The lysate was centrifuged at 10 000 rpm at 4°C for 5 min and the supernatant was collected for rgLh determination in series at0h,24h,48h,72h,96h, and 120h postinfection. Using BCA methods (Biorad), the rgLh in supernatant was quantitated and then used for in vivo and in vitro assays. It was also analyzed on the SDS-PAGE and PAGE; the PAGE was stained for glycoprotein using the silver staining method.
Detection of gnrh, cyp19a1a and cyp19a1b, lhb and cga mRNA Expression
The hCG (1 IU/bw-g), rgLh (5X10~3, 5X10~2, and 5X10~Vg/bw-g), physiological salt solution (PS) (1 il/bw-g), and the negative control (NC) (0 ig/bw-g), which derived from the insect cells transformed with an expression vector not containing rgLh, were injected intraperitoneally into 2-yr-old orange-spotted groupers (six fish per treatment), respectively. At 18 h postinjection, fish were first anesthetized by ice, blood was collected from caudal vein, and the serum was stored at —20°C for radioimmunoassay. After that, total RNA sample was harvested from pituitary, gonad, and hypothalamus using Trizol reagent (Invitrogen) for the further determination of mRNA levels of gnrh, cyp19a1a and cyp19a1b, cga, and Ihb by RT-PCR.