人源受体相关蛋白的原核表达及功能检测

Prokaryotic expression and functional examination of human receptor associated protein

目的研究人源受体相关蛋白(RAP)的表达并鉴定表达产物的功能。方法用IPTG诱导培养含有人源RAP重组表达载体(pET-28 a(+)-RAP)的BL21转化菌,利用融合蛋白中6个组氨酸纯化标签,经N i+-NTA树脂亲和层析、SDS-PAGE分离鉴定表达产物,并经脱盐、浓缩、冻干处理获得RAP融合蛋白冻干品。将RAP融合蛋白与富含LDLR家族受体的巨噬细胞进行结合实验,分析RAP融合蛋白的功能。结果经亲和层析分离纯化后获得可溶性表达的RAP融合蛋白。实验显示,表达的RAP融合蛋白可竞争性抑制VLDL与巨噬细胞的结合。结论实验获得的RAP融合蛋白具有正常的结合活性,可用于相关研究,为RAP蛋白生产开发奠定了良好的基础。

Objective To obtain RAP fusion protein with biological function. Methods Different hosts and different inducing culture time were tested to find the optimal expression condition in transformed E. coli which contains recombinant human RAP expression plasmids （pET-28a^（^） -RAP）. Ni^＋ -NTA resin was used for affinity chromatography to purify the RAP fusion protein by means of its six-histidine tag. The biological function was examined by assaying its binding character to mouse macrophage RAW264. 7 which expresses receptors of LDLR family abundantly. Results The high expression of soluble RAP was obtained in BL21 （DE3） strain after IPTG-inducing culture. The function assay showed that the RAP could bind with RAW264. 7, and inhibit the binding of VLDL to the cells. Conclusion The expressed RAP fusion protein had biological function and could be applied in research.