重组人E1A激活基因阻遏子/myc-His融合糖蛋白的纯化及功能鉴定

目的:纯化重组人E1A激活基因阻遏子(hCREG)糖蛋白,验证重组hCREG糖蛋白具有抑制体外培养的人胸廓内动脉平滑肌细胞(HITASY)增殖的生物学功能。方法:根据6×His亲和层析原理,应用Ni-NTA柱纯化重组hCREG蛋白,纯化后蛋白通过HiTrap脱盐柱脱盐。用流式细胞周期分析研究添加0.5μg/ml、1μg/ml及2μg/ml重组hCREG/myc-His融合糖蛋白对体外培养HITASY细胞增殖的影响。用BrdU掺入方法研究重组蛋白对体外培养HITASY细胞增殖的影响。结果:根据6×His亲和层析原理用Ni-NTA纯化重组hCREG蛋白,浓缩并脱盐后的重组hCREG蛋白浓度为1.6mg/ml,纯度为92%,此蛋白仍保留了糖基化形式。流式细胞仪细胞周期分析结果提示重组hCREG蛋白可抑制体外培养的HITASY细胞增殖,且低浓度组的抑制效应要高于高浓度组。BrdU掺入实验结果提示,添加2μg/ml重组hCREG蛋白组与对照组相比可显著抑制体外培养的HITASY细胞增殖,组间比较有统计学差异(P0.05)。结论:具有生物学功能的重组hCREG/myc-His糖蛋白的成功纯化,为hCREG蛋白的功能研究及生物工程制备提供了实验平台。

Purification and functional identification of the recombinant human CREG/myc-His glycoprotein

Objective： To purify the recombinant human cellular repressor of EIA stimulated gene （hCREG）/myc-His glycoprotein and confirm the biological function of hCREG/myc-His which could inhibit the proliferation of human internal thoracic artery smooth muscle cells （HITASY） cultured in vitro. Methods： The recombinant hCREG/myc-His protein was purified with Ni-NTA column according to 6× His affinity chromatographic theory. The recombinant hCREG/myc-His protein was desalted by HiTrap Desalting Column. The effect of recombinant hCREG/myc-His glycoprotein of different concentration（0.5μg/ml ,1μg/ml and 2μg/ml） on proliferation of HITASY cells was stu- died by flow cytometic analysis and the effect of recombinant protein on proliferation of HITASY cells was confimled by BrdU incorporation method. Results： The recombinant hCREG protein was purified with Ni-NTA column according to 6x His affinity, chrolnatographic theory. The concentration of recombinant hCREG protein which has been concentrated and desalted was determined to be 1.6 mg/mg and the purity of recombinant protein reached 92%. The protein was identified to be glycosylated. The recombinant hCREG protein was identified to inhibit the proliferation of HITASY cells cultured in vitro and the inhibition effect was stronger in low-dosage group than that in high-dosage group by flow cytometic analysis. The proliferation of HITASY cells cultured in vitro with 2 μg/ml recombinant hCREG protein was inhibited significantly compared with that in control group according to the BrdU incorporation result. There was statistical difference among the groups（ P 〈 0.05）. Conclusion： The pmification of recombinant hCREG/myc-His glycoprotein with biological activity provides an experiment platform for function study and engineering production of hCREG protein.