Preparation Of Mutant Human HMGB1 Proteins And Primary Research On Their Function

Severe burn or trauma can induce initial inflammatory reaction of organism. The inflammation may get dilated and out of control because of the water fall effect caused by multiple mediators of inflammation. And eventually, SIRS(Systemic Inflammation Response Syndrome) characterized by cellular autogenous destruction occurs. Over inflammatory reaction of organism or dropout of inflammation lead to sepsis. Sepsis, septic shock and MODS(Multiple organ dysfunction syndrome) are a series of a dynamic process, which reflects pathological change and severity of pathogenetic condition.They are results of intensification and aggravation of SIRS. Glucocorticoid, antiendotoxin antibody, TNF(Tumor Necrosis Factor) antagonist, IL-1 receptor antagonist were regarded to be able to block this inflammatory cascade reaction. However, many clinical trials confirmed that these antiinflammatory therapy failed to achieve the expected results. The reason is most inflammatory factors are released within 6 hours after inflammation occurred. Therefore, the therapeutic window targeting at these early inflammatory factors is quite narrow. A little delay will lead to failure of therapy. Hence, researchers started looking for late inflammatory factors. In 1999, Wang et al discovered that HMGB1 may participate in the development of sepsis as a late inflammatory mediator. HMGB1 is a highly conserved protein which is extensively expressed in eucaryotic cells. It was identified as a DNA-binding protein at first. It can regulate transcription and gene expression.It was reported that HMGB1 could be released by activated immunologic cells, such as macrophage and monocyte, and mediate sepsis as a potential inflammatory factor. And what is more significant is that HMGB1 is a late inflammatory mediator. This suggests HMGB1 creates a wider therapeutic window than other early inflammatory factors do. TNF is produced several minutes after stimulation, and its circulation concentration returns to basal level in the first few hours. Therefore, the clinical intervention window aiming at these inflammatory factors is relatively narrow. On the contrary, HMGB1 is secreted by macrophage 20 hours after stimulation. Because of its key role and its wider therapeutic window, HMGB1 has become a significant target for preventing and treating SIRS and sepsis.At present, there are two kinds of antiinflammatory agents targeting at HMGB1, anti-HMGB1 antibody and ethyl pyruvate. Anti-HMGB1 antibody must be humanized when applied to humanbeings, otherwise it will cause human anti-heterogenetic antibody reaction, even allergic response. The exact mechanism of ethyl pyruvate inhibiting the release of HMGB1 is still unclear. And its toxic reaction when applied through vain is a problem. Hence, there are some disadvantages with these two HMGB1 inhibitors respectively. It is quite necessary to improve them and to exploit new HMGB1 inhibitor. Based on the analysis above, we have designed, prepared and purified six mutant HMGB1 proteins. The mutants which can combine with HMGB1 receptors but cannot produce inflammation were selected. Their competitive inhibition to the inflammation effect of HMGB1 was detected. This research aims at preparing and searching for mutants which can inhibit inflammatory response of HMGB1 through competitive inhibition. This may lead to the creation of a new potential agent for anti-inflammation therapy .Objectives:To design and prepare 6 recombinant mutant HMGB1s(Human High Mobility Group Box 1 Protein) and select the mutants which can combine with HMGB1 receptors but cannot produce inflammation. To detect their competitive inhibition effect on the inflammation effect of HMGB1.Materials and Methods:1,Cloning of human HMGB1. Total RNA was extracted from human tonsil and HMGB1 cDNA was amplified by RT-PCR. Then the amplified product was inserted into pUC18 and sent to be sequenced.2,Six mutant HMGB1 cDNAs were constructed by one-step reverse PCR and then were inserted into pUC18. They were named as pUC18/HMGB1 M1, pUC18/HMGB1 M2, pUC18/HMGB1 M3, pUC18/HMGB1 M4, pUC18/HMGB1 M5, pUC18/HMGB1 M6, and sent to be sequenced.3,Construction of the prokaryotic expression vectors. After sequenced, the target DNA fragments were seperately cloned into pQE-80L. The recombinant expression vectors were named as pQE-80L/HMGB1, pQE-80L/HMGB1 M1, pQE-80L/HMGB1 