藤黄微球菌Rpf活性蛋白的制取及其对红球菌VBNC菌体的复苏作用

Cloning and expression of Micrococcus luteus IAM 14879 Rpf and its role in the recovery of the VBNC state in Rhodococcus sp.DS471

摘要:

摘要:【目的】克隆藤黄微球菌Micrococcus luteus IAM 14879 (=NCIMB 13267)的复苏促进因子Rpf(resuscitation promoting factor) 的基因，在大肠杆菌中表达获取基因重组蛋白，考察对近缘高GC 革兰氏阳性菌红球菌Rhodococcus sp.DS471 活的非可培养VBNC (viable but non-culturable)菌体的复苏促进生长能力。【方法】抽提制备藤黄微球菌的DNA，确定rpf 基因引物进行PCR 扩增，利用pET15b 质粒载体并转化大肠杆菌DE3 表达，以SDS-PAGE 检验获取纯化重组蛋白;在培养基中添加Rpf，以MPN (most probable number) 法计数、评价对VBNC 状态菌体的复苏促进生长效果。【结果】基因测序证实获得藤黄微球菌的rpf 基因并在大肠杆菌中表达;SDS-PAGE 分析表明获得rpf 基因的重组蛋白;该蛋白对处于VBNC 状态的红球菌具有近100倍的复苏促进生长能力。【结论】成功克隆了藤黄微球菌的rpf 基因，在大肠杆菌中获得了表达，表明了Rpf 蛋白对处于VBNC 状态的红球菌具有复苏促进生长效果。

Abstract:

Abstract:［Objective］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.［Methods］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． ［Results］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．［Conclusion］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.