问号钩端螺旋体LipL32-LipL41融合基因的克隆与原核表达
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摘要
目的构建钩端螺旋体融合基因lip L32-lip L41重组表达质粒,并获得重组表达的融合蛋白。方法设计引物,聚合酶链反应(PCR)从各参考标准株中扩增Lip L32、Lip L41基因,连接引物PCR法构建Lip L32-Lip L41融合基因,连接到大肠杆菌表达载体p ET28a上,构建p ET28a-Lip L32-Lip L41重组质粒,并转化到大肠杆菌DE3菌株中进行诱导表达,聚丙烯酰氨凝胶电泳(SDS-PAGE)及Western blot鉴定融合蛋白的表达。结果扩增获得的Lip L32-Lip L41融合基因为1 900 bp,原核表达重组质粒p ET28a-Lip L32-Lip L41经酶切鉴定含有融合基因片段,在大肠杆菌中表达出Mr 90 000左右的重组融合蛋白Lip L32-Lip L41。结论成功构建钩体Lip L32-Lip L41融合基因,并在大肠杆菌中成果表达融合蛋白,为进一步评价Lip L32-Lip L41作为候选抗原诊断钩端螺旋体病的价值奠定基础。
Objective To construct Leptospira interrogans(L. interrogans)Lip L32-Lip L41 fusion gene and express the fusion protein. Methods Lip L32 or Lip L41 was amplified by polymerase chain reaction(PCR) with the specific primers from genomic DNA of L. interrogans reference strains. Lip L32-Lip L41 fusion gene was constructed by PCR with overlap primers. The recombinant plasmid p ET28a-Lip L32-Lip L41 was constructed followed by digestion of the fusion gene and p ET28 a plasmid with endonucleases Eco RI and Xho I, ligation and transformation. The recombinant plasmid was identified by single or double endonuclease digestion. The fusion protein Lip L32-Lip L41 was expressed in E. coli DE3 by induction with adding isopropyl-β-thiogalactoside(IPTG) to the culture and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot with anti-6 ×His tag monoclonal antibody as the prime antibody. Results The codinglength of fusion gene Lip L32-Lip L41 was 1 900 bp. The recombinant plasmid p ET28a-Lip L32-Lip L41 was confirmed to include the target gene. Lip L32-Lip L41 fusion protein was expressed in prokaryotic expression system and its molecular mass was about 90 000. Conclusion The L. interrogans lip L32-lip L41 fusion gene was constructed and expressed in E.coli successfully. Our work was the foundation for evaluating the value of the fusion protein as a candidate diagnostic antigen.
Objective To construct Leptospira interrogans(L. interrogans)Lip L32-Lip L41 fusion gene and express the fusion protein. Methods Lip L32 or Lip L41 was amplified by polymerase chain reaction(PCR) with the specific primers from genomic DNA of L. interrogans reference strains. Lip L32-Lip L41 fusion gene was constructed by PCR with overlap primers. The recombinant plasmid p ET28a-Lip L32-Lip L41 was constructed followed by digestion of the fusion gene and p ET28 a plasmid with endonucleases Eco RI and Xho I, ligation and transformation. The recombinant plasmid was identified by single or double endonuclease digestion. The fusion protein Lip L32-Lip L41 was expressed in E. coli DE3 by induction with adding isopropyl-β-thiogalactoside(IPTG) to the culture and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot with anti-6 ×His tag monoclonal antibody as the prime antibody. Results The codinglength of fusion gene Lip L32-Lip L41 was 1 900 bp. The recombinant plasmid p ET28a-Lip L32-Lip L41 was confirmed to include the target gene. Lip L32-Lip L41 fusion protein was expressed in prokaryotic expression system and its molecular mass was about 90 000. Conclusion The L. interrogans lip L32-lip L41 fusion gene was constructed and expressed in E.coli successfully. Our work was the foundation for evaluating the value of the fusion protein as a candidate diagnostic antigen.
引文
[1]Guerra M.Leptospirosis:public health perspectives[J].Biol2013,41(5):295-297.
[2]刘波,丁凡,蒋秀高,等.2006-2010年中国钩端螺旋体病流行病学分析[J].疾病监测,2012,27(1):46-50.Liu B,Ding F,Jiang XG,et al.Epidemiology of leptospirosis in China,2006-2010[J].Disease Surveillance,2012,27(1):46-50.
