Construction Of Bone Matrix Non-collagen Osteopontin Experimental Tool

Osteopontin(OPN) is one of the non-collagen proteins in bone matrix,which plays an important role in bone growth,development and remodeling together with other non-collagen proteins.Previous studies have suggested that OPN plays a negative role in the mineralization of bone matrix,but with the deepening of research,the molecular mechanism of OPN in the mineralization of bone matrix remains unclear.Although there have been many studies on the mechanism of non-collagen proteins regulation of mineralization in vivo,due to the complex dynamics of the extracellular matrix microenvironment,the complex life process can only be explained at the molecular level.Therefore,our laboratory disassembles and reassembles the basic components of bone matrix to study the intermolecular relationships and their contribution to mineralized bone remodeling.This study was responsible for two of these,including the construction of cell line with OPN knocked out by CRISPR/Cas9,and the expression and purification of OPN in recombinant E.coli,to construct a research tool for studying the molecular mechanism of OPN regulating the mineralization of bone matrix.My major research responsibilities include:(1)CRISPR/Cas9 gene editing technology was applied to the knockout of spp1gene in osteocyte-like cell line MLO-Y4 and osteoblastic cell line MC3T3-E1.PCR and sequencing results showed that the deletion mutation of the OPN gene in the second exon of the spp1 gene was successfully achieved,the osteocyte line spp1^-/--MLO-Y4and the pre-osteoblast line spp1^-/--MC3T3-E1 were successfully constructed.(2)The recombinant plasmid expressing OPN protein was transformed into E.coli Stellar strain.After induced by IPTG,the protein was purified by cobalt ion affinity chromatography,Western Blot analysis showed that the purified protein could be recognized and specifically bound by the antibody.Mass spectrometric tests showed that the purified product contained OPN.Cultivation conditions and purification steps will continue to be optimized to increase the yield of OPN and meet the research needs.In summary,stable osteopontin-knockout cell lines were successfully constructed,which provides tool cell for further research on osteogenic differentiation.The recombinant OPN protein was obtained.It would provide the material for further study of molecular binding in vitro.In this study,cell models and recombinant protein tools were constructed,which laid a good foundation for further study on the molecular mechanism of bone matrix mineralization.