title: [Prokaryotic expression and immunological activity of human neutrophil gelatinase associated lipocalin].
abstract: OBJECTIVE
  To construct a prokaryotic expression vector of human neutrophil gelatinase associated lipocalin (NGAL) and identify the bioactivity of the fusion protein.
  
  
  METHODS
  The cDNA of human NGAL obtained from GenBank was linked to a cloning vector to construct the prokaryotic expression vector pCold-NGAL. Then the vector was transformed into E.coli BL21(DE3) plysS. Under the optimal induction condition, the recombinant NGAL (rNGAL) was expressed and purified by Ni Sepharose 6 Fast Flow affinity chromatography. The purity and activity of the rNGAL were respectively identified by SDS-PAGE and Western blotting combined with NGAL reagent (Latex enhanced immunoturbidimetry).
  
  
  RESULTS
  Restriction enzyme digestion and nucleotide sequencing proved that the expression vector pCold-NGAL was successfully constructed. Under the optimal induction condition that we determined, the rNGAL was expressed in soluble form in E.coli BL21(DE3) plysS. The relative molecular mass of the rNGAL was 25 000, and its purity was more than 98.0%. Furthermore, Western blotting and immunoturbidimetry indicated that the rNGAL reacted with NGAL mAb specifically.
  
  
  CONCLUSIONS
  Human rNGAL of high purity and bioactivity was successfully constructed in E.coli BL21(DE3) plysS using the expression vector pCold-NGAL.
authors.affiliation: National Engineering Laboratory of Rapid Diagnostic Test, Guangzhou Wondfo Biotech Co., Ltd. Guangzhou 510663, China.
authors.forename: Jianwei
authors.initials: J
authors.lastname: Wu
authors.affiliation: National Engineering Laboratory of Rapid Diagnostic Test, Guangzhou Wondfo Biotech Co., Ltd. Guangzhou 510663, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Cai
authors.affiliation: National Engineering Laboratory of Rapid Diagnostic Test, Guangzhou Wondfo Biotech Co., Ltd. Guangzhou 510663, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Qian
authors.affiliation: National Engineering Laboratory of Rapid Diagnostic Test, Guangzhou Wondfo Biotech Co., Ltd. Guangzhou 510663, China.
authors.forename: Liyuan
authors.initials: L
authors.lastname: Jiao
authors.affiliation: National Engineering Laboratory of Rapid Diagnostic Test, Guangzhou Wondfo Biotech Co., Ltd. Guangzhou 510663, China.
authors.forename: Jiangfeng
authors.initials: J
authors.lastname: Li
authors.affiliation: National Engineering Laboratory of Rapid Diagnostic Test, Guangzhou Wondfo Biotech Co., Ltd. Guangzhou 510663, China.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Song
authors.affiliation: National Engineering Laboratory of Rapid Diagnostic Test, Guangzhou Wondfo Biotech Co., Ltd. Guangzhou 510663, China.
authors.forename: Jihua
authors.initials: J
authors.lastname: Wang
mesh_terms: D000209:Acute-Phase Proteins / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D000071068:Lipocalin-2; D054834:Lipocalins / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000209:Acute-Phase Proteins; C489506:LCN2 protein, human; D000071068:Lipocalin-2; D054834:Lipocalins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins