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Expression of biologically active red seabream gonadotropins in a baculovirus system

具有生物活性的红鲷促性腺激素在杆状病毒系统中的表达

基本信息

批准号：
16580163
负责人：
GEN Koichiro
金额：
$ 2.37万
依托单位：
National Research Institute of Aquaculture, Fisheries Agency
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

Recent advances in recombinant gene technology have enabled the production of biologically active fish gonadotropins (GTHs : FSH and LH). However, an efficient expression method for the production of GTHs has not yet been developed. In this study, we attempted to produce red seabream GTHs in silkworm larvae using two different baculovirus expression systems. To synthesize recombinant red seabream heterodimeric GTHs, two strategies were employed. The first utilized a dual vector expression strategy, in which each GTH subunit (α, FSHβ and LHβ) was expressed from a separate comparative vector in a single host. The second method used a bicistronic expression strategy, in which both GTH subunit cDNAs were expressed from the vector. Either a dual or a bicistronic expression vector inoculated into silkworm larvae of the 5th instar early stage. Five days after inoculation, the hemolymph of the larvae containing recombinant FSH and LH was collected, and examined of biochemical property and biological activity. Western blot analysis revealed that by using a dual vector expression system, each subunit of FSH and LH was successfully synthesized and secreted into the hemolymph, allowing the synthesis of heterodimeric molecules. In the case of the bicistronic vector coexpressing GTH subunits, the yields of FSHα and LHα subunits was very low, whereas those of both FSHβ and LHβ subunits were high. The biological activities of GTHs produced by a dual vector system were comparable to those of native GTHs in in vitro steroid hormone production. Both recombinant FSH and LH stimulated the in vitro 11-ketotestosterone production in sliced testis of red seabream. Furthermore, recombinant LH stimulated the in vitro production of estradiol-17β by ovarian follicles in a concentration-dependent manner. Conversely, GTHs derived from a bicistronic vector system had no effect on in vitro steroid hormone production.

重组基因技术的最新进展使具有生物活性的鱼类促性腺激素（GTH：FSH和LH）的生产成为可能。然而，尚未开发用于生产GTH的有效表达方法。在这项研究中，我们试图用两种不同的杆状病毒表达系统在家蚕幼虫中产生红海鸟GTH。为了合成重组红藻异二聚体GTH，采用两种策略。第一种利用双载体表达策略，其中每个GTH亚基（α、FSHβ和LHβ）在单个宿主中从单独的比较载体表达。第二种方法使用双顺反子表达策略，其中两种GTH亚基cDNA均从载体表达。将双顺反子或双顺反子表达载体接种到5龄早期的家蚕幼虫中。接种后5d，收集含重组FSH和LH的幼虫血淋巴，测定其生化特性和生物活性。Western blot分析显示，通过使用双载体表达系统，FSH和LH的每个亚基成功地合成并分泌到血淋巴中，允许异二聚体分子的合成。在共表达GTH亚基的双顺反子载体中，FSHα和LHα亚基的产量很低，而FSHβ和LHβ亚基的产量都很高。在体外类固醇激素生产中，由双载体系统产生的GTH的生物活性与天然GTH的生物活性相当。重组FSH和LH均能刺激红石斑鱼睾丸组织11-酮睾酮的产生。此外，重组LH以浓度依赖的方式刺激卵泡体外产生雌二醇-17 β。相反，来自双顺反子载体系统的GTH对体外类固醇激素产生没有影响。