新基因NBEAL1的克隆、表达、纯化及其与胶质瘤病理分级的相关性

Cloning,expression and purification of novel gene NBEAL1 and its relationship with pathological grades of glioma

目的:构建新基因neurobeachin like1(NBEAL1)的表达载体,研究NBEAL1的表达与胶质瘤病理分级的相关性。方法:提取人脑胶质瘤细胞U251的总RNA,构建pGEX-KG/NBEAL1表达载体,转化大肠杆菌BL21,IPTG诱导NBEAL1重组蛋白的表达,GST亲和纯化,Western blotting鉴定重组蛋白的纯度;以免疫组化法用纯化蛋白制备的单克隆抗体分析NBEAL1表达与胶质瘤病理分级的相关性。结果:NBEAL1基因片段被成功地克隆入pGEX-KG表达载体,测序证实克隆片段序列正确。NBEAL1融合蛋白在大肠杆菌BL21包涵体中表达,表达量占菌体总蛋白的30%以上,纯化的重组蛋白纯度95%以上。Western blotting证明所纯化的蛋白含有GST标签与NBEAL1肽段。免疫组化法检测显示NBEAL1蛋白的表达在正常脑组织中较低,而在低级别的脑胶质瘤组织中表达明显上调,但随着恶性程度增加NBEAL1的表达程度降低,两者呈负相关。结论:成功地克隆、表达及纯化NBEAL1蛋白,NBEAL1蛋白在人脑胶质瘤组织中的表达与其恶性程度呈负相关。

Objective：To construct the expression plasmid of a novel gene human NBEAL1 （neurobeachin like 1）,and to study its relationship with the pathological grades of glioma.Methods：Total RNA of human glioma cell line U251 was extracted.NBEAL1 expression plasmid pGEX-KG/NBEAL1 was constructed and transferred into E.coli BL21.Recombinant NBEAL1 protein was induced by IPTG and further purified by GST affinity chromatographic column.The purity of recombinant NBEAL1 protein was examined by Western blotting analysis.A NBEAL1 protein specific monoclonal antibody was prepared and was used to study the relationship of NBEAL1 expression with pathological grades of glioma.Results：The NBEAL1 gene fragment was successfully cloned into pGEX-KG expression plasmid and verified by DNA sequencing.The recombinant NBEAL1 protein was expressed in inclusion bodies,with a yield of more than 30% of total bacterial proteins;the purity of purified NBEAL1 protein was above 95%.Western blotting analysis confirmed that the purified protein containing GST tag and NBEAL protein.NBEAL1 protein was lowly expressed in normal brain tissues and highly expressed in low grade glioma tissues;and the expression of NBEAL1 decreased with the increase of glioma malignancy.Conclusion：The NBEAL1 protein has been successfully cloned,expressed and purified.NBEAL1 protein expression in glioma tissues is negatively associated with the pathological grades of glioma.