棉铃虫蜕皮调节转录因子在大肠杆菌中的重组表达及其包涵体纯化

Recombinant expression and purification of inclusion bodies of the molt-regulating transcription factor HHR3 from Helicoverpa armigera (Lepidoptera: Noctuidae)

蜕皮调节转录因子 (hormonereceptor 3,HR3)在昆虫蜕皮过程中启动蜕皮相关早期基因簇表达 ,并抑制蜕皮相关晚期基因簇表达 ,对昆虫蜕皮级联反应起着关键的调控作用。利用合成的特异性引物通过RT_PCR扩增了棉铃虫Helicoverpaarmigera蜕皮调节转录因子 (HHR3) ,并与pGEX_4T_1载体连接 ,在大肠杆菌EscherichiacoliDH5α内进行扩增 ,经过PCR筛选获得了HHR3_pGEX_4T_1重组质粒。用该质粒转化大肠杆菌表达菌株BL2 1并进行诱导表达 ,获得了与谷胱甘肽_S转移酶 (GST)融合表达的HHR3包涵体 ,分子量在 94kD左右 ,通过无离子去垢剂CAPS(3_[cyclohexylamino]_1_propanesulfonicacid)变性、复性后获得了可溶性GST_HHR3融合蛋白 ,经凝血酶裂解和SDS_PAGE分离得到纯化的HHR3,经蛋白质N_端测序确认表达正确。用重组表达的HHR3免疫家兔 ,制备了兔抗HHR3多克隆抗体 ,免疫印迹检测显示该抗体对HHR3有特异性识别能力 ,可以用于HHR3功能与调控等下游研究。免疫印迹检测结果还表明 ,HHR3在 5龄向 6龄蜕皮的幼虫脂肪体中高表达 ,在进入 6龄 2 4h的幼虫脂肪体中含量明显下降 ,在 6龄 72h的幼虫中肠中没有检测到HHR3表达 ;成虫卵巢中有HHR3表达。

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 formed 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 instar molting larvae and declined at 24 h post molting. HHR3 was also detected from ovaries of the adult H. armigera, but not detectable from midguts the 6th instar larvae of H. armigera at 72 h after molting.