摘要
【目的】c-Myc是近年来研究较多的转录因子,也是受Wnt/β-catenin信号通路调节的重要靶标。本研究旨在克隆棉铃虫Helicoverpa armigera c-Myc基因,从核酸水平初步调查c-myc在滞育和非滞育蛹脑中的表达情况,同时制备其蛋白的多克隆抗体。【方法】通过RACE方法克隆棉铃虫c-myc基因的c DNA,运用RT-PCR方法比较滞育和非滞育蛹脑中Har-c-myc基因的表达情况。根据获取的序列构建原核表达载体,在大肠杆菌Escherichia coli中进行表达,纯化后免疫新西兰兔,制备了多克隆抗体。【结果】克隆了棉铃虫c-myc基因,核酸水平的研究表明滞育蛹脑中c-myc表达水平明显低于非滞育蛹脑。成功地在大肠杆菌中表达了c-Myc部分肽段并通过镍柱纯化获得了较纯的重组蛋白。制备的c-Myc抗体效价达到了1∶125 000。【结论】滞育蛹脑中Har-c-myc的表达下调。获得了抗棉铃虫c-Myc的多克隆抗体。本研究的成果为后续进一步深入研究棉铃虫Wnt/β-catenin信号通路在棉铃虫发育中的作用奠定了基础。
[ Aim] c-Myc is an intensively studied transcription factor and also an important downstream target of Wntβ-catenin signaling pathway. The objective of this research was to clone the c-myc cDNA from Helicoverpa armigera, to investigate the expression of Har-c-myc in mRNA level, and to prepare polyclonal antibody against Har-c-Myc. [ Methods] Har-c-myc was cloned by RACE. The mRNA levels of Har-c-myc in the brain of non-diapause and diapause pupae were investigated by RT-PCR. Prokaryotic expression vector of Har-c-myc was constructed and the recombinant protein was expressed in Escherichia coli. The purified recombinant protein was injected into a New Zealand rabbit to generate polyclonal antibody. The antibody titer was determined by ELISA. [ Results ] Har-c-myc cDNA was cloned successfully from H. armigera. The mRNA levels of Har-c-Myc were significantly lower in the brain of diapause pupae than in the brain of non-diapause pupae. Recombinant Har-c-Myc was successfully expressed in E. coli and purified by Ni-NTA agarose column. The titer of the antibody against Har-c-Myc was estimated by ELISA as high as 1 ： 125 000. [ Conclusion] The expression of Har-c-myc is down- regulated in the brain of diapause pupae. Antibody against Har-c-Myc was obtained. This study lays a foundation for further investigating the function of Wnt/β-catenin in the diapause of H. armigera.
出处
《昆虫学报》
CAS
CSCD
北大核心
2015年第2期115-121,共7页
Acta Entomologica Sinica
基金
国家自然科学基金项目(31230066)
国家重点基础研究发展计划项目(2012CB114101)
