title: [Cloning, prokaryotic expression, and antigenicity identification of extracellular domain of mouse CD40].
abstract: OBJECTIVE
  To construct a prokaryotic expression plasmid for extracellular domain of mouse CD40 (mCD40), express the mCD40/GST recombinant protein in E.coli, purify the mCD40/GST recombinant protein and characterize its antigenicity.
  
  
  METHODS
  Extracellular domain of mouse CD40 was amplified by PCR from cell line DC2.4 and then was cloned into prokaryotic expression vector pGEX-6P-1 to construct the recombinant expression vector pGEX-6P-1-mCD40. The expression vector was transformed into E.coli BL21 (DE3) and the fusion protein mCD40/GST was induced by IPTG. The fusion protein was purified through sepharose 4B. Then antigenicity of the purified mCD40/GST protein was verified by Western blotting and ELISA.
  
  
  RESULTS
  The PCR product was verified by DNA sequencing to be consistent with the sequence of mouse CD40 on GenBank. The recombinant plasmid was identified by double digestion successfully. SDS-PAGE analysis showed the relative molecular mass of the fusion protein induced by IPTG was 45 000. Western blotting and ELISA demonstrated that the purified mCD40/GST protein had a good antigenicity.
  
  
  CONCLUSIONS
  The prokaryotic expression plasmid pGEX-6P-1-mCD40 was constructed successfully. In E.coli BL21 (DE3) transformed with the plasmid, the mCD40/GST fusion protein was expressed by IPTG induction. The purified mCD40/GST fusion protein had a high antigenicity, which provides a strong support for the future study of CD40.
authors.affiliation: Department of Urology, PLA General Hospital, Beijing 100853, China.
authors.forename: Renli
authors.initials: R
authors.lastname: Tian
authors.forename: Jingying
authors.initials: J
authors.lastname: Zhu
authors.forename: Xiaotao
authors.initials: X
authors.lastname: Yin
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Xiaoliang
authors.initials: X
authors.lastname: Lin
authors.forename: Yuanji
authors.initials: Y
authors.lastname: Xu
authors.forename: Jinqi
authors.initials: J
authors.lastname: Yan
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Jiangping
authors.initials: J
authors.lastname: Gao
authors.forename: Jiyun
authors.initials: J
authors.lastname: Yu
mesh_terms: D000818:Animals; D019013:CD40 Antigens / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D005110:Extracellular Space / Q000378:metabolism*; D015870:Gene Expression; D051379:Mice; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D019013:CD40 Antigens; D011993:Recombinant Fusion Proteins