GnRH is a neuropeptide known to regulate reproduction in vertebrates. The purpose of this study was to design and produce recombinant gonadotropin-releasing hormone associated peptide (rGnRH/GAP) as an alternative of the previous GnRHs and native extracted hormone from tissue, to induce final maturation in fish. Decapeptide as well as GAP area sequences were compared between GnRH1, GnRH2, and mGnRH from Acipenser sp and Huso huso, respectively. Considering the conserved amino acids and the replacement of un-stable amino acids with those that were more stable against proteolytic digestion as well as had a longer half-life, the sequence was designed. The sequences of decapeptide and GAP region were synthesized and then cloned on pET28a expression vector and transformed into expression host Escherichia coli BL21(DE3). The supernatant of cultured recombinant bacteria was used for purification using TALON Metal affinity resin. The purity of the GnRH/GAP was confirmed by single 8 kDa band on SDS-PAGE and Western blot. Bioinformatics studies were performed for evaluation of homology between GnRH protein sequences and prediction of 3D protein structure using Swiss Model. The result showed that the structure prediction of the recombinant GnRH decapeptide was relatively similar to decapeptide of GnRH2 from Beluga (Huso huso). The GAP structure was similar to GAP1 of Nile tilapia (Oreochromis niloticus) and sturgeon and GnRH2 of Chinese sturgeon (Acipenser sinensis). The mass analysis showed that the sequence was exactly the same as designated sequence. Biology activity of rGnRH/GAP was tested in mature goldfish (Carassius auratus) and results showed that rGnRH/GAP had a positive effect in final maturation. Indeed 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) was increased 17 h and 24 h after injection with rGnRH/GAP and spawning stemmed from that injection. These novel findings introduce the potential of utilizing rGnRH/GAP in aquaculture.

中文翻译：

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一种新型重组促性腺激素释放激素相关肽作为鱼类产卵诱导剂的设计，生产和纯化。

GnRH是已知可调节脊椎动物繁殖的神经肽。这项研究的目的是设计和生产重组促性腺激素释放激素相关肽（rGnRH / GAP），以替代以前的GnRHs和从组织中提取的天然激素，以诱导鱼的最终成熟。比较了分别来自Acipenser sp和Huso huso的GnRH1，GnRH2和mGnRH的十肽和GAP区域序列。考虑到保守的氨基酸和用对蛋白水解消化更稳定并且半衰期更长的氨基酸替换的不稳定氨基酸，设计了序列。合成十肽和GAP区域的序列，然后克隆到pET28a表达载体上，并转化到表达宿主大肠杆菌BL21（DE3）中。使用TALON Metal亲和树脂将培养的重组细菌的上清液用于纯化。GnRH / GAP的纯度通过SDS-PAGE上的单个8 kDa条带和Western blot证实。进行了生物信息学研究，以评估GnRH蛋白序列之间的同源性，并使用Swiss Model预测3D蛋白结构。结果表明，重组GnRH十肽的结构预测与来自白海豚的GnRH2十肽的结构预测相对相似。GAP的结构类似于尼罗罗非鱼（Neo tilapia）和中华st（Acipenser sinensis）的st鱼和GnRH2的GAP1。质谱分析表明该序列与指定序列完全相同。在成熟的金鱼（Car鱼）中检测了rGnRH / GAP的生物学活性，结果表明rGnRH / GAP在最终成熟中具有积极作用。实际上，在注射rGnRH / GAP后17 h和24 h产生的17α，20β-dihydroxy-4-pregnen-3-one（DHP）有所增加，并且该注射产生了卵。这些新颖的发现介绍了在水产养殖中利用rGnRH / GAP的潜力。