Production of recombinant Japanese eel gonadotropins by baculovirus in silkworm larvae.
Recombinant follicle-stimulating hormone (reFSH) and luteinizing hormone (reLH) of the Japanese eel Anguilla japonica were produced by baculovirus in silkworm Bombyx mori larvae. cDNAs encoding Japanese eel gonadotropin subunits (i.e., FSH beta, LH beta, and common alpha) were introduced into the baculovirus, which was infected into silkworm larvae after propagation of the recombinant virus in B. mori culture cells. A 100ml solution of pooled hemolymph from silkworm larvae containing reFSH or reLH were obtained from approximately 250 infected larvae. Ten milliliters of hemolymph were applied to Ni-affinity choromatography, and 5.6 and 3.5mg of partially purified reFSH and reLH were obtained, respectively. Using Western blot analysis concentrations of reFSH and reLH in the original hemolymph was estimated to be 2.2 and 1.1mg/ml, respectively. Biological activities of reFSH and reLH were assessed in vitro and in vivo. Purified reFSH and reLH induced eel oocyte maturation in vitro, and administration of hemolymph containing reFSH or reLH induced spermatogenesis in vivo in sexually immature Japanese eel. The present study indicates that a baculovirus-silkworm system could produce large amounts of biologically active recombinant fish gonadotropins for use in investigations in reproductive endocrinology and/or aquaculture of fish.
Kobayashi
Makito
M
Department of Life Science, International Christian University, Mitaka, Tokyo 181-8585, Japan. makito@icu.ac.jp
Hayakawa
Youichi
Y
Park
Woodong
W
Banba
Akira
A
Yoshizaki
Goro
G
Kumamaru
Katsuya
K
Kagawa
Hirohiko
H
Kaki
Hiroki
H
Nagaya
Hidekazu
H
Sohn
Young Chang
YC
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D012831:Bombyx / Q000254:growth & development / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004353:Drug Evaluation, Preclinical; D004524:Eels / Q000235:genetics*; D005260:Female; D005822:Genetic Vectors / Q000008:administration & dosage; D006062:Gonadotropins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D007814:Larva / Q000378:metabolism; D008297:Male; D008954:Models, Biological; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D009866:Oogenesis / Q000187:drug effects / Q000502:physiology; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D013091:Spermatogenesis / Q000187:drug effects / Q000502:physiology; D014161:Transduction, Genetic / Q000379:methods
D006062:Gonadotropins; D011994:Recombinant Proteins