鹅GnRH基因CDS区克隆、序列分析及原核表达

Cloning,sequence analysis and prokaryotic expression for CDS of goose GnRH gene

本研究旨在了解鹅GnRH基因的CDS区序列及其蛋白表达情况,研究鹅GnRH蛋白的生理功能,为进一步阐明GnRH蛋白影响鹅繁殖性能的调控机制提供理论基础。采用RT-PCR方法从溆浦鹅下丘脑扩增获得GnRH基因的CDS区序列,将测序正确的DNA序列定向克隆到pET28a(+),构建表达载体pET28a-GnRH,并转化至BL21(DE3)大肠杆菌表达目的蛋白,经IPTG诱导后进行SDS-PAGE检测和Western blot分析。结果显示:获得的溆浦鹅GnRH基因CDS区部分序列,含有270个核苷酸,编码前体蛋白中的90个氨基酸;溆浦鹅GnRH基因CDS区在大肠杆菌中得到高效表达,GnRH基因CDS区的目的蛋白为9 900,最终测得菌体湿重为0.6g/L,纯化后得到的蛋白为20~30mg/L;清晰检测到GnRH基因表达的蛋白特异带,试验获得的抗体对原核表达的GnRH蛋白具有特异性反应;鹅GnRH基因在动物进化中高度保守,研究得到的鹅GnRH前体蛋白可为进一步研究GnRH基因的生物功能以及其对鹅就巢、产蛋等繁殖性状的影响奠定基础。

RH gene CDS was cloned,and GnRH precursor protein was expressed. The CDS sequence of Gn- RH gene was amplified in the hypothalamus of goose by RT-PCR,DNA sequence was cloned into pET28a （＋）,the expression vector pET28a-GnRH was constructed and transformed into BL21 （DE3） from Escherichia coii expressing target protein. SDS-PAGE and Western Blot analysis were done after IPTG induced. The results showed that the GnRH gene CDS sequence of Xupu geese contained 270 nucleotides, encoded a precursor protein of 90 amino acids; GnRH gene CDS was highly expressed in Escherichia coli, the CDS region of the GnRH gene protein was 9 900 and wet weight of bacteria body was 0.6 g/L and purified protein was about 20-30 mg/L;The specific pro- tein band of GnRH gene expression were clearlf detected, the anfibody obtained had a specific re- sponse to GnRH protein of prokaryotic expression;Goose GnRH gene was highly conservative in animal evolution. The precursor protein of goose GnRH may lay the foundation for further study on the biological function of GnRH genes and its effect on the goose production traits such as nest and egg.