Study Of Antibacterial Mechanisms And Chemotactic Activity Of HMGN2

We reported a new antibacterial function of high mobility group chromosal protein N2 (HMGN2) firstly. The aim of this paper was to study the antibacterial mechanism of HMGN2 and explore whether it had chemotactic activity to neutrophil. The thesis was divided into three parts.The first part was to isolate and identify HMGN2 originated from various tissues and recombinant HMN2 plasmid. [Objective] To prepare HMGN2 molecule from tissues and recombinant HMGN2 vector. [ Method ] Homogenized sample of human or animal tissues was extracted with 5% perchloric acid. The extract was purified by RP-HPLC. The recombinant vector pET-32a-c(+)-HMGN2 transformed into E.coli was induced by IPTG. The bacteria suspension was treated with sonication, and the supernatant was purified through affinity chromatography and reversed-phase chromatography. The target molecule was identified with SDS-PAGE and western blot. The antibacterial activity of HMGN2 was tested through agarose gel diffusion assay. [Result] SDS-PAGE analysis showed that the molecular mass of the target molecules was about 18KD. That had a strong positive signal confirmed by western blot using HMGN2 antiserum as the first antiboy. The agarose gel diffusion assay demonstrated that purified molecule had a potent antibacterial activity against E. coli., and the activity was approximately equal to that of HNP. However, the molecule had no antibacterial activity to gram-positive bacteria. [Conclusion] The high-purity HMGN2 was prepared successfully from various tissues and recombinant HMGN2 plasmid. That laid the foundation for the followingresearches.The second was aimed at the antibacterial mechanism of HMGN2 molecule.[Objective] To study the antibacterial mechanism of HMGN2 . [Method] The membrane permeability and gel-retardation experiments were applied for studying the antibacterial mechanism of recombinant HMGN2. Biofilm was prepared by plate culture method. Silver stain and scanning electron microscope was used for observation bioform formation and disruption. [Result] The permeability experiment detection indicated the light absorption value of OD260nm and OD280nm of the supernatant in culture cells stimulated by HMGN2 was increased and had a significant difference compared to that in negative group. HMGN2 could bind to plasmid and chromosomal DNA of E. coli K12 in concentration-dependent manner, and the migration of DNA on an agarose gel was retarded. The silver staining results showed that the transparency of the filter membrane increased and no black staining region was seen on the membrane after the bacteria was co-cultured with HMGN2. The black staining faded with different degrees after the HMGN2 acted on early and mature biofilms. The scanning electron microscope observation displayed that there was no biofilm formation after bacterial co-cultured with HMGN2. And after the early and mature biofilm formed, HMGN2 could damage it; only scattered bacteria adhered on the membrane. [Conclusion] HMGN2 could inhibit E.coli K12 through two ways at least. One, bacterial membrane permeability was influenced by HMGN2, another the DNA transcription process was disturbed. HMGN2 also could damage biofilm that would result in the impaired adhesion, colonization, growth and reproduction of bacteria and enhanced HMGN2 ability against E.coli.The third part was to explore the activation and chemotactic activity of neutrophils by HMGN2. [Objective] To study whether neutrophils could be activated by HMGN2 and whether HMGN2 had chemotactic activity towards PMNs. [Method] PMN was isolated from human blood and peritoneal exudates of guinea pigs by density gradient centrifugation . The effect of HMGN2 activating neutrophil was measured by nitroblue tetrazolium reduction assay. The migration of neutrophils stimulated by HMGN2 was tested by agarose gel and transwell method. [Result]The results of nitroblue tetrazolium reduction assay displayed that the mean of positive cells was about 20%, with no significant difference compared with the control group, and the mean of the activated cells by LPS was about 65%, with significant difference compared with the control group. In Transwell experiment, the mean number of positive cells of HMGN2 group was about 47, and it was similar that to the negative controls, but the positive control (the casein group) was about 200. The index of chemotactic activity of the casein contol was 3.64±0.53 , negative group 0.13±0.25 and HMGN2 group 0.17±0.23, respectively. There was no significant difference between the HMGN2 group and the negative control. [ Conclusion ] HMGN2 molecule had no activation and chemotaxis effect to neutrophil.