Cloning, Expression Of Human Growth Differentiation Factor 5 And Preliminary Evaluation Of Its Inductive Activity

Growth differentiation factor 5 (GDF5) is a divergent member of the bone morphogenetic proteins(BMPs) subfamily which belongs to the transforming growth factor-p(TGF-p) superfamily and is predominantly expressed in cartilage tissue, it plays a very important role in the chondrogenesis and long bone development. GDF5 can promote the osteochondrogenic differentiation of mesenchymal progenitor cells in vitro and in vivo transplantation test has demonstrated that ectopic cartilage and bone formation in mouse thigh muscle could be seen after being induced by recombinant GDF5, and the majority of the tissue formed was cartilaginous in nature. In order to further elucidate the biological activities of GDF5 and to expand its applications in the cartilage and bone tissue engineering, we carried out the following research.We respectively cloned the mature peptide sequence and full-length cDNA of GDF5 gene using total RNA extracted from human fetal cartilage tissue by means of RT-PCR methods, and construct a prokaryotic expression vector named pET22b(+)-GDF5 and a eukaryotic expression vector named pEGFP-C2-GDF5. E. coli BL-21 was transformed with recombinant pET22b(+)-GDF5 plasmid and mature peptide of GDF5 was expressed after induction of IPTG. SDS-PAGE was used to check the induction kinetics and Western bloting analysis was utilized to identified the expressed protein. Proteins of interest were purified with sepharose chelated with nickel ions(Ni2+) and then implanted in mouse hindlimb muscle to evaluate the biological activities. At the same time mesenchymal stem cells(MSCs) wereisolated from human fetal bone marrow aspirate and surface antigen were judged by flow cytometry. MSCs were transfected with recombinant pEGFP-C2-GDF5 and their biological features (such as morphology, growth and proliferation, and so on) were observed, expression of GDF5 and other related matrix protein were analyzed by irnniunocytochemistry, western bloting and RT-PCR methods at the level of mRNA and protein to initially find out the inductive activities of GDF5 gene.The results show that we successfully construct prokaryotic expression vector pET22b(+)-GDF5 and eukaryotic expression vector pEGFP-C2-GDF5, SDS-PAGE and western bloting revealed that mature peptide was expressed in E.Coli at a level of 13% of the total bacterial proteins and purified with a homogeneity of more than 80% after gel scan analysis, implantation test showed that cartilage and bone tissue were formed in the mouse muscle. Isolated MSCs with high purity through evaluation of flow cytometry were transfected successfully with recombinant pEGFP-C2-GDF5 plasmid by the fact of detected expression of GDF5 at the level of mRNA and protein with inimunocytochemistry and western bloting analysis. There are no evident morphological changes in light microscope except that some transfected cells became large and more polygonal cells were observed, in electron microscope the plenty organells in the transfected cells hinted that they could have a higher secreted ability, there were no negative influence on cell proliferation and cell cycle by transfer of GDF5 gene. Collagen II mRNA expression and protein formation found in transfected MSCs suggested that they had obtained partial phenotype of chondrocyte. Absence of ALPase activity and nonexistent mRNA expression of Osteocalcin implied that the transfected MSCs did not show osteoblast phenotype.In conclusion, we expressed in E.Coli GDF5 protein with biological activities, successfully transfected MSCs with GDF5 gene and acquired stable expression in it, the transfected MSCs could be induced by GDF5 to differentiate into chondrogenic lineage, suggested its potential applicationprospect in cartilage and bone tissue engineering.