M2, pQE-80L/HMGB1 M3, pQE-80L/HMGB1 M4, pQE-80L/HMGB1 M5, pQE-80L/HMGB1 M6.4,Expression of the target proteins. The recombinant plasmids were transformed into E.coli DH5αrespectively, and the target proteins were expressed in the presence of IPTG. Then they were analyzed by SDS-PAGE and Western Blotting.5,Isolation and purification of the target proteins. Cells were harvested. Supernatant of the ultra sonicated E. coli was collected and flowed through the Ni2+-NTA chromatography to purify the target proteins.6,confocal microscopy. THP-1 cell was chosen as the target cell of HMGB1. THP-1 cells were incubated with purified mutant proteins M1-M6 respectively, and polymyxinB was added into the system to eliminate the influence of LPS. Positive control(HMGB1) and negative control(PBS) were set. After fixation and blockage, cells were incubated with anti-HMGB1 antibodies and FITC anti-goat antibodies respectively, and then put under confocal microscopy for observation.7,IL-1βELISA. THP-1 cells were divided into 8 groups. Mutant proteins M1-M6 were added into group 1 to group 6 respectively. HMGB1 was added into group 7 as positive control while PBS was added into group 8 as negative control. Supernatants of different groups at different time points were collected respectively to run ELISA.8,Competitive inhibition. THP-1 cells were divided into 7 groups.HMGB1 was added into every group except for negative control. The selected mutants which can combine with HMGB1 receptors but cannot produce inflammation were added into corresponding group respectively. Supernatants of different groups at different time points were collected respectively to run IL-1βELISA. The most effecitve mutants M1 and M2 were selected to run competitive inhibition experiment again. And samples were collected to run TNF-αELISA to validate the results of IL-1βELISA.Results:1,HMGB1 cDNA was successfully cloned by RT-PCR. And it was in accordance with that was reported by GenBank. 2,Six mutant HMGB1 cDNA were successfully constructed.3,Fragments about 648bps are visible on agarose gel after enzyme cutting of the recombinant expression plasmids, pQE-80L/HMGB1,pQE-80L/HMGB1 M1,pQE-80L/HMGB1 M2,pQE-80L/HMGB1 M3,pQE-80L/HMGB1 M4,pQE-80L/HMGB1 M5,pQE-80L/HMGB1 M6.It indicated the successful construction of expression plasmids.4,After IPTG induction, target proteins about 30kDa were seen on SDS-PAGE and Western Blotting.5,The target proteins were purified through Ni2+-NTA chromatography. SDS-PAGE analysis showed that the target proteins were effectively purified.6,Confocal Microscopy indicated apparent green fluorescence in positive control and low green fluorescence in negative control. Six groups of THP-1 incubated with six mutants respectively all displayed green fluorescence. Therefore, all six mutants could combine with the receptors on THP-1.7,IL-1βELISA suggested that the IL-1βconcentration of M4 group is apparently higher than the positive control. But the IL-1βconcentration of rest of the groups is lower than positive control at all time points.8,Competitive inhibition experiment indicated that the IL-1βconcentration of M6 group is apparently higher than the positive control at each time point. During time point 2h to 6h, there was no big difference between groups of mutants M1,M2,M3,M5 and positive control group(P>0.05). But during 6h to 8h, these groups could apparently inhibit the release of IL-1β. And this effect of HMGB1 M2 is the most prominent. Competitive inhibition experiment was carried out with the two most effective mutants selected, M1 and M2. TNF-αELISA suggested that at each time point, the TNF-αconcentration of groups M1 and M2 is lower than the positive control. And at the time point of 8h, TNF-αconcentration of M1 group is markedly lower than that of positive control(P<0.05). From 6h to 8h, the group of M2 inhibit the release of TNF-αevidently compared to positive control(P<0.05).Conclusions:This experiment is based on successful preparation and purification of HMGB1 and six of its mutants. The mutants which can combine with its receptor on the target cells were selected with confocal microscopy by immunofluorescence. The mutants which not only can combine with its receptor but also can not induce inflammation were selected by ELISA.And the competitive inhibition function of the selected mutants in vitro was further confirmed. This may lead to the creation of a new potential agent for anti-inflammation therapy .