一种新型大肠杆菌表达-输出载体，其含有碱性磷酸酶作为插入失活筛选系统。

A novel Escherichia coli expression-export vector containing alkaline phosphatase as an insertional inactivation screening system.

作者信息

Guzmán C A, Piatti G, Walker M J, Guardati M C, Pruzzo C

机构信息

Institute of Microbiology, University of Genova, Italy.

出版信息

Gene. 1994 Oct 11;148(1):171-2. doi: 10.1016/0378-1119(94)90254-2.

DOI:10.1016/0378-1119(94)90254-2

PMID:7926833

Abstract

An Escherichia coli expression-export vector was constructed (pCGV1, 6.3 kb) containing the alkaline phosphatase structural gene (phoA) located downstream from the phage lambda pR and pL promoters positioned in tandem and the cIts857 gene encoding lambda thermosensitive repressor. The phoA gene is fused to DNA encoding a hybrid signal sequence that contains the N-terminal portion of the beta-lactamase (Bla) signal sequence and the C-terminal region of the PhoA signal sequence. Within the DNA encoding hybrid signal sequence, a unique NheI restriction site is present where polymerase chain reaction (PCR)-amplified genes may be cloned. The 5' PCR primers reconstitute the C-terminal portion of either the PhoA or Bla signal sequences to restore an intact signal peptide. Recombinant phoA- clones are selected on indicator plates containing 5-bromo-4-chloro-3-indolyl phosphate.

摘要

构建了一种大肠杆菌表达-输出载体（pCGV1，6.3 kb），其含有串联排列在噬菌体λ pR和pL启动子下游的碱性磷酸酶结构基因（phoA）以及编码λ热敏阻遏物的cIts857基因。phoA基因与编码杂交信号序列的DNA融合，该杂交信号序列包含β-内酰胺酶（Bla）信号序列的N端部分和PhoA信号序列的C端区域。在编码杂交信号序列的DNA内，存在一个独特的NheI限制性酶切位点，可在此处克隆聚合酶链反应（PCR）扩增的基因。5' PCR引物可重构PhoA或Bla信号序列的C端部分，以恢复完整的信号肽。在含有5-溴-4-氯-3-吲哚磷酸的指示平板上筛选重组phoA-克隆。