Cloning, expression and activity identification of human osteoprotegerin-mycobacteria heat shock protein 70 fusion protein / 中国组织工程研究

ABSTRACT

AIM: To solve the two difficulties of bone resorption and inflammation in rheumatoid arthritis, clone the recombinant human osteoprotegerin (OPG) and mycobacteria heat shock protein 70 (HSP70) functional gene,and study the expression and activity of OPG-HSP70 fusion protein in E.coli. METHODS: Experiments were performed at the Laboratory of Department of Immunology, Capital Medical University from May 2006 to September 2007. Complementary DNA encoding full length OPG protein was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from human osteosarcoma cell line MG63 and cloned into pGEMT-Easy vectors. Then, using the recombinant plasmid as the template,the DNA encoding the fusion protein OPG-HSP 70 was amplified by PCR,and was inserted into prokaryotic expression vector pET-28a. Construct pET-28a-OPG-HSP 70 was used to transform into competent E.coli. BL21(DE3) which were induced by isopropyl B-D-thiogalactopyranoside (IPTG),and the fusion protein from above E.coli was collected. Sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western-blotting were performed to identify OPG-HSP 70 fusion protein. The experiments of osteoclast inhibition and restraining inflammation were used to detect the bioactivity of fusion protein. RESULTS: ①The complementary DNA encoding full length OPG protein was obtained. OPG-HSP 70 fusion gene obtained in this experiment was successfully inserted into pET-28a vector. OPG-HSP70 fusion protein was expressed when transformed into E.coli.BL21(DE3). ②SDS-PAGE indicated that the fusion protein was large expressed at the molecular weight of Mr22 000, but there were no band in the total lysate of bacteria harboring pET-28a-OPG-HSP70 without IPTG induction group. ③Western-blotting indicated that the OPG-HSP70 fusion protein could specifically react with anti-human OPG monoclonal antibodies. ④The osteoclast inhibition test demonstrated that the fusion protein could reduce the number of osteoclast, and had the ability to inhibit bone absorptions in vitro. ⑤The experiment of restraining inflammation showed that the fusion protein could significantly reduce the inflammation of delayed type hypersensitivity (DTH) mice, which explained that HSP70 in fusion protein had the inflammation inhibitory bioactivity. CONCLUSION: OPG-HSP70 fusion protein is expressed in E.coli.BL21(DE3),and function study in vitro illustrates the bioactivity of fusion protein.