基于RSF1010的穿梭载体，用于在多杀性巴氏杆菌中克隆和表达。

RSF1010-based shuttle vectors for cloning and expression in Pasteurella multocida.

作者信息

Lee M D, Henk A D

机构信息

College of Veterinary Medicine, University of Georgia, Athens 30601, USA.

出版信息

Vet Microbiol. 1997 Mar;54(3-4):369-74. doi: 10.1016/s0378-1135(96)01294-1.

DOI:10.1016/s0378-1135(96)01294-1

PMID:9100336

Abstract

The broad host-range cloning vectors, pJRD215 and pMMB67EH, were evaluated for stability and cloning efficiency in Pasteurella multocida. Transformation of P. multocida by electroporation was unreliable and poorly efficient regardless of whether the transforming DNA was isolated from E. coli or P. multocida. Both vectors contain a mob site that enabled transfer by conjugation from E. coli to P. multocida with high efficiency. Kanamycin, streptomycin, and ampicillin resistance encoded by the vectors were expressed in P. multocida. LacZ was cloned in pMMB67EH, an expression vector, and was transferred to P. multocida by conjugation. The transconjugants expressed a functional beta-galactosidase as determined by o-nitrophenyl-beta-D-galactopyranoside (ONPG) test. We propose the use of these cosmid and expression vectors as a shuttle vectors for cloning in P. multocida.

摘要

对广宿主范围克隆载体pJRD215和pMMB67EH在多杀性巴氏杆菌中的稳定性和克隆效率进行了评估。无论转化DNA是从大肠杆菌还是多杀性巴氏杆菌中分离得到，通过电穿孔对多杀性巴氏杆菌进行转化都是不可靠且效率低下的。这两种载体都含有一个mob位点，能够通过接合作用高效地从大肠杆菌转移到多杀性巴氏杆菌。载体编码的卡那霉素、链霉素和氨苄青霉素抗性在多杀性巴氏杆菌中得以表达。LacZ被克隆到表达载体pMMB67EH中，并通过接合作用转移到多杀性巴氏杆菌中。如通过邻硝基苯基-β-D-吡喃半乳糖苷（ONPG）试验所确定的，转接合子表达了功能性的β-半乳糖苷酶。我们建议将这些黏粒和表达载体用作在多杀性巴氏杆菌中进行克隆的穿梭载体。