流产布氏杆菌BPE123基因的克隆表达及生物信息学分析
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摘要
根据GenBank中流产布氏杆菌BPE123基因序列(登录号:NC_006933.1),设计引物,以布氏杆菌病疫苗菌A19全基因组DNA为模板扩增BPE123基因,将目的基因克隆入pEASY-T3载体,测序正确后,双酶切pEASY-T3-BPE123,目的片段BPE123连入pET-32a(+)表达载体,构建表达质粒pET-32a(+)-BPE123,转化大肠杆菌BL21(DE3),IPTG诱导表达;对BPE123基因进行生物信息学分析。结果显示,成功构建了重组菌pET-32a(+)-BPE123-BL21(DE3),诱导表达了融合蛋白BPE123。生物信息学分析显示,BPE123基因N端25个氨基酸是其信号肽,与其相互作用的蛋白大都与菌毛相关,它们是flgF、fliI、motA、BruAb2_0155、BruAb2_0121和BruAb2_0125。布氏杆菌属内BPE123基因序列的同源性高达99%。本研究获得了BPE123蛋白并预测了BPE123基因的相关生物学功能,为进一步探索该蛋白的免疫原性和在布氏杆菌胞内寄生过程中的作用奠定了基础。
According to Brucella abortus BPE123 gene sequence in GenBank,BPE123 gene was amplified by polymerase chain reaction,and then cloned into the pEASY-T3 vector.After sequenced for identification,the gene was cloned into the expression vector pET-32a(+)to construct a recombinant plasmid pET-32a(+)-BPE123.The recombinant plasmid pET-32a(+)-BPE123 was transformed into Escherichia coli BL21(DE3)and induced with IPTG to express the recombinant BPE123 protein.In addition,the BPE123 gene was analyzed by bioinformatics.Results showed that the recombinant plasmid pET-32a(+)-BPE123 was successfully constructed and highly purified protein was expressed.Bioinformatics analysis found that the first 25 amino acids in the N-terminus of BPE123 were essential for its secretory.The Universal Protein Database predicted that most proteins interacting with BPE123 were mostly associated with fimbriae,such as flgF,fliI,motA,BruAb2_0155,BruAb2_0121and BruAb2_0125.The homology of gene sequence of Brucella BPE123 is as high as 99%.In conclusion,this study obtained the BPE123 protein and forecasted its related biological functions.This research laid the foundation for further studies on BPE123protein's biofunction in intracellular parasitic and its immunogenicity.
According to Brucella abortus BPE123 gene sequence in GenBank,BPE123 gene was amplified by polymerase chain reaction,and then cloned into the pEASY-T3 vector.After sequenced for identification,the gene was cloned into the expression vector pET-32a(+)to construct a recombinant plasmid pET-32a(+)-BPE123.The recombinant plasmid pET-32a(+)-BPE123 was transformed into Escherichia coli BL21(DE3)and induced with IPTG to express the recombinant BPE123 protein.In addition,the BPE123 gene was analyzed by bioinformatics.Results showed that the recombinant plasmid pET-32a(+)-BPE123 was successfully constructed and highly purified protein was expressed.Bioinformatics analysis found that the first 25 amino acids in the N-terminus of BPE123 were essential for its secretory.The Universal Protein Database predicted that most proteins interacting with BPE123 were mostly associated with fimbriae,such as flgF,fliI,motA,BruAb2_0155,BruAb2_0121and BruAb2_0125.The homology of gene sequence of Brucella BPE123 is as high as 99%.In conclusion,this study obtained the BPE123 protein and forecasted its related biological functions.This research laid the foundation for further studies on BPE123protein's biofunction in intracellular parasitic and its immunogenicity.
引文
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[20]陆勇军,李向辉,曾咏伦.一个细菌的致命之旅——嗜肺军团菌分泌系统及效应蛋白的研究进展[J].遗传,2011,33(10):1093-1101.LU Yong-jun,LI Xiang-hui,ZENG Yong-lun.The lethiferous journey of a bacterium—The research progress of secretion systems and effectors in Legionella pneumophila[J].Hereditas,2011,33(10):1093-1101.(in Chinese)
[21]AMOR J C,SWAILS J,ZHU X,et al.The structure of RalF,an ADP-ribosylation factor guanine nucleotide exchange factor from Legionella pneumophila,reveals the presence of a cap over the active site[J].J Biol Chem,2005,280(2):1392-1400.
[22]CHEN J,REYES M,CLARKE M,et al.Host cell-dependent secretion and translocation of the LepA and LepB effectors of Legionella pneumophila[J].Cell Microbiol,2007,9(7):1660-1671.
[2]MORENO E,STACKEBRANDT E,DORSCH M,et al.Brucella abortus 16SrRNA and lipid A reveal a phylogenetic relationship with members of the alpha-2subdivision of the class Proteobacteria[J].J Bacteriol,1990,172(7):3569-3576.
