Ding Linxian, Zhang Pinghua, Hong Huachang, Lin Hongjun, Yokota Akira
College of Geography and Environmental Sciences, Zhejiang Normal University, Jinhua 321004, China.
Wei Sheng Wu Xue Bao. 2012 Jan;52(1):77-82.
The purpose of the present study was to produce the Rpf (resuscitation promoting factor) protein by cloning and expressing the rpf gene, secreted by Micrococcus luteus IAM 14879, in Escherichia coli and to evaluate its role in the recovery of the VBNC (viable but non-culturable) state in high-GC Gram-positive bacteria.
Genomic DNA was extracted from Micrococcus luteus IAM 14879 and the rpf gene was amplified by PCR using specific primers. The PCR products was purified, cloned into a pET15b expression vector, and transformed into Escherichia coli BL21 (DE3). Then the pET15b plasmid expression vector was used to confirm the purification of the recombinant proteins via SDS-PAGE. The VBNC state cells from the high-GC Gram-positive bacteria, Rhodococcus sp. DS471, were used to confirm the promotion and recovery of growth capacity. Rhodococcus sp. DS471 were isolated from soil and closely related to Micrococcus luteus IAM 14879.
The gene sequences confirmed that the rpf gene from Micrococcus luteus IAM 14879 that was expressed in Escherichia coli, was 672 bp. SDS-PAGE analysis showed that the recombinant Rpf protein was obtained successfully, and further studies showed it capable of promoting the recovery of the VBNC state by about 100-fold relative to the control.
Rpf of Micrococus luteus IAM 14879 can be successfully cloned and expressed in Escherichia coli and shows a strong ability to promote the recovery of the VBNC state of cells of Rhodococcus sp. DS471.
本研究的目的是通过克隆并在大肠杆菌中表达藤黄微球菌IAM 14879分泌的rpf（复苏促进因子）基因来生产Rpf蛋白，并评估其在高GC革兰氏阳性菌中VBNC（活的但不可培养）状态恢复中的作用。
从藤黄微球菌IAM 14879中提取基因组DNA，使用特异性引物通过PCR扩增rpf基因。将PCR产物纯化，克隆到pET15b表达载体中，并转化到大肠杆菌BL21（DE3）中。然后使用pET15b质粒表达载体通过SDS-PAGE确认重组蛋白的纯化。来自高GC革兰氏阳性菌红球菌属DS471的VBNC状态细胞用于确认生长能力的促进和恢复。红球菌属DS471从土壤中分离得到，与藤黄微球菌IAM 14879密切相关。
基因序列证实，在大肠杆菌中表达的来自藤黄微球菌IAM 14879的rpf基因长度为672 bp。SDS-PAGE分析表明成功获得了重组Rpf蛋白，进一步研究表明其能够将VBNC状态的恢复促进至相对于对照约100倍。
藤黄微球菌IAM 14879的Rpf能够在大肠杆菌中成功克隆和表达，并显示出强大的促进红球菌属DS471细胞VBNC状态恢复的能力。
