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）是一种在细胞表面表达的GPI锚定蛋白。 ALP分布在肠，胎盘，肝，肾脏，骨骼和某些器官中。同样事实，ALP在B细胞分化的B淋巴细胞的细胞表面暂时表达。但是，ALP在B细胞分化中的功能或作用仍然不清楚，因此我们试图澄清ALP的功能。我们试图从Burkitt的淋巴瘤细胞系ALP中净化ALP蛋白，以使1x1010 ALP表达细胞的细胞裂解，蜂窝裂解缓冲和提取缓冲区和脱水缓冲区和Vortex cice上。通过离子 - 交换色谱，凝胶化色谱和羟基磷灰石色谱法纯化细胞裂解物。我们获得了386mg纯化的蛋白质，包括ALP蛋白。在下一步中，我们使用纯化的ALP蛋白或ALP表达细胞系的全细胞进行免疫抑制的BALB/C小鼠，以产生抗人类ALP抗体。 2或3次免疫抑制后，将脾细胞与NS-1细胞（小鼠骨髓瘤细胞系）融合。现在，我们有3个候选者可能会产生抗ALP抗体，并正在与ALP表达细胞和其他非ALP表达细胞的反应性。我们将确认克隆是否实际产生抗ALP抗体。这是产生针对人淋巴细胞ALP抗体的最重要的事情之一，因为我们认为抗ALP抗体可以更容易地研究该功能和在生物学上进行免疫学和分子的结构。