[3]张瑞,王秀然,李晓艳,等.布鲁氏菌胞内寄生的研究进展[J].安徽农业科学,2012,40(9):5246-5248.ZHANG Rui,WANG Xiu-ran,LI Xiao-yan,et al.Research advance on Brucellaparasitism mechanism[J].Journal of Anhui Agricultural Sciences,2012,40(9):5246-5248.(in Chinese)
[4]SIEIRA R,COMERCI D J,SANCHEZ D O,et al.A homologue of an operon required for DNA transfer in Agrobacteriumis required in Brucella abortus for virulence and intracellular multiplication[J].J Bacteriol,2000,182(17):4849-4855.
[5]SIEIRA R,COMERCI D J,PIETRASANTA L I,et al.Integration host factor is involved in transcriptional regulation of the Brucella abortus virB operon[J].Mol Microbiol,2004,54(3):808-822.
[6]CELLI J.Surviving inside a macrophage:the many ways of Brucella[J].Res Microbiol,2006,157(2):93-98.
[7]CELLI J,DE CHASTELLIER C,FRANCHINI D M,et al.Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum[J].J Exp Med,2003,198(4):545-556.
[8]MARCHESINI M I,HERRMANN C K,SALCEDO S P,et al.In search of Brucella abortus typeⅣsecretion substrates:screening and identification of four proteins translocated into host cells through VirB system[J].Cell Microbiol,2011,13(8):1261-1274.
[9]LACERDA S L,SALCEDO P S,GORVEL P J.Brucella T4SS:the VIP pass inside host cells[J].Curr Opin Microbiol,2013,16(1):45-51.
[10]KHLER S,MICHAUX-CHARACHON S,PORTE F,et al.What is the nature of the replicative niche of a stealthy bug named Brucella?[J].Trends Microbiol,2003,11(5):215-219.
[11]ROSE A,SCHRAEGLE S J,STAHLBERG E A,et al.Coiled-coil protein composition of 22proteomes differences and common themes in subcellular infrastructure and traffic control[J].BMC Evol Biol,2005,5:66.
[12]GAZI A D,CHAROVA S N,PANOPOULOS N J,et al.Coiled-coils in typeⅢsecretion systems:structural flexibility,disorder and biological implications[J].Cell Microbiol,2009,11(5):719-729.
[13]ROOP R MⅡ,GAINES J M,ANDERSON E S,et al.Survival of the fittest:how Brucellastrains adapt to their intracellular niche in the host[J].Med Microbiol Immunol,2009,198(4):221-238.
[14]FRETIN D,FAUCONNIER A,KOHLER S,et al.The sheathed flagellum of Brucella melitensis is involved in persistence in a murine model of infection[J].Cell Microbiol,2005,7(5):687-698.
[15]王景龙,张瑞,王兴龙,等.细菌Ⅳ型分泌系统结构的研究进展[J].安徽农业科学,2011,39(23):13979-13980.WANG Jing-long,ZHANG Rui,WANG Xing-long,et al.Research advance of molecular architecture of bacterial typeⅣsecretion systems[J].Journal of Anhui Agricultural Sciences,2011,39(23):13979-13980.(in Chinese)
[16]FRONZES R,CHRISTIE P J,WAKSMAN G.The structural biology of typeⅣsecretion systems[J].Nat Rev Microbiol,2009,7(10):703-714.
[17]FRONZES R,REMAUT H,WAKSMAN G.Architectures and biogenesis of non-flagellar protein appendages in Gramnegative bacteria[J].EMBO J,2008,27(17):2271-2280.
[18]GHOSH P.Process of protein transport by the typeⅢsecretion system[J].Microbiol Mol Biol Rev,2004,68(4):771-795.
[19]LEONARD S,FEROOZ J,HAINE V,et al.FtcR is a new master regulator of the flagellar system of Brucella melitensis 16M with homologs in Rhizobiaceae[J].J Bacteriol,2007,189(1):131-141.
[20]陆勇军,李向辉,曾咏伦.一个细菌的致命之旅——嗜肺军团菌分泌系统及效应蛋白的研究进展[J].遗传,2011,33(10):1093-1101.LU Yong-jun,LI Xiang-hui,ZENG Yong-lun.The lethiferous journey of a bacterium—The research progress of secretion systems and effectors in Legionella pneumophila[J].Hereditas,2011,33(10):1093-1101.(in Chinese)
[21]AMOR J C,SWAILS J,ZHU X,et al.The structure of RalF,an ADP-ribosylation factor guanine nucleotide exchange factor from Legionella pneumophila,reveals the presence of a cap over the active site[J].J Biol Chem,2005,280(2):1392-1400.
[22]CHEN J,REYES M,CLARKE M,et al.Host cell-dependent secretion and translocation of the LepA and LepB effectors of Legionella pneumophila[J].Cell Microbiol,2007,9(7):1660-1671.