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.
Highlights
Lates calcarifer, known as barramundi, Asian sea bass or locally called siakap, is native to coastal Australia, Southeast and Eastern Asia, and India (Luna 2008)
Vitellogenin was purified from plasma of E2-treated juvenile L. calcarifer by gel filtration chromatography using Sepachryl HR-300 column (GE Healthcare BioScience)
A single symmetrical peak was obtained from purification of E2-treated fish indicating the presence of vtg molecule
Summary
Known as barramundi, Asian sea bass or locally called siakap, is native to coastal Australia, Southeast and Eastern Asia, and India (Luna 2008). This species are farmed in cages, as well as in fresh water and salt water ponds (Webster and Lim 2002). Sea bass has gained growing importance in aquaculture both as recreational and commercial fish, with a high and fairly stable price (Luna 2008). The reproductive biology of many fish species has been studied by analyzing the vitellogenin (vtg) levels in the blood plasma (Susca et al 2001)
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