关键词: 棉铃虫, 蜕皮调节转录因子, 重组表达, 包涵体纯化, 抗体制备

Abstract: Molt-regulating transcription factor 3 (hormone receptor 3，HR3) plays important roles in regulating expression of tissue-specific genes involved in insect molting and metamorphosis, which initiates expression of chitinase and proteases but represses expression of larval cuticular protein and eclosion hormone during molting. The cDNA encoding the molt-regulating transcription factor 3 was cloned with RT-PCR using gene specific primers from Helicoverpa armigera. A recombinant expression plasmid, HHR3-pGEX-4T-1, was constructed and the protein was expressed in BL21 strain of Escherichia coli. However, the expressed target protein fo rmed complete inclusion bodies of the molecular size 94 kD. The inclusion bodies were solubilized by being denatured with CAPS（3-［cyclohexylamino］-1-propanesulfonic acid）and refolded through dialysis against Tris-HCl buffer. After cleavage with thrombin, the target protein was separated by SDS-PAGE and confirmed with N-terminal amino acid sequencing. This recombinant HHR3 was used as an antigen for preparation of rabbit polyclonal antibody. The immunoblotting results indicated that the antibody was specific to HHR3 from H. armigera and can be used for further research on the function and regulation of HHR3 Moreover, HHR3 showed high expression in the fat bodies of 5th toward 6th inst ar molting larvae and declined at 24 h post molting. HHR3 was also detected from ovaries of the adult H. armigera, but not det ectable from midguts the 6th instar larvae of H. armigera at 72 h after molting.

Key words: Helicoverpa armigera, molt-regulating transcription factor, recombinant expression, purification, antibody production