Hmgbla-Box Cloning Expression, Purification And The Effect In Vitro And Vivo

The aim of this study was to investigate if transfusion of purification of human HMGB1A-box could exhibit beneficial effects on rheumatoid arthritis. A-box fragment of HMGB-1was cloned into E. coli and the expressed products were purified.HMGB1A-box injected into collagen-induced arthritis Wistar rats at a dose of0.62mg weekly for four weeks, methotrexate injected into collagen-induced arthritis Wistar rats at lmg· kg-1and pHysiological saline injected into collagen-induced arthritis at the same volume with HMGB1A-box and methotrexate for therapeutic potentials. The symptom scorings were documented weekly. During the4th week, the hind joints of the rats were pathologically examined and the X-ray was examined. DNA sequencing showed that human HMGB1A-box was successfully cloned and could be expressed efficiently in the E. coli. host, as SDS-PAGE showed that the target protein accounted for up to45%of the total protein. After purification with chromatograpHy, the expressed protein was found to react with an anti-human HMGB1polyclonal antibody and an anti-His-Tag polyclonal antibody, as shown by western blotting. Further, the addition of the purified A-box into the human MSC culture was found to promote MSCs to differentiate along the osteogenic pathway.On the fourth week, The results showed that arthritis symptoms occurred in all the tested rats and the scoring (7.25±0.70) was significantly higher than that of healthy controls (0.00±0.00, P<0.01). After four-week therapy, the symptom scores in CIA rats were7.77±0.44(n=12), greatly higher than those of rats that had received MTX (6.25±1.03, n=12, P<0.05) and A-BOX (6.12±0.99, P<0.01, n=12). Meanwhile, there was no difference between rats received MTX and A-box (P>0.05). Pathological examination revealed that the scores of synovial hyperplasia, new blood vessel formation and inflammation were0.00±0.00,1.14±0.69and1.71±0.75respectively, and those in rats that had treated by MTX were0.71±0.48,1.57±0.78and1.42±0.53, all the parameters of which were significantly lower than those in control rats (2.28±0.45,1.85±0.69,2.14±0.89, P<0.05). Furthermore, the scoring of synovial hyperplasia in A-BOX rats was lower than that of MTX rats (P<0.05), while there were little difference on the other two parameters between these two groups (P>0.05). Scoring on X-ray results showed that A-box rats (scores:1.85±1.06) restored better than MTX rats (2.14±0.37), which was lower than that of control rats (3.28±0.48).The results here imply that recombinant bacterial strain for expressing human HMGB1A-box with biological activities was constructed successfully. HMGB1A-box could exhibit more beneficial effect on rat CIA than MTX, enhancing the possibility of its application in clinical trials.