Synthesis of partial amino acid sequence of crustacean hyperglycemic hormone from Macrobrachium rosenbergii and antibody production of sequence analysis
1998
Nanthika Panchan | Paisarn Sithigorngul | Tirayut Vilaivan | Weerawan Sithigorngul | Amorn Petsom (Chulalongkorn Univ., Bangkok (Thailand). Inst. of Biotechnology and Genetic Engineering)
Heptapeptide (YANAVQV-NH2=T-) and octapeptide (YANAVQTV-NH2=T+), the putative C-terminal of crustacean hyperglycemic hormone (CHH) from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii, were synthesized by solid phase peptide synthesis and conjugated to bovine serum albumin, then used for immunization into swiss mice. Specificify and quality of the antisera against both peptides were determined by indirect immunoperoxidase ELISA. The best response antiserum agianst each peptide was used to detect the presence of the natural CHH in the eyestalk extract after separated by one step RP-HPLC with Dot-ELISA. The immunoreactive substances were found in fraction 30 with anti T- antiserum and in fraction 38 with anti T+ antiserum. However, the CHH bioactivity was found in only fraction 37-39. Immunocytochemical localization using the anti T-antiserum did not show any specific staining, but the anti T+ antiserum revealed staining on a group of 16-40 neurons in X-organ and their processes leading to the sinus gland. These evidences show strong indication that the anti T+ antiserum can bind to the natural CHH therefore this isoform of the CHH in M. rosenbergii consists of 72 residues and Threonine is likely to be present at position 71.
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