Expression and purification of growth differentiation factor 5 in E.coli and its effect on osteoarthritis
10.13200/j.cnki.cjb.004502
- VernacularTitle:生长分化因子5在大肠埃希菌中的表达、纯化及其对骨关节炎的作用
- Author:
WANG Xuan
- Publication Type:Journal Article
- From:
Chinese Journal of Biologicals
2025;38(06):674-684+690
- CountryChina
- Language:Chinese
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Abstract:
Objective To establish a protein preparation process for growth differentiation factor 5(GDF5),and explore its role in cartilage injury through in vivo and in vitro experiments.Methods The gdf5 gene was ligated with the pCZN1 vector to construct the recombinant plasmid pCZN1-hGDF5,which was then transformed into E.coli BL21(DE3) pLysS host bacteria.Single colonies were picked to screen for high-expression strains.The strains were induced by IPTG,and the protein expression form was detected by SDS-PAGE.A method for inclusion body washing,denaturation and dilution renaturation was established,and the protein was purified by Ni affinity column chromatography and DEAE ion exchange column chromatography.ATDC5 cells were used as the detection cell line,and the biological activity of rhGDF5 protein in promoting proliferation was determined by MTT method.A model of cell injury induced by interleukin-1β(IL-1β) was established.The expression of cartilage-related genes and proteins was detected by RT-qPCR and Western blot,and the effect of rhGDF5protein on chondrocyte differentiation was analyzed by Alcian blue staining and alkaline phosphatase(ALP) activity determination.Poloxamer 407(P407) and 188(P188) thermo sensitive gels were prepared by cold dissolution method,and detected for their rheological properties by a rheometer.A male C57BL/6J mouse model of osteoarthritis(OA) induced by destabilization of medial meniscus(DMM) was established.The mice were administered by intra-articular injection once a week for four consecutive weeks.The mice were sacrificed and their knee joint tissues were collected.After decalcification,paraffin embedding and sectioning,they were stained with safranin O and fast green and detected by immunohistochemistry,and the OARSI score was determined.Results The results of double enzyme digestion(Nco Ⅰ/BamH Ⅰ) showed that the recombinant plasmid pCZN1-hGDF5 was correctly constructed.The protein expression level of the selected rhGDF5 high-expression strain accounted for 35% of the total protein.The expressed protein mainly existed in the form of inclusion bodies.After refolding and column chromatography,an rhGDF5 homodimer protein with a relative molecular mass of approximately 25 000and an electrophoretic purity of over 95% was obtained.The rhGDF5 protein exhibited a good proliferation-promoting effect on ATDC5 cells within the dose range of 25 to 800 ng/mL,and no cytotoxicity was observed at high doses.The rhGDF5protein could effectively promote the increase in chondrocyte number and proteoglycan secretion,and significantly inhibit the expression of ALP in ATDC5 cells.The results of RT-qPCR and Western blot demonstrated that compared with the IL-1βgroup,the administration of rhGDF5 protein could effectively reverse the inhibition of collagen type 2(COL-2) and SRY-box transcription factor 9(SOX9)(COL-2:t=4.899 and 9.799,respectively,each P <0.05;SOX9:t=4.950 and 10.73,respectively,each P <0.05),and significantly inhibit the expression of matrix metalloproteinase 13(MMP13)(t=6.500 and23.51,respectively,each P <0.05).The poloxamer thermosensitive hydrogel containing 22% P407 and 5% P188 with rhGDF5 protein prepared by cold dissolution method showed the gelation temperature of(30.06±0.16)℃ and the gelation time of 92.67 s.The results of the DMM mouse OA model experiment showed that compared with the control group(0 ng/mL),the injection of rhGDF5 protein-loaded thermo sensitive hydrogel into the joint cavity effectively repaired the damaged joint,with the high-dose group having the best repair effect,with an OARSI score of 1.The medium-dose group had an OARSI score of 1.67,while the low-dose group had no significant effect,with an OARSI score of 5.33.The immunohistochemical results also showed that compared with the control group,the treatment with rhGDF5 protein increased the distribution of COL-2.Conclusion A simple and efficient preparation process for protein renaturation,dimerization promotion and purification of rhGDF5 inclusion body was established,and its effective effect on cartilage was proved through in vivo and in vitro experiments,which provides technical support for further development of OA treatment products.
