Luo J, Li W, Zhang T, Chai S, Wang H
Dept. of Biology, Zhongshan University Guangzhou.
Yi Chuan Xue Bao. 1994;21(1):74-80.
Promoter and signal sequence coding regions from B. subtilis were cloned in E. coli using a bifunctional and signal sequence selection plasmid pGPB14 as a vector. The Sau3A digested chromosome DNA was ligated with BamHI digested pGPB14. The ligated mixture was used to transform E. coli C600. Ampicillin and erythromycin resistant clones were selected. Recombinant plasmids were isolated from double resistant transformants. Restriction analysis showed that inserts of various length had been cloned and these inserts rendered the E. coli cells resistant to different concentrations of ampicillin. The recombinant plasmids were transformed and showed the same secretion function in B. subtilis. The amount and localization of beta-lactamase were determined in transformed E. coli and B. subtilis. The results indicate that the beta-lactamase activities of E. coli mainly in periplasm while the enzyme produced by B. subtilis secreted extracellularly. 10 of the cloned fragments were sequenced by Sanger's dideoxy chain termination method. The sequencing data show that all fragments contain promoter, ribosome binding site and signal sequence coding region.
使用双功能信号序列选择质粒pGPB14作为载体，将枯草芽孢杆菌的启动子和信号序列编码区克隆到大肠杆菌中。用Sau3A消化的染色体DNA与用BamHI消化的pGPB14连接。连接混合物用于转化大肠杆菌C600。选择对氨苄青霉素和红霉素有抗性的克隆。从双抗性转化体中分离重组质粒。限制性分析表明，已克隆了各种长度的插入片段，这些插入片段使大肠杆菌细胞对不同浓度的氨苄青霉素具有抗性。重组质粒被转化并在枯草芽孢杆菌中显示出相同的分泌功能。在转化的大肠杆菌和枯草芽孢杆菌中测定了β-内酰胺酶的量和定位。结果表明，大肠杆菌中的β-内酰胺酶活性主要存在于周质中，而枯草芽孢杆菌产生的酶则分泌到细胞外。通过桑格双脱氧链终止法对10个克隆片段进行了测序。测序数据表明，所有片段均包含启动子、核糖体结合位点和信号序列编码区。
