Isolation, Cloning and High- Level Expression of Neutrophil Gelatinase-Associated Lipocalin Lipocalin۲ by Baculovirus Expression System through Gateway Technology

Objective(s) Lipocalin ۲ (Lcn۲) is a ۲۵-kDa glycoprotein that has initially been extracted from neutrophil granules. Expression of Lcn۲ is induced under various pathophysiological conditions. It is also known as an early marker of kidney and heart injury. High-level expression of recombinant Lcn۲ neutrophil gelatinase-associated(NGAL) in insect cells was the aim of this study. Materials and Methods Lcn۲ gene was isolated from HepG۲ cell line. The PCR product was cloned into TOPO vector to construct TOPO-Lcn۲. Then Lcn۲ was transferred to Gateway adapted Baculovirus DNA by LR recombination reaction. The recombinant Baculovirus DNA was transfected into insect cell line. Expression of recombinant Lcn۲ was detected by RT-PCR, ELISA and western blot analysis. Results Insertion of Lcn۲ into pENTR/D-TOPO vector was confirmed by using PCR. The accuracy of the nucleotides sequence was verified by DNA sequencing. Transfer of the Lcn۲ cDNA into the Baculovirus destination vector by LR recombination reaction was confirmed by amplification of a band of about ۸۶۰ bp length by using forward Lcn۲ primer and V۵ reverse primer. Next, Lcn۲ protein was detected as a prominent band with approximate molecular weight of ۳۰ kDa in SDS-PAGE and western blot analysis. ELISA results revealed high-level expression of Lcn۲ by Spodoptera frugiperda (Sf۹) cells. Conclusion High-level expression of Lcn۲ protein in insect cells is promising for future potential applications. Recombinant Lcn۲ 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.