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 luteus,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 Escherichia coli BL21(DE3).Based on rpf nucleic acid sequence,the bioinformation of Rpf protein,such as protein 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 identity 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 - DOI:10.3969/j.issn.1000-1565.2016.04.012藤黄微球菌rpf基因的克隆表达及其结构预测张秀敏1,景凤霞1,王海强1,赵若玮1,张艳芬2(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)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.关键词:藤黄微球菌;复苏促进因子(Rpf);克隆;表达;结构预测中图分类号:Q939.13; 文献标志码:A 文章编号:1000-1565(2016)04-0402-10Cloning and expression of Micrococcus luteus rpf gene and structure prediction of Rpf proteinZHANG Xiumin1,JING Fengxia1,WANG Haiqiang1,ZHAO Ruowei1,ZHANG Yanfen2(1.College of Life Scineces,Hebei University,Baoding 071002,China;2.Office of Science and Technology,Hebei University,Baoding 071002,China)Abstract:In order to study the properties of resuscitation-promoting factor(Rpf)of Micrococcus luteus,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 Escherichia coli BL21(DE3).Based on rpf nucleic acid sequence,the bioinformation of Rpf protein,such as protein 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 identity 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 - 收稿日期:2016-04-13 基金项目: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) 第一作者:张秀敏(1970—),女,河北涞水人,河北大学教授,博士,主要从事微生物系统性及多样性研究.E-mail:zhxiumin1106@126.com 通信作者:张艳芬(1979—),女,河北邯郸人,河北大学讲师,主要从事分子生物学研究.E-mail:zhangjing@hbu.edu.cn第4期张秀敏等:藤黄微球菌rpf基因的克隆表达及其结构预测(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 explained 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.
[1] MUKAMOLOVA G V,KAPRELYANTS A S,YOUNG D I,et al.A bacterial cytokine[J].Proceedings of the National Academy of Sciences,1998,95(15):8916-8921.
[2] 丛延广,胡福泉.细菌复苏促进因子Rpf[J].生命的化学,2005,25(5):482-484. CONG Y G,HU F Q.Resuscitation-promoting factors(RPF)of bacteria[J].Chemistry of Life,2005,25(5):482-484.
[3] XU H S,ROBERTS N,SINGLETON F L,et al.Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment[J].Microbial Ecology,1982,8(4):313-323. DOI:10.1007/BF02010671.
[4] RAVAGNANI A,FINAN C L,YOUNG M.A novel firmicute protein family related to the actinobacterial resuscitation-promoting factors by non-orthologous domain displacement[J].BMC Genomics,2005,6:39.DOI:10.1186/1471-2164-6-39.
[5] DEWI P I,UEHARA M,KATAYAMA,et al.Resuscitation promoting factor(Rpf)from Tomitella biformata AHU 1821(T)promotes growth and resuscitates non-dividing cells[J].Microbes and Environments,2013,28(1):58-64.DOI:10.1264/jsme2.ME12122.
[6] HUANG W,QI Y,DIAO Y,et al.Use of resuscitation-promoting factor proteins improves the sensitivity of culture-based tuberculosis testing in special samples[J].Am J Respir Crit Care Med,2014,189(5):612-614. DOI:10.1164/rccm.201310-1899LE.
[7] 魏忠彬,李影,钱爱东,等.VBNC 乳酸杆菌的复苏[J].中国饲料,2006(18):27-28. WEI Z B,LI Y,QIAN A D,et al. Recovery of VBNC Lactobacill[J].Chinas Feed,2006(18):27-28.
[8] 丁林贤,张萍华,洪华嫦,等.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[J]. 微生物学报,2012,52(1):77-89. DING L X,ZHANG P H,HONG H C,et al.Cloning and expression of Micrococcus luteus IAM 14879 Rpf and its role in the recovery of the VBNC state in Rhodococcus sp.DS471[J].Acta Microbiologica Sinica,2012,52(1):77-89.
[9] COHEN G M,BARTHE P,BAGNERIS C,et al.The structure of a resuscitation-promoting factor domain from Mycobacterium tuberculosis shows homology to lysozymes[J].Nature Structural and Molecular Biology,2005,12(3):270-273.DOI:10.1038/nsmb905.
[10] 李伦,李元,白婧,等.细菌复苏促进因子[J].动物医学进展,2010,31(4):103-106. LI L,LI Y,BAI J,et al.Progress on resuscitation-promoting factor[J].Progress in Veterinary Medicine,2010,31(4):103-106.
[11] GARVEY K J,SAEDI M S,ITO J.Nucleotide sequence of Bacillus phage phi 29 genes 14 and 15:homology of gene 15 with other phage lysozymes[J].Nucleic Acid Res,1986,14:10001-10008.
