title: Isolation and partial characterization of Asian sea bass (Lates calcarifer) Vitellogenin.
abstract: A study was conducted to isolate, partial characterize Asian sea bass (Lates calcarifer) vitellogenin (vtg). Two-year-old juvenile L. calcarifer (n = 10) were given three intraperitoneal injections of 17-β estradiol (E2) at a dose of 2 mg/kg body weight to induce vitellogenesis. Blood was collected 3 days after the last injection, and plasma was purified through gel filtration chromatography. A broad single symmetrical peak consisting of vtg molecule was produced. Protein concentration was 0.059 mg/ml as determined by Bradfrod assay using bovine serum albumin as a standard. The protein appeared as one circulating form in Native PAGE considering the dimeric form of putative vtg with molecular weight of 545 kDa. In SDS-PAGE under reducing conditions, two major bands appeared at 232.86 and 118.80 kDa and minor bands at 100.60, 85.80 and 39.92 kDa, respectively. The purified vtg was used to generate a polyclonal antibody, and the specificity of antibody was assessed by Western blot analysis. Two major bands were immunoreacted, but no cross-reactivity was observed with plasma from non-induced males. The protein was characterized as phosphoglycolipoprotein as it positively stained for the presence of lipid, phosphorus and carbohydrate using Sudan Black B, methyl green and periodic acid/Schiff reagent solution, respectively. The amino acid composition was analyzed by high sensitivity amino acid analysis that showed high percentage of non-polar amino acids (~48 %). The results suggest the potential utilization of vtg as a basis tool to further study about reproductive physiology of this important economical species.
authors.affiliation: Department of Aquaculture, Faculty of Agriculture, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
authors.forename: N M R
authors.initials: NM
authors.lastname: Fazielawanie
authors.forename: S S
authors.initials: SS
authors.lastname: Siraj
authors.forename: S A
authors.initials: SA
authors.lastname: Harmin
authors.forename: M Y
authors.initials: MY
authors.lastname: Ina-Salwany
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001492:Bass / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western / Q000662:veterinary; D002850:Chromatography, Gel / Q000662:veterinary; D002851:Chromatography, High Pressure Liquid / Q000662:veterinary; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004958:Estradiol / Q000008:administration & dosage / Q000494:pharmacology; D008296:Malaysia; D014818:Vitellogenesis / Q000187:drug effects; D014819:Vitellogenins / Q000097:blood / Q000302:isolation & purification*
chemical_list: D000596:Amino Acids; D014819:Vitellogenins; D004958:Estradiol