Prokaryotic Expression Optimization And Biological Function Identification Of Recombinant Human Amelogenin

Objective:The enamel matrix protein secreted by ameloblasts is closely related to the formation of tooth enamel,which has also been shown to promote the proliferation and osteogenic differentiation of periodontal related cells.Amelogenin(Am)is the main component of enamel matrix protein,and porcine amelogenin has been successfully developed as a marketed drug for periodontal disease in foreign countries.Natural amelogenin must be extracted from tooth germs,and the extraction rate is low,so recombinant amelogenin has become a new research trend.Our research team has successfully constructed a prokaryotic expression system for human full-length amelogenin,but the desired expression has not been obtained.This study hopes to optimize the expression of amelogenin,and to carry out the preliminary identification of the biological function of the obtained protein,so as to lay the foundation for the subsequent study of amelogenin.Methods and Results:1.The PET-42 a,PET-3c,PET-28 a,Pmal-C4 X plasmids were used to express the human Am gene,and PET-28 a was selected as the optimal carrier after considering the expression level and labeling factors.It was also found that the length and position of the histidine tag can affect the expression of rh Am.The PET-28a-Am recombinant plasmid was transformed into Escherichia coli BL21,through optimization of culture medium,temperature,induction time,inducer concentration and other conditions,we determined the best conditions: fermentation with TB medium,IPTG concentration of 0.5 m M,induced at 28 ℃ for 20 h.2.The rh Am protein with N-terminal histidine tag was purified by three methods of metal ion affinity chromatography,ion exchange chromatography and acetic acid one-step method.The rh Am protein with N-terminal and C-terminal labeled with histidine was purified,and the rh Am protein was successfully obtained.3.The biological functions of the purified rh Am protein were identified by HS-5cells and h PDLF cells.The results show that rh Am can promote the proliferation and migration of the two cell lines,enhance the intracellular ALP activity,and stimulate the generation of CollagenⅠin h PDLF cells,but has no significant effect on the adhesion of h PDLF cells.At the same time,rh Am can significantly inhibit the production of TNFα,IL-6 and IL-1β in h PDLF cells induced by LPS,indicating that rh Am has a certain anti-inflammatory effect.And rh Am can effectively activate the MKK3-P38 MAPK cascade pathway,indicating that rh Am may also promote the osteogenic differentiation of h PDLF cells through this pathway.Conclusion:In this study,a prokaryotic expression plasmid of recombinant human full-length amelogenin was constructed,and a strain capable of expressing soluble rh Am was obtained after optimization.Cell experiments show that the purified rh Am can effectively promote the biological activity of HS-5 cells and h PDLF cells,and has good anti-inflammatory activity,so it can be a candidate for the development of amelogenin.