用人肝细胞cDNA文库制备Dig-Fas探针

为探索SLE发生、发展与Fas表达的关系 ,本文采用降落PCR技术、分子克隆技术和Fas特异性引物 ,将从人肝细胞cDNA文库中扩增的FascDNA插入T -easyvector中 ,转染JM10 9,经耐药性和lacZ插入失活筛选 ,快速细胞裂解法、限制性内切酶酶切片段和PCR扩增试验分析鉴定 ,确证获阳性转化子。然后采用地高辛化学连接标记和查见方法 ,标记扩增、分离和纯化的FascDNA ,经斑点免疫实验证实获高滴度Dig -FascDNA探针

Preparation of Dig-labeled Fas cDNA probe with human liver cDNA l ibrary

To investigate the relation between the expression of Fas and the course of Systemiclupus Erythematosus (SLE), the TD Polymerase Chain Reaction and the specific primers of Fas were used to amplify Fas cDNA in Humam liver cDNA library. The amplified products were inserted into the PGEM-T easy vector and the recombinants were transferred into JM109 competent cells. It was confirmed by screening with the genes of resistant drug and lacZ , as well as the size of the fragments cut with EcoRI that the positive clones contained Fas cDNA had been gained. The Fas cDNA isolated from positive clones was labeled with Dig and Dig-labeled Fas cDNA probe was detected by anti-Dig-AP Conjugation.