Production of recombinant orange-spotted grouper (Epinephelus coioides) luteinizing hormone in insect cells by the baculovirus expression system and its biological effect.
The cDNA sequence encoding orange-spotted grouper lhb (LHbeta) and cga (GTHalpha) subunits were cocloned into baculovirus transfer vectors and expressed in insect Sf9 cells. The results showed that two bands of 15.6 kDa and 11.4 kDa could be detected by SDS-PAGE and a band of 30 kDa could be detected by native PAGE. The recombinant grouper Lh (rgLh) could stimulate the secretion of testosterone (T) and estradiol-17beta (E2) from the gonad in a static incubation system in a time-dependent, but not a dose-dependent, manner. Using in vivo bioassay, the mRNA levels of two aromatases (cyp19a1a [P450aromA] and cyp19a1b [P450aromB]), gnrh (GnRH), lhb, and cga in the pituitary, gonad, and hypothalamus were determined in different groups of orange-spotted groupers treated respectively with rgLh, human chorionic gonadotropin (hCG), and a culture medium of insect cells transformed with an expression vector without lhb and cga subunits. The mRNA levels of cyp19a1a and cyp19a1b rose dramatically after injecting rgLh intraperitoneally, which was consistent with the secretion of sex steroid hormones. Interestingly, the mRNA levels of gnrh dropped in the pituitary, hypothalamus, and gonad, and the mRNA levels of lhb and cga in the pituitary of the experimental group expressed at a higher level than that of the hCG group. These results are in accord with the long positive feedback loop of Lh on gonad sex steroid hormones and the short negative feedback loop of Lh on gnrh mRNA levels. These results indicate that the rgLh is successfully expressed by the baculovirus-insect expression system and that the rgLh has biological activity.
Cui
Miao
M
State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, People's Republic of China.
Li
Wensheng
W
Liu
Wen
W
Yang
Kai
K
Pang
Yi
Y
Haoran
Lin
L
D000818:Animals; D001141:Aromatase / Q000235:genetics / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D001492:Bass / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D015292:Glycoprotein Hormones, alpha Subunit / Q000096:biosynthesis / Q000235:genetics; D006066:Gonads / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007313:Insecta / Q000166:cytology; D007986:Luteinizing Hormone / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D037101:Luteinizing Hormone, beta Subunit / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D013739:Testosterone / Q000378:metabolism; D014162:Transfection / Q000379:methods
D018076:DNA, Complementary; D015292:Glycoprotein Hormones, alpha Subunit; D037101:Luteinizing Hormone, beta Subunit; D011994:Recombinant Proteins; D013739:Testosterone; D007986:Luteinizing Hormone; D001141:Aromatase