Expression And Purification Of Anti-bacterial/anti-LPS Peptide Gloverin, And Study On Its Bioactivity

Pulpitis is one of very commonly oral diseases. It is generally considered that gram-negative bacteria is the major pathogen in infective pulpitis, and endotoxin (Hpopolysaccharide, LPS) is the key factor. LPS induces monocytes, macrophages and fibroblasts to release many pro-inflammatory cytokines such as IL-1, IL-6 and IL-8, which could trigger inflammatory cascade. Pulpitis happens due to elevated vascular penetrability, and increased exudates and pressure in pulp cavity induced by pro-inflammatory cytokines. So it is important to study anti-LPS methods to lessen the toxic effects of LPS on pulp cells for the treatment of pulpitis and protection of vital-pulp.Gloverin is an inducible antibacterial protein that is active exclusively against gram-negative bacteria isolated from pupae ofgiant silk moth Hyalophora. This 13.8KD basic protein could bind to LPS and block its biological effects. Thus, gloverin is an antibacterial peptide with killing gram-negative bacteria and neutralizing LPS activities.In our previous study, a recombinant plasmid contained gloverin cDNA was constructed and transfected into COS-7 cells, the cell supernatant had the activity of killing E.coli J5. Since the quantity of recombinant gloverin from COS-7 cells is not sufficient, E.coli was used to acquire sufficient gloverin in this study. Here, our objective is to express gloverin in E.coli, and investigate the antibacterial and neutralizing LPS effects.Firstly, gloverin cDNA was amplified by PCR. Then the fragment was cloned into expression vector pIVEX2.3 after digested by Sma I and Nco I endonucleases. The recombinant plasmid was named as pIVEX2.3-G. pIVEX2.3-G was used as template in RTS500 system according to the manufacturer's instructions. The expression products were separated by SDS-PAGE, and a 14KD band could be detected by anti-His 6 IgG. There was a high affinity agent to lipid A in expression product by Affinity Sensor.E.coli BL21 (DE3) was used in order to get more gloverin. pIVEX2.3-G was transformed into BL21. Cells were harvested after induced by IPTG. Cells were sonicated, and the supernatant was separated by SDS-PAGE and stained with Coomassie Brilliant Blue. The protein band was identified with anti-His6 antibody by western blot. The protein was purified by Ni2+-NTA-agarose affinity chromatography column according to the manufacturer's instructions. After the concentration and purity of gloverin weretested by Lowry method and HPLC, the neutralization LPS effect of gloverin was assayed with Tachypleus Amebocyte Lysate (TAL). Our data supported the expressed and purified protein did be gloverin.Finally, inhibition of gloverin on IL-1 P release from primary pulp cells induced by LPS were observed. The recornbinant gloverin could inhibit IL-1 3 release induced by LPS in a dose-dependent manner.In summary, recornbinant gloverin plasmid was successfully constructed in this study. Gloverin was expressed, and the expression products had affinity to lipid A and could neutralize LPS in vitro. Meaningful, IL-1 3 release from pulp cells induced by LPS could be inhibited by gloverin in a dose-dependant manner.