棉铃虫激活增强子结合蛋白AP-4基因的原核表达、多克隆抗体制备、表达谱分析及功能鉴定  

Prokaryotic expression, polyclonal antibody preparation, expression profiling and functional characterization of the activating enhancer binding protein AP-4 gene from the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

作　　者：王婷婷 索倩[1,2,3] 孙晓燕 马跃敏 刘凯于 彭建新[1,2,3] 彭蓉[1,2,3] WANG Ting-Ting;SUO Qian;SUN Xiao-Yan;MA Yue-Min;LIU Kai-Yu;PENG Jian-Xin;PENG Rong(School of Life Sciences, Central China Normal University, Wuhan 430079, China;Institute of Entomology, Central China Normal University, Wuhan 430079, China;Hubei Provincial Key Laboratory of Genetic Regulation and Integrative Biology, Institute of Evolution and Ecology, Central China Normal University, Wuhan 430079, China)

机构地区：[1]华中师范大学生命科学学院,武汉430079 [2]华中师范大学昆虫学研究所,武汉430079 [3]华中师范大学进化与生态学研究所,遗传调控与整合生物学湖北省重点实验室,武汉430079

出　　处：《昆虫学报》2019年第9期1028-1037,共10页Acta Entomologica Sinica

基　　金：国家重点研发计划(2017YFD0200400);国家自然科学基金项目(31401762,31672355);武汉市青年科技晨光计划项目(2017050304010320)

摘　　要：【目的】激活增强子结合蛋白4(activating enhancer binding protein 4, AP-4)是近年来备受关注的一类功能广泛的转录因子,参与Wnt/β-catenin信号通路的调控。本研究旨在研究棉铃虫Helicoverpa armigera HaAP-4基因的原核表达并制备多克隆抗体,明确HaAP-4基因的时空表达谱,初步探究HaAP-4对棉铃虫胆固醇载体蛋白2(sterol carrier protein-2, SCP-2)基因(HaSCP-2)表达的调控作用。【方法】通过PCR扩增棉铃虫HaAP-4基因片段,将其克隆至pET-28a原核表达载体,转化至大肠杆菌Escherichia coli BL21,经IPTG诱导表达,采用镍柱纯化重组蛋白,免疫新西兰兔制备多克隆抗体。运用RT-qPCR检测HaAP-4基因在棉铃虫不同发育阶段(卵、幼虫、预蛹、蛹和成虫)以及5龄幼虫和预蛹不同组织(中肠、脂肪体、头和表皮)中的表达水平。设计并合成HaAP-4 siRNA,转染棉铃虫Ha细胞,通过RT-qPCR检测和分析使用HaAP-4 siRNA进行RNA干扰后Ha细胞中HaAP-4和HaSCP-2基因mRNA的表达情况。【结果】成功构建pET-28a-HaAP-4重组表达质粒,在大肠杆菌细胞中获得高效表达的可溶性蛋白,目的蛋白大小约为55 kD,经纯化获得了纯度较高的重组蛋白,免疫新西兰兔成功制备了多克隆抗体,通过Western blotting检测显示抗体具有较好的特异性,ELISA分析表明抗体效价较高。发育阶段特异性表达结果显示,HaAP-4基因在棉铃虫不同发育阶段均有表达,其中在卵期、5龄幼虫期和预蛹期的表达量较高。组织表达结果表明,HaAP-4基因在5龄幼虫和预蛹不同组织中均有表达,且在中肠和头部具有高表达,而在表皮和脂肪体中表达量较低。RNA干扰实验发现,HaAP-4基因的敲降对HaSCP-2基因转录有显著影响,HaSCP-2基因mRNA表达水平下降了约55%。【结论】利用pET-28a原核表达系统能有效地在体外表达可溶性的HaAP-4,并制备了较好的多克隆抗体。RNAi实验结果提示,在中肠内高表达的HaAP-4基因能促进H【Aim】 Activating enhancer binding protein 4(AP-4), which is involved in the Wnt/β-catenin signaling pathway, is an important transcription factor that has attracted extensive attention in recent years. This study aims to express HaAP-4 gene of Helicoverpa armigera by using prokaryotic expression system, to prepare the polyclonal antibody, to determine the spatio-temporal expression profiles of HaAP-4 and to investigate the possible role of HaAP-4 in regulating the expression of sterol carrier protein-2 gene(HaSCP-2) of H. armigera.【Methods】 The HaAP-4 gene of H. armigera was amplified by PCR and cloned into prokaryotic expression vector pET-28 a. The recombinant vector was transformed into Escherichia coli BL21 strain. The recombinant protein was expressed by IPTG induction and purified by Ni-NTA column. The polyclonal antibody was prepared by immunizing New Zealand rabbits with the purified recombinant protein. The expression levels of HaAP-4 in different developmental stages(egg, larva, prepupa, pupa and adult) and different tissues(midgut, fat body, head and epidermis) of the 5 th instar larva and prepupa of H. armigera were determined by RT-qPCR. HaAP-4 siRNA was synthesized and transfected into Ha cells of H. armigera. The mRNA levels of HaAP-4 and HaSCP-2 in Ha cells after RNAi with HaAP-4 siRNA were detected by RT-qPCR.【Results】 The recombinant expression vector pET-28 a-HaAP-4 was successfully constructed. The soluble HaAP-4 protein of about 55 kD was effectively expressed in E. coli. The highly purified recombinant protein was obtained by purification, and the polyclonal antibody was successfully prepared using the purified recombinant protein. Western blotting results showed that the antibody had high specificity, and ELISA results revealed that the antibody had high titer. Developmental stage-specific expression profiles revealed that HaAP-4 was expressed in all developmental stages, with high expression levels in the egg, 5 th larval and prepupal stages. Tissue expression profiles reveale

关 键 词：棉铃虫 激活增强子结合蛋白 原核表达 多克隆抗体 表达谱 RNA干扰 

分 类 号：Q966[生物学—昆虫学]