Nakanishi S, Nakajima T, Tazumi A, Matsubara K, Moore J E, Millar B C, Matsuda M
Laboratory of Molecular Biology, Graduate School of Environmental Health Sciences, Azabu University, Sagamihara 252-5201, Japan.
Br J Biomed Sci. 2013;70(1):15-21. doi: 10.1080/09674845.2013.11669924.
A recombinant molecule of the full-length urease gene operon was constructed in vitro from the Japanese urease-positive thermophilic Campylobacter (UPTC) CF89-12 isolate and expressed in Escherichia coli cells. Several large deletion recombinant variants of urease subunit genes were also constructed and expressed in E. coli cells. A positive urease reaction with the log-phase cultured E. coli JM109 cells in the NiCl2-containing medium transformed with pGEM-T vector carrying the recombinant molecule of the full-length operon was detected with isopropyl-beta-D-thiogalactoside. Among the several deletion recombinant variants, each ureA-, ureB-, ureE-, ureF-, ureG- and ureH-large deficient, only ureE-large deletion variant (63% deficient) showed a positive urease reaction (approximately 15-fold). In addition, a ureE-complete deletion recombinant variant (100% deficient) constructed also showed a positive reaction of urease (approximately 18-fold). Recombinant urease subunits A and B were immunologically identified by Western blot analysis with anti-urease alpha (A) and beta (B) raised against Helicobacter pylori.
从日本脲酶阳性嗜热弯曲菌（UPTC）CF89 - 12分离株体外构建了全长脲酶基因操纵子的重组分子，并在大肠杆菌细胞中表达。还构建了几种脲酶亚基基因的大缺失重组变体，并在大肠杆菌细胞中表达。用异丙基 - β - D - 硫代半乳糖苷检测到，在含有NiCl₂的培养基中，对数期培养的用携带全长操纵子重组分子的pGEM - T载体转化的大肠杆菌JM109细胞出现阳性脲酶反应。在几种缺失重组变体中，每个ureA -、ureB -、ureE -、ureF -、ureG - 和ureH - 大缺失变体中，只有ureE - 大缺失变体（缺失63%）显示出阳性脲酶反应（约15倍）。此外，构建的ureE - 完全缺失重组变体（100%缺失）也显示出脲酶阳性反应（约18倍）。通过用针对幽门螺杆菌产生的抗脲酶α（A）和β（B）进行蛋白质免疫印迹分析，对重组脲酶亚基A和B进行了免疫学鉴定。
