Purification of alkaline phosphatase protein and clarifying of the function of alkaline phosphatase on B lymphocyte

碱性磷酸酶蛋白的纯化及碱性磷酸酶对B淋巴细胞作用的阐明

• 批准号: 10670169
• 负责人: ONO Nobutaka
• 金额: $ 1.86万
• 依托单位: Fukushima Medical University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670169/
• 关键词: alkaline phosphatase; anti ALP antibody; B lymphocyte; B cell differentiation; PCR; アルカリフォスファターゼ; 悪性リンパ腫; ハイドロキシアパタイト; cDNAクローニング; ALP発現ヒトリンパ腫細胞; ALP-transfected細胞; クロマトグラフィー; 悪性リンパ腫細胞株

Alkaline phosphatase (ALP) is a kind of the GPI-anchoring protein expressed on the cell surface. ALP is distributed in intestine, placenta, liver, kidney, bone, and some organs. It is also a fact that ALP is temporarily expressed on the cell surface of B lymphocytes in B cell differentiation. However, the function or role of ALP in B cell differentiation remains still unclear, so that we try to clarify the function of ALP.We tried to purify ALP protein from a Burkitt's lymphoma cell line bearing ALP.To make cell lysate from 1x1010 ALP-expressing cells, Cell Lysis Buffer ＆ Extraction Buffer were mixed and vortex vigously on ice. The cell lysate was purified by Ion-exchanging chromatography, Gel-filtarting chromatography and Hydroxyapatite chromatography. We got 386mg purified protein including ALP protein.In next step, We immunized BALB/c mouse with purified ALP protein or whole cells of ALP expressing cell line to produce anti-human ALP antibody. After 2 or 3 times immunization, spleen cells were fused with NS-1 cell (mouse myeloma cell line). Now We have got 3 candidates which might produce anti ALP antibody and are checking reactivity with ALP expressing cells and other non-ALP expressing cells. We are going to confirm whether the clones actually produce anti ALP antibody.It is one of the most important thing to produce an antibody against human lymphocyte ALP because we think anti ALP antibody makes easier to investigate the function and the structure immunologically and molecular biologically.

碱性磷酸酶（ALP）是在细胞表面表达的GPI锚定蛋白ALP在B细胞分化中的作用仍然不清楚ALP的功能。我们试图从伯基特的淋巴瘤细胞系中净化ALP蛋白，该细胞系带有ALP。从1x1010表达ALP的细胞中，细胞裂解物，细胞裂解缓冲液和提取缓冲液为冰上的混合和涡流是由离子 - 交换色谱纯化的，凝胶化的色谱和羟基磷灰石色谱法。与NS-1细胞（小鼠骨髓瘤细胞系）融合，HABE有3种可能会产生ALP抗体，并且与ALP Expressinin和其他非ALP表达细胞进行了混乱对于ANT来说，这是抗体ALP副作用的重要性之一，因为我们认为抗体更容易调查功能和结构免疫学和分子生物学。