Expression Of Eriocheir Japonica Sinensis Molt-inhibiting Hormone-1 (Ers-MIH1) Fusion Protein In E.coli

Molt-inhibiting hormone belongs to the CHH/MM/GIH (VIH) family that includes the crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), vitellogenesis/gonad-inhibiting hormone (VIH/GIH) and mandibular organ-inhibiting hormone (MOIH). These peptides have common amino acid compositions and organizations. All members of this family generally have molecular weights of 7-9 kD and consist of approximately 70-78 amino acids and three disulfide bridges.MIH is produced in the medulla terminalis of the X-organ (MTXO) and stored in the sinus gland of the eyestalk. MIH negatively regulates the synthesis of ecdysteroids in Y-organs and plays an important role in the regulation of growth and development.Although recent progress has been made in understanding the structure and function of MIH, important questions remain unanswered. The detail mechanism of MIH actions is unknown, and the expression of MIH during development has not been investigated. As an initial step towards answering remaining questions regarding MIH structure, function and mechanism, we report expression of Eriocheir japonica sinensis molt-inhibiting hormone-1 (Ers-MIHl) fusion protein in E. coli.Chapter 1: Structure and function of crustacean neuropeptidesA variety of neuropeptides have been found in the sinus gland, pericardial organ, nervous and other endocrine organs from different crustaceans, for example CHH/MIH/VIH (GIH) neuropeptide family, crustacean cardio active peptide, allatostatin-related peptide, tachykinin, FMRF-amide related peptides (FaRPs) and chromatophorotropins etc. These neuropeptides play important roles in the regulation of reproduction, development, molting, carbohydrate metabolism, muscle contractility and pigment moving etc. This chapter reviewed the structure and function of presently known curstacean neuropeptides.Chapter 2: PCR cloning of the molt-inhibiting hormone-1 gene from the mitten crab, Eriocheirjaponica sinensisA primer pair was designed based on the complete sequence of molt-inhibiting hormone-1 (Ers-MIHl) gene of the mitten crab {Eriocheir japonica sinensis) obtained by Song et al of this lab. The mature peptide-coding cDNA fragment of Ers-MIHl was amplified 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-MIHl gene coding region reported by Song et al.Chapter 3: Cloning of the coding region fragment of the Ers-MIHl gene from genomic DNA by SOE methodUsing the genomic DNA extracted from muscle tissues of the mitten crab, Eriocheir japonica sinensis as template, the two coding region fragments of the Ers-MIHl gene were linked by SOE method. Two pairs of primers were designed based on the complete sequence of molt-inhibiting hormone-1 (Ers-MIHl) gene of the mitten crab and a part of linker peptide gene sequence was added on 3'and 5' terminals respectively. The PCR products were mixed and re-amplified and the coding region cDNA fragment was spliced by overlap extension. The product was inserted into the vector pMD1 8-T. The analysis of the sequence data shows that it is identical with that of the mature peptide coding cDNA fragment of Ers-MIH1 gene.Chapter 4: Construction of prokaryotic expression vector of Ers-MIHl geneThe cDNA fragment of Ers-MIHl gene was cloned into the pMDl 8-T vector to produce the new construct, pMD18-T-MIHl. The Ers-MIHl fragments of the construction plasmids pMD18-T-MIHl were digested with BamH I + Xho I and purified via agarose-gel electrophoresis. These fragments were inserted into the prokaryotic expression vector pGEX-4T-l which had been cleaved with the same enzymes and purified to produce the new plasmid, pGEX-4T-MIHl. Then the construction plasmid was identified using PCR, double enzymes digestion and gene sequencing. The results showed that the expression plasmid pGEX-4T-MIHl was successfully reconstructed.Chapter 5: Expression of Eriocheir...