Molecular Cloning And Expression Analysis Of Crustacean Hyperglycemic Hormone From Swimming Crab (Portunus Trituberculatus)

The eyestalk X-organ -sinus gland complex (XO-SG) is the main control center of neuroendocrine in crustacean,which Synthesis and secretion of hyperglycemic hormone family of neuropeptides, including crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), gonad-inhibiting hormone (GIH) and mandibular organ-inhibiting hormone (MOIH) , which cooperatively regulate the complicated physiological and biochemical process, such as growth, reproduce and molt in crustacean.Sequence alignment of CHH/MIH/GIH/MOIH indicate that the CHH precursor-related peptide(CPRP) which between the signal peptide and mature peptide was only found in CHH cDNA, while the signal peptide and mature peptide in the cDNA sequence of MIH / GIH / MOIH are directly connected to ench other. Thus, according to the genetic structure, the CHH peptide family can be divided into two CHH sub-family :CHH and MIH / GIH / MOIH, or named CHHâ… family and CHHâ…¡Family.In this paper, a full-length of crustacean hyperglycemic hormone cDNA from XO-SG in Portunus trituberculatus (Pot-CHH) was cloned by the reverse transcription-polymerase chain reaction (RT-PCR), with degenerate nucleotide primers that designed based on the amino acid sequence of other crustacean CHHs, which is 1952bp of full length and the GenBank No. is EU395808. Sequence analysis showed that Pot-CHH sequence has 86bp of 5'uncoding region, 417bp of open-reading frame and 1449bp of 3'uncoding region . The Signal 3.0 Server predicted result showed that the signal peptide has 27 amino acid residues, CPRP has 35 amino acid residues and the CHH mature peptide has 75 amino acid residues respectively. The cleavage site of CPRP and mature peptide is the KR conservative sites. 3 -'uncoding region contains a conserved polyadenylation signal (AGTAAA) which is 51bp in distance with downstream Poly (A). Sequence alignment results showed that Pot-CHH has the highest homology identity with Charybdis japonica and Scylla olivacea,at least 80%;and has exceeded 60% homology identity with other crustacean CHH. Tissue Expression studies showed that Pot-CHH was detected in the XO-SG, thoracic ganglion, abdominal ganglion, Y-organ and gut,while it was not detected in retina, heart, testis, ovary, muscle, brain, gills and hepatopancrea. This result indicated that Pot-CHH has specific tissue expression which maybe associated with physiological functions of CHH in crustacean.We used pET-28a vector to construct the Pot-CHH recombinant protein expression vector: pET-CHH and His-pET-CHH, transformed to BL21 (DE3) and then inducted byIPTG, successfully obtained two kinds of recombinant proteins (Pot-CHH recombinant peptide and His-Pot-CHH recombinant peptide). And the polyclonal antibody was prepared through the injection of the Pot-CHH purified recombinant protein injection to ICR mouse, which not only specific binding to Pot-CHH recombinant protein, but also with the recombinant His-Pot-CHH recombinant peptide. The preparation of CHH recombinant and polyclonal antibody lay a foundation of more accurate study of variety of physiological functions of CHH in the future.