人酪氨酸酶相关蛋白-2的cDNA克隆及表达

Cloning and expression of human tyrosinase related protein-2 cDNA

目的 克隆酪氨酸酶相关蛋白 - 2 (TRP- 2 )编码基因 ,表达 TRP- 2蛋白 .方法 从培养的人黑素细胞中提取总RNA,反转录成 c DNA,通过 PCR方法扩增 TRP- 2编码基因 ,克隆至 p UC19载体并测序 ,再亚克隆至谷胱甘肽巯基转移酶 (GST)融合表达载体 p GEX- 4T- 1,转化大肠杆菌 ,IPTG诱导表达 TRP- 2 /GST融合蛋白 .结果 从培养的人黑素细胞中扩增出编码 TRP- 2的 c DNA,序列测定证实与文献报道的序列一致 ;成功构建了 GST融合表达载体 p GEX- 4T- 1/TRP- 2 ;表达了 TRP- 2 /GST融合蛋白 .结论 成功克隆了人 TRP- 2基因 ,构建了 GST融合表达载体并表达了 TRP- 2

AIM To clone the cDNA of human tyrosinase related protein 2 (TRP 2) and express the corresponding protein. METHODS Total RNA was isolated from cultured human pigmental cells and mRNA was reversly transcribed intocDNA. Then PCR was used to amplify the TRP 2 coding region. The PCR product was cloned into pUC19 plasmid and sequenced, then subcloned into vector pGEX 4T 1. The TRP 2 protein was expressed in E. coli of DH5α as fusion protein with glutathione S transferase (GST) induced by IPTG. RESULTS TRP 2 coding region was cloned into pUC19 and the sequence was confirmed; Fusion expressing vector of pGEX 4T 1/TRP 2 was successfully constructed; TRP 2/GST fusion protein was correctly expressed. CONCLUSION Human TRP 2 gene is successfully cloned and TRP 2/GST fusion expressing vector is constructed. TRP 2/GST fusion protein is correctly expressed in E. coli .