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

碱性磷酸酶（Alkaline phosphatase，ALP）是一种表达于细胞表面的GPI锚定蛋白。ALP分布于肠、胎盘、肝、肾、骨和某些器官中。在B细胞分化中，ALP在B淋巴细胞的细胞表面暂时表达也是事实。然而，ALP在B细胞分化中的功能或作用仍然不清楚，因此我们试图阐明ALP的功能。我们试图从携带ALP的伯基特淋巴瘤细胞系中纯化ALP蛋白。为了从1 × 1010个ALP表达细胞制备细胞裂解液，将细胞裂解缓冲液和提取缓冲液混合并在冰上剧烈涡旋。细胞裂解液经离子交换层析、凝胶过滤层析和羟基磷灰石层析纯化。本实验获得了386 mg含ALP蛋白的纯化蛋白，并将其免疫BALB/c小鼠，制备了抗人ALP抗体。免疫2 ~ 3次后，取脾细胞与小鼠骨髓瘤细胞系NS-1融合。现在我们已经得到了3个可能产生抗ALP抗体的候选细胞，并正在检查与ALP表达细胞和其他非ALP表达细胞的反应性。因此，制备抗人淋巴细胞ALP的抗体是目前最重要的研究课题之一，因为我们认为，抗ALP抗体的制备有利于从免疫学和分子生物学的角度研究ALP的功能和结构。

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.