Production of recombinant Japanese eel gonadotropins by baculovirus in silkworm larvae
Production of recombinant Japanese eel gonadotropins by baculovirus in silkworm larvae
复制标题
DOI：
10.1016/j.ygcen.2010.01.003
复制
发表时间：
2010-07-01
影响因子：
2.7
通讯作者：
Sohn, Young Chang
中科院分区：
文献类型：
--
作者：
Kobayashi, Makito;Hayakawa, Youichi;Sohn, Young Chang
日本鳗鲡的重组卵泡刺激素（reFSH）和黄体生成素（reLH）是由家蚕Bombyx mori幼虫中的杆状病毒产生的。将编码日本鳗鱼促性腺激素亚基（即 FSH β、LH β 和普通 α）的 cDNA 引入杆状病毒中，重组病毒在 B. mod 培养细胞中繁殖后感染蚕幼虫。从大约 250 只受感染的幼虫中获得了 100 ml 含有 reFSH 或 reLH 的蚕幼虫合并血淋巴溶液。将10毫升血淋巴进行Ni亲和层析，分别获得5.6和3.5毫克部分纯化的reFSH和reLH。使用蛋白质印迹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 的生物活性。纯化的reFSH和reLH在体外诱导鳗鱼卵母细胞成熟，并且给予含有reFSH或reLH的血淋巴在性成熟的日本鳗鱼体内诱导精子发生。本研究表明杆状病毒-蚕系统可以产生大量具有生物活性的重组鱼促性腺激素，用于鱼类生殖内分泌学和/或水产养殖的研究。 (C) 2010 Elsevier Inc. 保留所有权利。
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. mod culture cells. A 100 ml 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 3.5 mg 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 1.1 mg/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. (C) 2010 Elsevier Inc. All rights reserved.