[3]Sabina G,Juan P,Elba H,et al.Usefulness of real-time PCR assay targeting lipL32 gene for diagnosis ofhuman leptospirosis in Uruguay[J].J Infect Dev Ctries,2013,7(12):941-945.
[4]Sabrina T,Carolina L,Albert K,et al.Identification of immunodominant antigens incanine leptospirosis by MultiAntigen PrintImmunoAssay[J].BMC Vet Res,2014,8(10):288-296.
[5]Timothy D,Azad E,Jarlath E,et al.Post-translational Modification of LipL32 duringLeptospira interrogans Infection[J].PLOS NTD,2013,8(10):3280-3288.
[6]Alejandra H,Patricia A,Noelia O,et al.Increased immunogenicity to LipL32 of leptospira interroganswhen expressed as a fusion protein with the cholera toxin BSubunit[J].Curr Microbiol,2011,10(62):526-531.
[7]Lo Y,Hsu S,Ko Y,etal.Essential Calcium-binding cluster of Leptospiral LipL32 protein for inflammatory responses through the Toll-like receptor pathway[J].Biol Chem,2013,288(17):12335-12344.
[8]Xu L,Sun H,Ruan P,etal.Characterization of Conserved Combined T and B Cell Epitopes in leptospira interrogans major outer membrane proteins ompL1 and LipL41[J].BMC Microbiol,2011,8(11):21-29.
[9]King A,Bartpho T,Sermswan R,et al.Leptospiral outer membrane protein LipL41is not essential for acute leptospirosisbut requires a small chaperone protein,LEP,forstable expression[J].Infect Immun,2013,81(8):2768-2776.
[10]Carolina L,Camila B,Jozelyn P,et al.Identification of seroreactive proteins of leptospirainterrogans serovar copenhageni using a high-density protein microarray approach[J].PLOSNTD,2013,7(10):2499-2511.
[2]刘波,丁凡,蒋秀高,等.2006-2010年中国钩端螺旋体病流行病学分析[J].疾病监测,2012,27(1):46-50.Liu B,Ding F,Jiang XG,et al.Epidemiology of leptospirosis in China,2006-2010[J].Disease Surveillance,2012,27(1):46-50.
[3]Sabina G,Juan P,Elba H,et al.Usefulness of real-time PCR assay targeting lipL32 gene for diagnosis ofhuman leptospirosis in Uruguay[J].J Infect Dev Ctries,2013,7(12):941-945.
[4]Sabrina T,Carolina L,Albert K,et al.Identification of immunodominant antigens incanine leptospirosis by MultiAntigen PrintImmunoAssay[J].BMC Vet Res,2014,8(10):288-296.
[5]Timothy D,Azad E,Jarlath E,et al.Post-translational Modification of LipL32 duringLeptospira interrogans Infection[J].PLOS NTD,2013,8(10):3280-3288.
[6]Alejandra H,Patricia A,Noelia O,et al.Increased immunogenicity to LipL32 of leptospira interroganswhen expressed as a fusion protein with the cholera toxin BSubunit[J].Curr Microbiol,2011,10(62):526-531.
[7]Lo Y,Hsu S,Ko Y,etal.Essential Calcium-binding cluster of Leptospiral LipL32 protein for inflammatory responses through the Toll-like receptor pathway[J].Biol Chem,2013,288(17):12335-12344.
[8]Xu L,Sun H,Ruan P,etal.Characterization of Conserved Combined T and B Cell Epitopes in leptospira interrogans major outer membrane proteins ompL1 and LipL41[J].BMC Microbiol,2011,8(11):21-29.
[9]King A,Bartpho T,Sermswan R,et al.Leptospiral outer membrane protein LipL41is not essential for acute leptospirosisbut requires a small chaperone protein,LEP,forstable expression[J].Infect Immun,2013,81(8):2768-2776.
[10]Carolina L,Camila B,Jozelyn P,et al.Identification of seroreactive proteins of leptospirainterrogans serovar copenhageni using a high-density protein microarray approach[J].PLOSNTD,2013,7(10):2499-2511.