Human PTP1B Expressed In The COS-7 Cell And In Vitro Inhibitors Experiment

Objective:To clone protein tyrosine phosphatase 1B(PTP1B) cDNA into the pcDNA3.1/myc-His-B vector and construct pcDNA3.1-hPTP1B,transfect the recombinant plasmid-hPTP1B into the COS-7 cell line.Expressed fusion protein was tested with the enzyme activity and Na₃VO₄ inhibition after purified through the method of affinity chromatography.Method:Human PTP1B full length cDNA was amplified using RT-PCR from the total RNA of the peripheral blood of the T2DM patient(BMI＞28kg·m^-2),then subcloned into pcDNA3.1/myc-His-B,transfected into the COS-7 cells.After screening with 400mg·L^-1G-418 for two weeks,we got the purified recombinant PTP1B through the Ni²⁺ affinity chromatography.With the purified proteins,we identified the correlation between PTP1B activity and the reaction condition factors including:the concentration of PTP1B,the concentration of substrate(pNPP-Na2),temperature, tested the inhibition characteristics of the specific competitor of PTP1B(Na₃VO₄), and classified the category of Na₃VO₄ inhibition effects,judged whether the traditional anti-diabetes drugs like metformin and pioglitazone have the inhibition effects on PTP1B,and observed the inhibition effect of total flavonoids of litsea coreana leve.var(TFLC),a vulgar extract from traditional Chinese medicine called Laoying tea.Result:The whole sequence PTP1B was obtained and testified by DNA sequencing and BamH I and EcoR I digestion.RT-PCR and Western blotting showed the successful transfection into COS-7 cells.Enzyme activity test and inhibitor studies showed:1.The PTP1B activity enhanced with the increase of the enzyme concentration,the substrate concentration;2.The temperature around 35℃provide the conditions of best enzyme activity;3.The enzyme activity decreased with the increased concentration of a specific inhibitor(Na₃VO₄),And further we put the Na₃VO₄ under uncompetitive inhibition from the drawn graphics of double reciprocal plots(Lineweaver-Burk);4.No clues show there be any reaction between the traditional anti-diabetes drugs(like Metformin,pioglitazone) and PTP1B.But with the vulgar extract from the traditional Chinese medicine {total flavonoids of litsea coreana leve.var(TFLC)},there definitely have an effect on PTP1B.The inhibition effect enhanced with the increase of extracts concentration.Conclusion:the construction and eukaryotic expression of the pcDNA3.1-hPTP1B has been successfully established,and the purified rhPTP1B fusion protein could be used for the inhibitor research.In the process of inhibitor research,we explored the condition factors of PTP 1B enzyme with substrate reaction,and further we interpret the inhibiting characteristics of some typical inhibitors.