藤黄微球菌rpf基因的克隆表达及其结构预测  

Cloning and expression of Micrococcus luteus rpfgene and structure prediction of Rpf protein

作　　者：张秀敏[1] 景凤霞[1] 王海强[1] 赵若玮 张艳芬[2] 

机构地区：[1]河北大学生命科学学院,河北保定071002 [2]河北大学科学技术处,河北保定071002

出　　处：《河北大学学报（自然科学版）》2016年第4期402-411,共10页Journal of Hebei University(Natural Science Edition)

基　　金：国家自然科学基金资助项目(31270053);河北省自然科学基金资助项目(C2014201141);河北省生物工程重点学科建设经费资助项目(1050-5030023);河北省高等学校科学技术研究资助项目(Z2015013)

摘　　要：为了了解藤黄微球菌(Micrococcus luteus)复苏促进因子(resuscitation-promoting factor,Rpf)的性质,用自行设计的引物,以藤黄微球菌ACCC41016基因组DNA为模板,扩增rpf基因.随后,构建pET-28a(+)-rpf表达载体,在Escherichia coli BL21(DE3)中进行IPTG诱导表达,并以克隆测得的rpf基因核酸序列为基础,对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.结果表明:该rpf基因与文献报道的藤黄微球菌IAM 14879 rpf基因核苷酸序列一致性为69.72%;该rpf基因所编码蛋白的氨基酸序列与文献报道的Rpf蛋白氨基酸序列一致性为63.44%.在E.coli BL21(DE3)中成功表达了Rpf目的蛋白.初步了解了Rpf蛋白的理化性质及生物学功能.解释了Rpf蛋白能够使细菌复苏并促进其生长的理论,为进一步探讨Rpf蛋白的作用机理奠定了基础.In order to study the properties of resuscitation-promoting factor （Rpf） of Micrococcus lute- us ,rpf gene （813 pb）was amplified by using genome DNA of M. luteus ACCC41016 as templates.pET-28a （＋）-rpf expression vector was constructed and Rpf protein was induced using IPTG to express in Esche- richia coli BL21（DE3）.Based on rpf nucleic acid sequence,the bioinformation of Rpf protein,such as pro- tein amino acid sequence, hydrophobic, signal peptide, phosphorylation sites, tertiary structure, structure and functional domains,were predicted by using bioinformatics method.The results showed that the identi- ty of nucleotide sequences between the cloning rpf gene and the reportorial rpf gene was 69.72%.And the identity of amino acids sequences between the protein encoded by the cloning rpf gene and the reportorial Rpf protein was 63.44%.SDS-PAGE results confirmed that the expression of rpf gene in the E.coli BL21 （DE3）was successful.According to the bioinformatics and homology analysis of Rpf protein,the physical and chemical properties and biological function of Rpf protein were preliminary elucidated.The study ex- plained the theory of the Rpf protein can make the bacteria recovery and promote its growth.This laid the groundwork for further mechanistic studies of Rpf protein.

关 键 词：藤黄微球菌 复苏促进因子(Rpf) 克隆 表达 结构预测 

分 类 号：Q939.13[生物学—微生物学]