人促性腺激素释放激素及其转运肽基因的克隆、表达与纯化

研究根据GenBank中已发表的人GnRH2基因mRNA序列以及转运肽基因核苷酸序列,借助Oli-go4.1设计了一对寡核苷酸引物,以引物3'末端的短互补序列退火形成小段双链,从而互为模板和引物,通过引导合成长达90bp的目的序列GnRH/TRS。将其克隆到pMD18-T载体上,构建重组质粒pYC1,经酶切鉴定筛选出阳性克隆pYC1,进一步的序列分析确认其中GnRH/TRS序列的正确性。pYC1经BamHI和EcoRI酶切得到GnRH/TRS片段,然后将此片段克隆到表达载体pGEX-6p-1上,构建重组质粒pYC2。经酶切鉴定筛选出阳性克隆pYC2,并转化到大肠杆菌BL21(ED3)plyS实现原核表达。IPTG诱导后成功表达出与预期大小相符的约28ku的融合蛋白,光密度扫描对表达产物进行初步定量,表达产物约占菌体总蛋白的33.3%,表达产物经GlutathioneSepharose4B层析纯化后,得到了纯度较高的GST-GnRH/TRS融合蛋白,为进一步科研和实际应用奠定基础。

Cloning and prokaryotic expression and purification of GnRH and transporter gene sequence

A pair of primers were designed according to the Published sequence of GnRH2′s gene mRNA and transporter gene with Oligo version 4.1 software, they were annealed by their 3′terminal brief complementary base sequence to form small segment double chain. They serve mutually as template and primer, and their extension were carried out to synthesize 90 bp'GnRH/TRS. GnRH/TRS gene was cloned into the pMD18-T vector, the recombinant pYC1 plasmid was identified and analyzed by corresponding restriction endonuclease and nucleotid sequence. It is indicated that the GnRH/TRS gene has been cloned successfully, then this gene was cloned into the expression vector pGEX-6p-1. The recombinant pYC2 plasmid was identified and analyzed by corresponding restriction endonuclease. It is indicated that the GnRH/TRS gene has been cloned successfully. The recombinant plasmid pYC2 was transformed into the BL21(ED3) plysS prokaryotic expression system. The expression product was induced with IPTG. SDS-PAGE analysis showed an induced expression product band about 28 ku, which correspond to the sizes of GST-GnRH/TRS, reported in the literature. The amount of the goal protein was evaluated by densitometric scanning. It indicated that the product of the GST-GnRH/TRS gene was 33% of total bacterial protein of BL21(ED3). Then the product was purified by Glutathione Sepharose 4B chelation affinity chromatography, and upper purified GST-GnRH/TRS fusion proteins was received for further the foundations established in the scientific research and the real application.