Cheng X B, Chen T M, Ma X K, Huang C F
Institute of Biotechnology, Academy of Military Medical Sciences, Beijing.
Chin J Biotechnol. 1989;5(1):11-7.
Plasmid pJY11 of the Escherichia coli H10407 strain was isolated and completely digested with restriction endonuclease PstI in order to isolate the heat-labile enterotoxin (LT) operon. The resulting fragments were then separated by electrophoresis on agarose gel, and the location of the LT region in pJY11 was determined by Southern hybridization. The LT region was found to be located on a 5.3 kb fragment of pJY11. This fragment was recovered and subsequently ligated to PstI-linearized pUC8 DNA. After transformation and selection, a clone that effectively expressed LT was obtained. Biological and immunological assays showed the LT synthesized by this clone to be biologically and immunologically identical to that synthesized by the parent E. coli strain H10407; the LT production level of this recombinant strain was 15 times greater.
分离出大肠杆菌H10407菌株的质粒pJY11，并用限制性内切酶PstI将其完全消化，以分离热不稳定肠毒素（LT）操纵子。然后将所得片段在琼脂糖凝胶上进行电泳分离，并通过Southern杂交确定pJY11中LT区域的位置。发现LT区域位于pJY11的一个5.3 kb片段上。回收该片段，随后将其连接到用PstI线性化的pUC8 DNA上。经过转化和筛选，获得了一个有效表达LT的克隆。生物学和免疫学分析表明，该克隆合成的LT在生物学和免疫学上与亲本大肠杆菌菌株H10407合成的LT相同；该重组菌株的LT产生水平高15倍。
