title: [Preparation and identification of recombinant human neutrophil elastase].
abstract: OBJECTIVE
  To prepare a purified recombinant human neutrophil elastase (HNE) using genetic engineering technology, and pave the way for the preparation of the antibody to HNE and establishment of semen HNE detection methods.
  
  
  METHODS
  HNE mRNA was obtained from human peripheral blood granulocytes with specific HNE primers, and the cDNA of HNE was cloned into the plasmid pGEX-2T to derive a recombinant plasmid pGEX-2T/HNE. After PCR identification, double-enzyme digestion and gene sequencing, the recombinant plasmid was transferred into competent Escherichia coli DH5alpha and further induced to express the recombinant fusion protein GST/HNE by isopropyl beta-D-thiosulfate galactosidase (IPTG). The recombinant fusion protein was cleaved by thrombin and further purified with glutathione agarose beads to obtain purified recombinant HNE.
  
  
  RESULTS
  The recombinant plasmid pGEX-2T/HNE was successfully prepared and transferred into E. coli DH5o; the expression of the recombinant fusion protein GST/ HNE was successfully induced by IPTG at 18 degrees C overnight; and the purified recombinant protein HNE was successfully obtained by thrombin cleavage and purification of glutathione agarose beads.
  
  
  CONCLUSIONS
  The acquirement of purified recombinant HNE has prepared the ground for the preparation of the antibody to HNE and establishment of semen HNE detection methods.
authors.affiliation: Department of Laboratory Science, Nanjing Hospital, Jiangsu Corps of the Armed Police Force, PLA, Nanjing, Jiangsu 210028, China. lujc@androl.cn
authors.forename: Jin-Chun
authors.initials: JC
authors.lastname: Lu
authors.forename: Kun-Gang
authors.initials: KG
authors.lastname: Lu
authors.forename: Hong-Ye
authors.initials: HY
authors.lastname: Zhang
authors.forename: Jian
authors.initials: J
authors.lastname: Gao
authors.forename: Rui-Xiang
authors.initials: RX
authors.lastname: Feng
mesh_terms: D017931:DNA Primers; D005818:Genetic Engineering; D006801:Humans; D019272:Leukocyte Elastase / Q000096:biosynthesis* / Q000235:genetics; D009504:Neutrophils / Q000201:enzymology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*
chemical_list: D017931:DNA Primers; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019272:Leukocyte Elastase