京海黄鸡促性腺激素释放激素1基因克隆与原核表达研究

本试验根据GenBank公布的原鸡（Gallus gallus）促性腺激素释放激素1(gonadotropin releasing hormone 1，GnRH1)基因mRNA序列设计1对引物，采用RT-PCR方法，从京海黄鸡下丘脑组织克隆GnRH1基因编码区序列，对其进行生物信息学分析，并构建原核表达载体pET32a-GnRH1，在大肠杆菌BL21感受态细胞中表达。最终获得了包括编码区、大部分启动子区和部分3′区域在内的长度为352 bp京海黄鸡GnRH1基因序列，该核酸序列与火鸡、鸽子、人、牛、野猪、山羊、绵羊、藏羚羊、马和大鼠的GnRH1基因序列同源性分别为93%、81%、54%、58%、61%、76%、76%、59%、76%和66%。酶切测序鉴定结果显示，成功构建了原核表达载体pET32a-GnRH1；SDS-PAGE检测结果显示，构建的重组质粒在大肠杆菌BL21感受态细胞中成功表达，分子质量为25～28 ku，且上清表达量高于沉淀；Western blotting检测结果显示，在大肠杆菌BL21感受态细胞中表达的蛋白为目的蛋白，且主要为可溶性表达。结果表明，成功克隆了京海黄鸡GnRH1基因，构建了GnRH1原核表达体系，并成功表达目的蛋白，为后续相关研究打下了基础。

Cloning and Prokaryotic Expression of Gonadotropin Releasing Hormone 1 Gene in Jinghai Yellow Chicken

Based on the published mRNA sequence of Gallus gallus gonadotropin releasing hormone 1 （GnRH1） gene, a pair of primers was designed to clone the GnRH1 gene coding sequence of Jinghai Yellow chicken by RT-PCR. GnRH1 gene was cloned in to pET32a vector for expression in E.coil BL21 and bioinformatics analysis. Finally, a 352 bp gene was cloned, which contained CDS region, promoter region and part of the 3′region. It was found that the GnRH1 gene nucleotide sequence shared 93%, 81%, 54%, 58%, 61%, 76%, 76%, 59%, 76% and 66% identities with Meleagris gallopavo, Columba livia, Homo sapiens, Bos taurus, Babirussa, Capra hircus, Ovis aries, Pantholops hodgsonii, Equus caballus and Rattus norvegicus, respectively. Restriction and sequencing analysis confirmed that prokaryotic expression vector pET32a-GnRH1 was successfully constructed. SDS-PAGE analysis showed that the recombinant plasmid was successfully expressed in E.coli BL21 and the molecular weight was approximate 25 to 28 ku. The expression quantity in supernatant was higher than that in the precipitant. Western blotting analysis confirmed that the protein expressed in E.coli BL21 was the interest protein and expressed in soluble form with high activity. The results showed that GnRH1 gene of Jinghai Yellow chicken was cloned and GnRH1 prokaryotic expression system was constructed successfully. The interest protein was expressed successfully, which laid the foundation for further studies.