中华绒螯蟹蜕皮抑制激素1(Ers-MIH1)-GST融合蛋白在大肠杆菌中的表达

Expression of Eriocheir japonica sinensis molt-inhibiting hormone-1-GST fusion protein in E. coli

蜕皮抑制激素(molt-inhibitinghormone,MIH)属甲壳动物高血糖激素(crustaceanhyperglycemichormone,CHH)家族。MIH抑制Y 器官蜕皮激素的合成。以中华绒螯蟹(Eriocheirjaponicasinensis)眼柄总RNA为模板,根据中华绒螯蟹蜕皮抑制激素1(Eriocheirjaponicasinensismolt inhibitinghormone 1,Ers MIH1)基因序列设计引物,用RT PCR的方法获得了Ers MIH1基因成熟肽的cDNA片段。序列分析表明,Ers MIH1基因成熟肽编码区含有228bp,编码75个氨基酸残基,与已发表的序列一致。将该cDNA片段插入到中间载体pMD18 T中,酶切后再将该片段插入到pGEX 4T 1表达载体中,构建成表达质粒pGEX 4T MIH1。在BL21细胞中经IPTG诱导表达,得到GST MIH1的融合蛋白。SDS PAGE分析表明,经0.1mmol/LIPTG诱导4h,发现大量GST MIH1融合蛋白表达,在分子量(Mr)为34kD处有1条特异的蛋白质条带。中华绒螯蟹蜕皮抑制激素1融合蛋白的成功表达为进一步深入研究MIH在中华绒螯蟹蜕皮过程中的作用机制奠定了基础。

Molt-inhibiting hormone (MIH) is a member of CHH (crustacean hyperglycemic hormone) family. MIH negatively regulates the synthesis of ecdysteroids in Y-organs and plays an important role in the regulation of growth and development. In this study, a primer pair was designed based on the complete sequence of molt-inhibiting hormone-1 (Ers-MIH1) gene of mitten crab (Eriocheir japonica sinensis) obtained by Song et al of this lab. The mature peptide-coding cDNA fragment of Ers-MIH1 gene was amplified by RT-PCR using the total RNA extracted from eyestalk neural tissues as a template. The analysis of the sequence data indicated that the coding region of the cDNA fragment, which encoded 75 amino acid residues, is 228 bp in size. The sequence is identical with that of Ers-MIH1 coding region reported by Song et al. The PCR product was cloned into the pMD18-T vector to produce the new construct pMD18-T-MIH1. The cloned Ers-MIH1 fragment was cut out again with two restriction enzymes and was inserted into the prokaryotic expression vector, pGEX-4T-1, to produce the expression vector pGEX-4T-MIH1. The recombinant plasmid was transformed into E.coli BL21(DE3). GST-MIH1 fusion protein was obtained after the addition of IPTG into the growth media. SDS-PAGE analysis revealed that the GST-MIH1 fusion protein was highly expressed after the induction with IPTG for 4 h. A protein band of 34 kD Mr appeared on SDS-PAGE gel. It is anticipated that the fusion protein will be proved useful for studies on structure and function of MIH.