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

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

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 armigera） c-Myc（Ha-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.