摘要
目的克隆斑马鱼促性腺激素释放激素(GnRH)基因,构建重组质粒pQE30-GnRH并获得表达蛋白。方法从斑马鱼脑组织提取总RNA,应用RT-PCR方法获得斑马鱼GnRHcD-NA。将该cDNA插入质粒pQE30中,并使其在大肠杆菌RB791中表达。经IPTG诱导后SDS-PAGE电泳检测蛋白的表达情况。结果斑马鱼GnRHcDNA长为207bp,编码的GnRH前体为69个氨基酸残基。与鲤鱼、鲫鱼、拟鲤、黑头软口鲦等淡水鲤科鱼类的氨基酸序列同源性比较,分别为74.4%,69.8%,73.3%和73.3%。电泳结果显示一新的分子量约为14.4 kDa的特异蛋白带。结论本研究所克隆的斑马鱼GnRH基因属于GnRH-Ⅱ。构建斑马鱼GnRH原核表达质粒不仅获得大量的GnRH蛋白,还可对该蛋白做进一步分离纯化以及生物活性方面的研究。
Objective To amplify the gonadotropin- releasing hormone （GnRH） gene in Danio rerio and to construct the recombinant plasmid pQE30 - GnRH for obtaining expressed protein. Methods The total RNA was extracted from brain of Danio rerio. The GnRH cDNA was amplified by RT - PCR method. The GnRH cDNA fragment was inserted to plasmid pQE30 and made it expression in E. coli RB791 cells. The expression protein was assayed by SDS - PAGE after IPTG inducing. Results The GnRH eDNA fragment of Danio rerio was 207bp in length and encoded GnRH precursor possess 69 amino acids residues. Sequence homology comparison showed 74.4%, 69.8%, 73.3% and 73.3% homology at amino acid level with fresh water fish of Cyprinidae such as C. carpio, C. auratus, R. rutilus, P. promelas, respectively. The results of electrophoresis showed that a new specific protein band was found with molecular mass of about 14.4 kDa. Conclusion The GnRH gene reported in this study belongs to GnRH - Ⅱ. The construction of expressed plasmid in prokaryote not only obtained abundant protein but done research further for its separation, purification and biological activity.
出处
《青海医学院学报》
CAS
2009年第1期9-13,共5页
Journal of Qinghai Medical College
基金
青海省高校日元贷款人才培养项目
