Zhang T, Zhang G X, Han K P, Tang Y, Wang J Y, Fan Q C, Chen X S, Wei Y, Wang Y J
College of Animal Science and Technology, Yangzhou University, Yangzhou, China.
College of Animal Science and Technology, Yangzhou University, Yangzhou, China
Genet Mol Res. 2015 Mar 31;14(1):2831-49. doi: 10.4238/2015.March.31.14.
The gonadotropin-releasing hormone (GnRH) plays an important role in the control of reproductive functions. Recent studies have reported the occurrence of GnRH molecular variants in numerous species. In this study, the GnRH1 gene from Jinghai yellow chicken was cloned by reverse transcriptase-polymerase chain reaction and transformed into BL21 (DE3) competent cells. The GnRH1 gene and amino acid sequences were subjected to bioinformatic analyses. The GnRH1 gene nucleotide sequence was discovered to be 352 bp long, containing a coding, promoter, and section of the 3'-regions. The GnRH1 gene shared 93, 81, 54, 58, 61, 76, 76, 59, 76, and 66% sequence identity with Meleagris gallopavo, Columba livia, Homo sapiens, Bos taurus, swines, Capra hircus, Ovis aries, Pantholops hodgsonii, Equus caballus, and Rattus norvegicus, respectively. The GnRH1 gene showed conserved domains. The GnRH1 protein was a secreted protein comprising 92 amino acids, with a molecular weight of 10205.6 Da and a theoretical pI of 5.67. Most of the amino acid residues were observed to be hydrophilic, indicating water solubility. The predicted secondary structures of proteins included α-helices (h; 23.08%), β-extensions (e; 10.92%), and random coils (c; 66.0%). The successful construction of prokaryotic expression vector pET32a-GnRH1 was confirmed by restriction and sequence analysis. SDS-PAGE analysis showed the successful expression of recombinant plasmid in Escherichia coli BL21 (molecular weight = 25-28 kDa). Larger quantities of protein were expressed in supernatant, indicating greater expression in soluble form. Western blot analysis confirmed the expression of the target protein.
促性腺激素释放激素（GnRH）在生殖功能的调控中发挥着重要作用。最近的研究报道了许多物种中GnRH分子变体的存在。在本研究中，通过逆转录聚合酶链反应克隆了静海黄鸡的GnRH1基因，并将其转化到BL21（DE3）感受态细胞中。对GnRH1基因和氨基酸序列进行了生物信息学分析。发现GnRH1基因核苷酸序列长352 bp，包含一个编码区、启动子和3'区域的一部分。GnRH1基因与火鸡、家鸽、人类、牛、猪、山羊、绵羊、藏羚羊、马和大鼠的序列同一性分别为93%、81%、54%、58%、61%、76%、76%、59%、76%和66%。GnRH1基因显示出保守结构域。GnRH1蛋白是一种分泌蛋白，由92个氨基酸组成，分子量为10205.6 Da，理论pI为5.67。观察到大多数氨基酸残基是亲水性的，表明其具有水溶性。预测的蛋白质二级结构包括α螺旋（h；23.08%）、β延伸（e；10.92%）和无规卷曲（c；66.0%）。通过酶切和序列分析证实了原核表达载体pET32a-GnRH1的成功构建。SDS-PAGE分析表明重组质粒在大肠杆菌BL21中成功表达（分子量 = 25 - 28 kDa）。在上清液中表达了大量蛋白质，表明以可溶性形式表达量更高。蛋白质印迹分析证实了目标蛋白的表达。
