Stable Expression And Characterization Of Human SLPI In Drosophila S2Cell Lines

Object: Human secretory leukocyte protease inhibitor (SLPI), previously known asmucous protease inhibitor (MPI) or antileukoproteinase (ALP), is a non-glycosylatedsingle chain polypeptide protein secreted by neutrophils, macrophages and mucosalepithelial cells, etc., and has the ability to anti-bacterial, anti-fungal, anti humanimmunodeficiency virus (HIV), anti-inflammatory, induction of cell proliferation anddifferentiation, et al. Data display SLPI also has important role in tissue trauma repair.Recently, more attention has been paid to the potential role of mucosal adjuvant of SLPI.Therefore, in this research, we try to establish stable polyclonal cell lines to expressSLPI protein by Drosophila expression system and characterize the expressed SLPIbiologically. This work will provide an important foundation for further understandingthe activity of SLPI as an immune adjuvant.Methods: The cDNA was synthesized from total RNA of A549cells by RT-PCR. Theopen reading frame (ORF) of SLPI gene was produced by PCR from the above cDNAwith a paired primers containing restriction endonucleoase of,KpnⅠand EcoRⅠ,respectively.. The resultant gene of SLPI ORF was cloned into expression vectorpMT/V5-His A. The constructed recombinant plasmid was contransfected into S2cellswith a selection vector of pCoHygro containing hygromycin-resistant gene. After3-4weeks’ selection with complete medium containing300g/ml Hygromycin B, thestable S2cell lines espressing SLPI would be produced. The established cell lines wereconfirmend by RT-PCR and Western blot following induction with500M CuSO4for 48hours. Characterization of the expressed SLPI was identified further by its inhibitionon trypsin.Result: The ORF of SLPI gene was obtained from A549cells by RT-PCR. Afterdigested by EcoRⅠand KpnⅠ, SLPI gene was inserted into pMT/V5-His A vectorsuccefully. We established stable S2cell lines expressing SLPI, designated S2-SLPI,following S2cells cotransfected by plasmids of pMT/V5-SLPI and pCoHygro as wellas screened by Hygromycin B. The induced recombinant SLPI was confirmed byRT-PCR and Western blot, and displayed evident inhibition activity on trypsin.Conclusion: The stable S2cell lines expressing SLPI are successfully established. Thisreserch provides an important foundation to fully characterize SLPI’s influence onadaptive immunity and virus infection.