摘要
目的 克隆TRP 1编码基因 ,表达并纯化TRP 1蛋白。方法 通过RT PCR方法扩增TRP 1编码基因 ,克隆至 pUC19载体并测序 ,再亚克隆至GST融合表达载体 pGEX 4T 1,转化大肠杆菌 ,IPTG诱导表达TRP 1/GST融合蛋白 ,分析鉴定表达蛋白的可溶性 ,并用谷氨酰胺树脂柱纯化表达的蛋白。结果 ①从培养的人黑素细胞中扩增出编码TRP 1的cDNA序列 ,序列测定证实与文献报道的序列一致 ;②成功构建了GST融合表达载体 pGEX 4T 1/TRP 1;③表达了TRP 1/GST融合蛋白 ;④得到纯化的TRP 1/GST融合蛋白。结论 人TRP 1基因的克隆成功及TRP 1/GST融合蛋白的表达与纯化 ,为进一步探索TRP
Objective To clone human TRP 1 code gene,express and purify TRP 1 protein.Methods TRP 1 gene was amplified by RT PCR and the fragments of the DNA were cloned into vector pUC19 and checked by sequencing.Then subcloned the fragments of the TRP 1 gene into vector pGEX 4T 1.The TRP 1 protein was expressed in E coli as fusion protein with glutathione S transferase(GST) induced by IPTG.The fusion protein was purified by glutathione resin column.Results ①TRP 1 code gene was amplified by RT PCR and the sequence was confirmed by sequencing;②Fusion expressing vector of pGEX 4T 1/TRP 1 was successfully constructed;③TRP 1/GST fusion protein was correctly expressed;④Purified fusion protein was obtained.Conclusion We successfully cloned the human TRP 1,constructed TRP 1/GST fusion expressing vector.TRP 1/GST fusion protein was correctly expressed in E coli and we purified the fusion protein.What we have done will surely be useful to the study of the antigenic epitope of human TRP 1.
出处
《中国皮肤性病学杂志》
CAS
北大核心
2001年第6期368-370,共3页
The Chinese Journal of Dermatovenereology
基金
国家教委出国留学人员启动资金资助 (HG970 0 1 )
