摘要
人胎盘核糖核酸酶抑制因子(HRI)是一种存在于细胞浆中的50kDa的酸性蛋白质,富含亮氨酸和半胱氨酸。作为胞浆蛋白可保护细胞不受外来的胰RNase的侵袭,同时做为RNase抑制剂在各实验室广为应用。HRI主要结构是由7个富含亮氨酸的重复序列组成,7个亮氨酸重复单位有规律环状排列使N-末端、C-末端在空间上较为接近。本文用PCR的方法在HRIcDNA5'端去除30个碱基,并将此缺失突变的HRI的cDNA片段构建于质粒pPIC9K,PCR和双酶切鉴定结果为阳性,测序结果证实在HRIcDNA5'端成功引入30个碱基的缺失突变。电击转化入毕赤酵母(Pichiapastores)GS115中,进行分泌型表达。对表达产物进行亲和层析纯化。经SDS-PAGE和Western-blot免疫印迹杂交,证实表达产物为N-端缺失突变的核糖核酸酶抑制因子。
The human placental ribonuclease inhibitor (HRI) is an acidic protein of Mr～50kDa with an amino acid composition characterized by unusually high levels of leucine and cysteine. It is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonuclease. HRI is composed of 7 leucine-rich repeats units as either a circle, a helix, or a line. The circular arrangement may bring the N and C terminals into close proximity. In present paper, 5'-termini of 30 bp deletion-mutated HRI cDNA was constructed into plasmid pPIC9K to show positive clonies and then transformed Pichia pastoris GS115 by electroporation. After screening the colony, the bacterium was cultured and its product was purified with affinity chromatography. Purified HRI were examined by SDS-PAGE and Western-blot.
出处
《水产科学》
CAS
北大核心
2004年第9期19-22,共4页
Fisheries Science
