奥利亚罗非鱼促性腺激素释放激素及相关肽基因的克隆与表达

Cloning and expression of tilapia Oreochromis aurea gonadotropin-releasing hormone and GnRH associated peptide in Escherichia coli

采用RT-PCR方法从奥利亚罗非鱼丘脑中扩增出长约386 bp的目的序列GnRH/GAP,先将其克隆到T载体上,通过酶切鉴定、序列测定分析,确认序列的正确性后,将此片段克隆到表达载体pMAL-c2x中构建重组表达质粒pMAL-GnRH/GAP,再转化到大肠杆菌TB1中,经IPTG诱导后成功表达出与预期大小相符的相对分子量约56 000的融合蛋白。

The cDNA encoding gonadotropin -releasing hormone （GnRH） and GnRH associated peptide （GAP） was amplified from total RNA from tilapia Oreochromis aurea pituitary glands by reverse transcri - ption polymerase chain reaction （RT- PCR） method, and then blasted against other GnRH cDNA sequences in the GenBank. The analysis of the sequence data indicated that the coding region of the cDNA fragment, which encoded 89 amino acid redidues, is about 400 bp in size. The amplified cDNA fragment was cloned into the prokaryotic expression vector, pMAL- c2x, to produce the expression vector pMAL- GnRH. The recombinant plasmid was transformed into Escherichia coli TB1. GnRH - MBP fusion protein was obtained after the addition of IPTG into the growth media. SDS - PAGE analysis revealed that the GnRH - MBP was expressed after induction with IPTG for 4 h. A protein band of 56 000 was observed on SDS - PAGE gel. It is anticipated that the fusion protein will be proved useful for further research.