.
    [26] KIM MS, LAN Q. Sterol carrier protein-x gene and effects of sterol carrier protein-2 inhibitors on lipid uptake in Manduca sexta[J]. BMC Physiology, 2010, 10:9.
    [27] DU X, MA HH, ZHANG X, LIU KY, PENG JX, LAN Q, HONG HZ. Characterization of the sterol carrier protein-x/sterol carrier protein-2 gene in the cotton bollworm, Helicoverpa armigera[J]. Journal of Insect Physiology, 2012, 58(11):1413-1423.
    [28] PENG R, FU Q, HONG HZ, SCHWAEGLER T, LAN Q. THAP and ATF-2 regulated sterol carrier protein-2 promoter activities in the larval midgut of the yellow fever mosquito, Aedes aegypti[J]. PLoS One, 2012, 7(10):e46948.
    [29] ZHANG LL, LI D, XU R, ZHENG SC, HE HW, WAN J, FENG QL. Structural and functional analyses of a sterol carrier protein in Spodoptera litura[J]. PLoS One, 2014, 9(1):e81542.
    [30] MA HH, MA YM, LIU XH, DYER DH, XU PY, LIU KY, LAN Q, HONG HZ, PENG JX, PENG R. NMR structure and function of Helicoverpa armigera sterol carrier protein-2, an important insecticidal target from the cotton bollworm[J]. Scientific Reports, 2015, 5:18186.
    [31] 何华伟, 王叶菁, 赵敏健, 位曙光, 赵朋, 蒋文超, 刘莉娜, 赵萍. 家蚕精氨酸激酶原核表达纯化、结构与活性分析[J]. 生物工程学报, 2017, 33(7):1109-1123. HE HW, WANG YJ, ZHAO MJ, WEI SG, ZHAO P, JIANG WC, LIU LN, ZHAO P. Prokaryotic expression, purification and characterization of arginine kinase of Bombyx mori[J]. Chinese Journal of Biotechnology, 2017, 33(7):1109-1123(in Chinese).
    [32] JOSHI M, AGARWAL HC. Site of cholesterol absorption in some insects[J]. Journal of Insect Physiology, 1977, 23(3):403-404.
    [33] LASSER NL, CLAYTON RB. The intracellular distribution of sterols in Eurycotis floridana and its possible relation to subcellular membrane structures[J]. Journal of Lipid Research, 1966, 7(3):413-421.
    引证文献
    网友评论
    网友评论
    分享到微博
    发 布
引用本文

索倩,孙晓燕,张莹,王育婧,刘凯于,杨红,洪华珠,彭建新,彭蓉. 棉铃虫c-Myc基因的原核表达纯化、抗体制备、时空表达谱测定与功能分析[J]. 生物工程学报, 2023, 39(7): 2730-2742

复制
分享
文章指标
  • 点击次数:257
  • 下载次数: 1185
  • HTML阅读次数: 739
  • 引用次数: 0
历史
  • 收稿日期:2022-09-23
  • 录用日期:2023-02-13
  • 在线发布日期: 2023-07-11
  • 出版日期: 2022-07-25
文章二维码
您是第5992685位访问者
生物工程学报 ® 2025 版权所有

通信地址:中国科学院微生物研究所    邮编:100101

电话:010-64807509   E-mail:cjb@im.ac.cn

技术支持:北京勤云科技发展有限公司