Expression of biologically active red seabream gonadotropins in a baculovirus system

重组基因技术的最新进展使得生产具有生物活性的鱼类促性腺激素（GTHs: FSH和LH）成为可能。然而，目前还没有一种高效的表达GTHs的方法。在本研究中，我们尝试使用两种不同的杆状病毒表达系统在家蚕幼虫中产生红鲷GTHs。为了合成重组红鲷异二聚体GTHs，采用了两种策略。第一种方法采用双载体表达策略，其中每个GTH亚基（α， FSHβ和LHβ）在单个宿主中通过单独的比较载体表达。第二种方法使用双链表达策略，其中两个GTH亚基cdna都从载体上表达。用双或双双表达载体接种5龄早期家蚕幼虫。接种后5 d，采集含有重组FSH和LH的幼虫血淋巴，检测其生化特性和生物活性。Western blot分析显示，通过双载体表达系统，FSH和LH的每个亚基都被成功合成并分泌到血淋巴中，从而合成异二聚体分子。在共表达GTH亚基的双频载体中，FSHα和LHα亚基的产率很低，而FSHβ和LHβ亚基的产率都很高。双载体系统产生的GTHs在体外产生类固醇激素方面的生物活性与天然GTHs相当。重组卵泡刺激素和黄体生成素均能刺激红鲷切片睾丸体外11-酮睾酮的产生。此外，重组LH以浓度依赖的方式刺激卵巢卵泡体外产生雌二醇-17β。相反，从双电子载体系统衍生的GTHs对体外类固醇激素的产生没有影响。

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.