title: Isolation, Cloning and High- Level Expression of Neutrophil Gelatinase-Associated Lipocalin Lipocalin2 by Baculovirus Expression System through Gateway Technology.
abstract: OBJECTIVE
  Lipocalin 2 (Lcn2) is a 25-kDa glycoprotein that has initially been extracted from neutrophil granules. Expression of Lcn2 is induced under various pathophysiological conditions. It is also known as an early marker of kidney and heart injury. High-level expression of recombinant Lcn2 neutrophil gelatinase-associated (NGAL) in insect cells was the aim of this study.
  
  
  METHODS
  Lcn2 gene was isolated from HepG2 cell line. The PCR product was cloned into TOPO vector to construct TOPO-Lcn2. Then Lcn2 was transferred to Gateway adapted Baculovirus DNA by LR recombination reaction. The recombinant Baculovirus DNA was transfected into insect cell line. Expression of recombinant Lcn2 was detected by RT-PCR, ELISA and western blot analysis.
  
  
  RESULTS
  Insertion of Lcn2 into pENTR/D-TOPO vector was confirmed by using PCR. The accuracy of the nucleotides sequence was verified by DNA sequencing. Transfer of the Lcn2 cDNA into the Baculovirus destination vector by LR recombination reaction was confirmed by amplification of a band of about 860 bp length by using forward Lcn2 primer and V5 reverse primer. Next, Lcn2 protein was detected as a prominent band with approximate molecular weight of 30 kDa in SDS-PAGE and western blot analysis. ELISA results revealed high-level expression of Lcn2 by Spodoptera frugiperda (Sf9) cells.
  
  
  CONCLUSIONS
  High-level expression of Lcn2 protein in insect cells is promising for future potential applications. Recombinant Lcn2 might be used for producing monoclonal or polyclonal antibodies and as potential therapeutic agent. Large scale expression and purification are next steps that are on the way.
authors.affiliation: Blood Transfusion Research Centre, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.
authors.forename: Mahdi
authors.initials: M
authors.lastname: Rouhbakhsh
authors.forename: Raheleh
authors.initials: R
authors.lastname: Halabian
authors.forename: Nasser
authors.initials: N
authors.lastname: Masroori
authors.forename: Mahshid
authors.initials: M
authors.lastname: Mohammadi Pour
authors.forename: Parisa
authors.initials: P
authors.lastname: Bahmani
authors.forename: Amaneh
authors.initials: A
authors.lastname: Mohammadi Roush
authors.forename: Ali
authors.initials: A
authors.lastname: Jahanian-Najafabadi
authors.forename: Mehryar
authors.initials: M
authors.lastname: Habibi Roudkenar
keywords: Baculoviridae; Lipocalin 2 (Lcn2); Neutrophil gelatinase-associated lipocalin (NGAL); Recombination; Vector