白色放线动孢菌DSM 45114^(T)rpf基因的克隆表达及结构预测

Cloning and expression of Actinokineospora alba DSM 45114^(T) rpf gene and structure prediction of Rpf protein

复苏促进因子(Rpf)广泛存在于高G+C含量的革兰氏阳性细菌中,与休眠细胞的复苏有关,且不同细菌Rpf的种类和数量有一定差异.为了解不同物种中rpf基因的差别,对白色放线动孢菌(Actinokineospora alba)DSM 45114 T的3个Rpf蛋白的氨基酸序列、疏水性、信号肽、磷酸化位点、三级结构以及结构功能域进行生物信息学预测分析.设计了白色放线动孢菌(A.alba)DSM 45114^(T)rpf基因的特异性引物,从其基因组中扩增出了3个不同的rpf基因,随后构建pET-28a(+)-rpf表达载体,在Escherichia coli BL21(DE3)中进行诱导表达,成功表达了Rpf-2蛋白.将重组Rpf-2蛋白加入到活的非可培养(viable but non culturable,VBNC)状态的藤黄微球菌(Micrococcus luteus)和滨海红球菌(Rhodococcus marinonascens)中,结果表明,重组Rpf-2蛋白可以促进其复苏,添加量在体积分数为10%时复苏效果最好.本研究为后续探讨白色放线动孢菌(A.alba)Rpf蛋白的功能奠定了基础.

Resuscitation-promoting factor(Rpf)is widely present in gram-positive bacteria with high G+C content and is related to the resuscitation of dormant cells.There are some differences in the types and quantities of Rpf among different bacteria.In order to understand the difference of rpf genes in different species,bioinformatics analysis was conducted on the amino acid sequence,hydrophobicity,signal peptide,phosphorylation site,tertiary structure and structural functional domain of three Rpf proteins in Actinokineospora alba DSM 45114^(T).The specific primers for the three rpf genes of A.alba DSM 45114^(T) were designed,and corresponding genes were amplified.Subsequently,pET-28a(+)-rpf expression vectors were constructed and induced in Escherichia coli BL21(DE3),Rpf-2 protein was successfully expressed.Adding the recombinant Rpf-2 protein to the cells in viable but non culturable(VBNC)state of Micrococcus luteus and Rhodococcus marinonascens,the result showed that it could significantly promote their resuscitation,and the resuscitation effect was the most significant when 10% Rpf-2 protein was added.This laid a foundation for the subsequent study on the function of Rpf-2 protein in A.alba.