OBJECTIVE: To express the recombinant SPLUNC1 protein in HNE1 cells and to study its function of bactericidal and binding to lipopolysaccharide (LPS). METHODS: Full length of SPLUNC1 gene was cloned into pCMV-tag4A vector and stably transfected into HNE1 cell lines, the supernatant of cell cultures was collected. After being treated with the supernatant, the Pseudomonas aeruginosa was seeded to LB soft agar plate, and the bacteria clones were counted and analyzed. For in vitro LPS binding assay, LPS was coated to 96-well plates. We incubated in the plate with SPLUNC1 protein, and detected the binded SPLUNC1 protein by ELISA. Incubating the FITC-LPS with the SPLUNC1 stably transfected or control cells, the intracellular intensity of fluorescence was observed under the fluorescence microscope. RESULTS: SPLUNC1 inhibited the bacteria clone formation obviously. Although the binding efficiency of LPS and SPLUNC1 in vitro was very low, more FITC-LPS entered into the SPLUNC1 stably transfected cells. CONCLUSION: SPLUNC1 can inhibit the growth of Pseudomonas aeruginosa and bind LPS, and play an important defensive role in innate immunity of the upper airway.
OBJECTIVE: To express the recombinant SPLUNC1 protein in HNE1 cells and to study its function of bactericidal and binding to lipopolysaccharide (LPS). METHODS: Full length of SPLUNC1 gene was cloned into pCMV-tag4A vector and stably transfected into HNE1 cell lines, the supernatant of cell cultures was collected. After being treated with the supernatant, the Pseudomonas aeruginosa was seeded to LB soft agar plate, and the bacteria clones were counted and analyzed. For in vitro LPS binding assay, LPS was coated to 96-well plates. We incubated in the plate with SPLUNC1 protein, and detected the binded SPLUNC1 protein by ELISA. Incubating the FITC-LPS with the SPLUNC1 stably transfected or control cells, the intracellular intensity of fluorescence was observed under the fluorescence microscope. RESULTS:SPLUNC1 inhibited the bacteria clone formation obviously. Although the binding efficiency of LPS and SPLUNC1 in vitro was very low, more FITC-LPS entered into the SPLUNC1 stably transfected cells. CONCLUSION:SPLUNC1 can inhibit the growth of Pseudomonas aeruginosa and bind LPS, and play an important defensive role in innate immunity of the upper airway.
Authors: Clemente J Britto; Naiqian Niu; Sara Khanal; Luai Huleihel; Jose D Herazo-Maya; Alison Thompson; Maor Sauler; Martin D Slade; Lokesh Sharma; Charles S Dela Cruz; Naftali Kaminski; Lauren E Cohn Journal: Am J Physiol Lung Cell Mol Physiol Date: 2018-11-21 Impact factor: 5.464
Authors: Yang Liu; Jennifer A Bartlett; Marissa E Di; Jennifer M Bomberger; Yvonne R Chan; Lokesh Gakhar; Rama K Mallampalli; Paul B McCray; Y Peter Di Journal: Am J Pathol Date: 2013-03-15 Impact factor: 4.307