| From the study results of protein tyrosine phospharases (PTPs), we can find that this kind of enzyme is related with human diseases more or less. It's very useful to study this enzyme better for curing human diseases. PTP-MEG2 is widely expressed, it must have some regulation function in different human tissues, but till now the study on PTP-MEG2 is so limited. We designed two parts of experiments to study the enzyme characteristics and physiologic function.We constructed a soluble expression plasmid by cloning the catalytic domain of PTP-MEG2. This plasmid was expressed and then PTP-MEG2 with high activity and purity was obtained by purification. The characterization was carried out to study the enzyme property of PTP-MEG2. In detail, we cloned the catalytic domain of PTP-MEG2 from pBluescriptII KS-MEG2 and linked it to pT7, so we got pT7-△PTP-MEG2 expression plasmid. After this plasmid was expressed in E.coli Rosetta DE3, we found that△PTP-MEG2 had two expression forms, soluble and insoluble, and mostly soluble. We used Q Sepharose and SP Sephadex to purify the enzyme and got the pure△PTP-MEG2 with purity of 94.8% and specific activity of 54606 U/mg. The characterization results showed that the optimal enzymatic reaction conditions are 26℃, pH4.5, no ionic strength and reaction time less than 45min. Its Km value is 8.965mM, so this enzyme has strong bonding force with substrates. At optimal reaction conditions, we tested the inhibition of several substances towards△PTP-MEG2, among them, ratinoic acid (RA) had no inhibition towards△PTP-MEG2. So we could deplete the impact of modeling drug to PTP-MEG2 activity. The results we got above laid a good foundation for screening the inhibitors of PTP-MEG2.In addition, we focused on studying the relationship between PTP-MEG2 and osteoporosis by the osteoporosis model and Western blot. First we used the purified△PTP-MEG2 protein as antigen to immune rabbits and obtain the anti-serum of△PTP-MEG2. Through PVDF affinity chromatograph the antibodies were purified. From the results of ECL experiments, the titer of them was 1:4000, and the sensitivity of them was 1ng. The purified△PTP-MEG2 antibody could recognize PTP-MEG2 in tissues of rats. We produced osteoporosis model rats by ratinoic acid intragastric administration. After becoming osteoporosis model, the ALP and ACP level of model rats dramatically ascended, while the control rats didn't. Moreover, shank bones of model rats appeared more fragile. After modeling successfully, we extracted tissues of rats and carried out Western blot assay, the results showed that PTP-MEG2 was expressed in every tissues determined, meanwhile the PTP-MEG2 expression in kidney of model rats increased, while in muscle of model rats decreased. It suggested that PTP-MEG2 may have some relationships with muscle activity and kidney injury. We also extracted bone marrows of rats shank bones, and cultured BMSCs, and then the expression level of PTP-MEG2 in bone marrow cells, red cells and BMSCs of model and control rats were compared by Western blot with anti-△PTP-MEG2 antibodies. We found that bone marrow cells of model rats had more PTP-MEG2, but in red cells and BMSCs of model rats this didn't happen. It means overexpression only happened in mature bone marrow cells. Above all, we think that PTP-MEG2 may participate in signal transduction during the development process of osteoporosis.
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