title: [Expression and activity identification of a fusion protein for promoting adenovirus infection efficiency of dendritic cells].
abstract: OBJECTIVE
  To construct a prokaryotic expression plasmid for CT40L, express the target protein in E. coli, purity the CT40L fusion protein and verify its antigenicity.
  
  
  METHODS
  Gene sequences of Coxsackie and adenovirus receptor (CAR), bacteriophage T4 fibritin and mouse CD40L were found out in GenBank. Then functional domains of three molecules were linked to form a fusion sequence which was then optimized for prokaryotic expression. The optimized sequence was cloned into prokaryotic expression vector pET42a(+) to construct the recombinant expression vector pET42a-CT40L. The recombinant vector was transformed into BL21 (DE3) and the fusion protein CT40L/GST was induced by IPTG. The fusion protein was then subjected to purification using GST affinity chromatography and to identification of the immune activity using Western blotting and ELISA.
  
  
  RESULTS
  The recombinant expression vector was verified correct by double digestion with Nco I and EcoR I. After IPTG induction, SDS-PAGE showed that the relative molecular mass of the fusion protein was about 78 kDa and that the purity of the purified protein reached 90%. Western blotting and ELISA demonstrated that the purified fusion protein had a valid antigenicity.
  
  
  CONCLUSIONS
  The prokaryotic expression plasmid pET42a-Ct40L was successfully constructed and expressed in E. coli, and the purified fusion protein was proved to have a good antigenicity.
authors.affiliation: Department of Urology, General Hospital of PLA, Beijing 100853; Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
authors.forename: Renli
authors.initials: R
authors.lastname: Tian
authors.affiliation: Department of Urology, General Hospital of PLA, Beijing 100853; Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
authors.forename: Xiaotao
authors.initials: X
authors.lastname: Yin
authors.affiliation: Department of Urology, General Hospital of PLA, Beijing 100853; Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: Department of Urology, General Hospital of PLA, Beijing 100853; Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
authors.forename: Xiaoliang
authors.initials: X
authors.lastname: Lin
authors.affiliation: Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
authors.forename: Yuanji
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
authors.forename: Jinqi
authors.initials: J
authors.lastname: Yan
authors.affiliation: Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Department of Urology, General Hospital of PLA, Beijing 100853, China.
authors.forename: Jiangping
authors.initials: J
authors.lastname: Gao
authors.affiliation: Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
authors.forename: Jiyun
authors.initials: J
authors.lastname: Yu
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000276:immunology / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019013:CD40 Antigens / Q000276:immunology / Q000378:metabolism; D023201:CD40 Ligand / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism* / Q000821:virology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000276:immunology; D054884:Host-Pathogen Interactions / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D019013:CD40 Antigens; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C057839:fibritin protein, Enterobacteria phage T4; D023201:CD40 Ligand