一种有效的基因转移技术——原生质体融合法 Ⅰ 大肠杆菌的原生质体制备

PROTOPLAST FUSION METHOD-AN EFFECTIVE TECHNIQUE WITH HIGH EFFICIENCY Ⅰ. PREPARATION OF PROTOPLAST IN E. COLI

原生质体融合法系一有效的基因转移技术,其先决条件是有效地除去细菌的胞壁,制备好原生质体。本文对大肠杆菌形成原生质体的条件进行了初步探讨。结果表明:对含质粒pSV_2 gpt的大肠杆菌HB101而言,以0.3％浓度的溶菌酶,37℃,作用45min,即可制备理想的原生质体,为进一步进行基因转移打下基础。

Protoplast fusion is a method for direct transferring cloned DNA from bacteria to mammalian cells with high efficiency. This technique does not require isolation or purification of the cloned DNA sequence and special regent and equipment. Recently the transferring of exogenous gene to human cells by means of protoplast fusion has provided new tool for gene transfer in vitro. The prereguisite for this method is to digest the bacterial cell walls with lysozyme, thus to produce qualified protoplast. The present study assesses the conditions of protoplast formation in E. Coli. Our preliminary results show that digestion with 0.3 % lysozyme at 37癈 for 45 minutes would be favorable for cell wall removal of E. Coli HB 101 containing pSV2gpt, to produce protoplast for gene transferring.