Department of Life Science, International Christian University, Mitaka, Tokyo 181-8585, Japan.
Gen Comp Endocrinol. 2010 Jul 1;167(3):379-86. doi: 10.1016/j.ygcen.2010.01.003. Epub 2010 Jan 11.
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.
重组日本鳗鲡（Anguilla japonica）促卵泡激素（reFSH）和促黄体生成素（reLH）由杆状病毒在桑蚕（Bombyx mori）幼虫中产生。日本鳗鲡性腺激素亚基（即 FSHβ、LHβ和共同α）的 cDNA 被引入杆状病毒中，该病毒在 B. mori 培养细胞中繁殖后感染桑蚕幼虫。从大约 250 只感染的幼虫中获得了含有 reFSH 或 reLH 的 100ml pooled 血淋巴溶液。10ml 血淋巴应用于 Ni 亲和层析，分别获得了 5.6 和 3.5mg 的部分纯化的 reFSH 和 reLH。使用 Western blot 分析，估计原始血淋巴中 reFSH 和 reLH 的浓度分别为 2.2 和 1.1mg/ml。在体外和体内评估了 reFSH 和 reLH 的生物学活性。纯化的 reFSH 和 reLH 诱导鳗鲡卵母细胞体外成熟，并且含有 reFSH 或 reLH 的血淋巴在体内给药可诱导性未成熟的日本鳗鲡的精子发生。本研究表明，杆状病毒-桑蚕系统可以大量生产具有生物活性的重组鱼类促性腺激素，用于生殖内分泌学和/或鱼类水产养殖的研究。
