摘要
从人胚胎肝组织中分离纯化出总ＲＮＡ，在总ＲＮＡ中提取ｍＲＮＡ，经逆转录得到ｃＤＮＡ第一条链，并以之为模板和相应寡聚脱氧核苷酸为引物，进行ＰＣＲ合成人蛋白质二硫键异构酶（Ｐｒｏｔｅｉｎｄｉｓｕｌｆｉｄｅｉｓｏｍｅｒａｓｅ，ＰＤＩ）ｃＤＮＡ基因，将所得ｃＤＮＡ克隆到质粒ｐＵＣ１８上，转化大肠杆菌ＤＨ５α细胞，进行诱导表达筛选和活性筛选。将筛选的基因克隆到载体ｐＢＶ２２０上，在大肠杆菌细胞中表达，产物以溶解形式在胞浆中表达。表达产物经硫酸铵分级沉淀和ＤＥＡＥＳｅｐｈａｒｏｓｅＦａｓｔＦｌｏｗ柱层析分离，产物的纯度与活性分别为９０％、１１００ｕ／ｇ。
The mRNAs ,isolated from the total RNAs of a Chinese fetal liver tissue,were reverse transcribed to the first strands of cDNA.The human protein disulfide isomerase (PDI) gene was amplified from the first strands of cDNA by PCR,and was inserted into plasmid pUC18 for screening and DNA sequencing.The PDI cDNA gene was subcloned into the expression vector pBV220,the expression product in E.coli DH5α was a cytoplasmic soluble protein.Through ammonium fractional precipitation and DEAE-Sepharose Fast Flow Chromatography,the recombinant PDI was 90% purity and the isomerase activity was 1100u/g.
出处
《生物工程学报》
CAS
CSCD
北大核心
1999年第3期349-354,共6页
Chinese Journal of Biotechnology
基金
北京市自然科学基金
北京市科技新星计划
