Production of autoantibodies and an animal model of autoimmune disease using a knockoutmouse

1)抗磺基糖脂单克隆抗体的制备：通过用纯硫苷脂免疫糖脂磺基转移酶敲除小鼠获得抗磺基糖脂单克隆抗体DI 8。DI 8抗体与硫苷脂以及胆固醇脂反应，但不与中性糖脂、神经节苷脂和其他硫酸化碳水化合物链反应。DI 8可以识别非还原性末端Gal 3-硫酸酯结构，因为它不与SM 2反应，SM 2的硫酸酯基团连接到内部Gal残基。在分离DI 8 IgG的重链和轻链的可变区的基因后，通过接头将它们连接。在噬菌体展示系统中表达所得的scFv基因。最后，证明可溶性scFv抗体保留了原始DI 8抗体的特异性。2）识别包括脂筏上的磺基糖脂的分子复合物的单克隆抗体的产生：从洗涤剂不溶性微粒中回收谷脂，并将其用于制备抗磷脂抗体。 ...更多信息 结果表明，生精细胞质膜的脂筏中含有精磷脂。然后用生精细胞的DIM组分免疫同系小鼠产生单克隆抗体。基于它们对DIM反应强烈，但对胆固醇脂反应较弱，选择了三个克隆。全部为IgM。Western blotting分析表明，三个克隆中有两个能与特异性蛋白条带反应。另一个克隆（#1）对任何条带都没有反应，这表明它可能识别三维结构。发现#1Ab识别分子复合物结合硫苷脂，但不结合半乳糖神经酰胺。3）产生抗磺基糖脂的自身免疫性疾病的动物模型：用硫苷脂免疫糖脂磺基转移酶敲除小鼠，并确认抗硫苷脂的抗血清活性提高后，从免疫小鼠中分离出spreen细胞并移植到同基因野生型小鼠中。结果，受体小鼠中抗硫苷脂抗体滴度未增加，未显示异常。少

1)Production of an anti-sulfoglycolipid monoclonal antibody: An anti-sulfoglycolipid monoclonal antibody, DI8, was obtained by immunizing glyolipid sulfotransferase-knockout mice with pure sulfatide. The DI8 antibody reacted to sulfatide as well as seminolipid, but not to neutral glycolipids, gangliosides and other sulfated carbohydrate chains. DI8 may recognize the non-reducing terminal Gal 3-sulfate structure, because it did not react to SM2, whose sulfate group is attached to the inner Gal residue. After isolation of the genes of the variable regions of the heavy chain and light chain of DI8 IgG, they were connected through a linker. The resultant scFv gene was expressed in a phage display system. Finally, it was demonstrated that the soluble scFv antibody preserves the specificity of the original DI8 antibody.2)Production of monoclonal antibodies that recognize molecular complexes including sulfoglycolipids on lipid rafts: Seminolipid was recovered from the detergent insoluble micr … More odomain(DIM) fraction of spermatogenic cells, suggesting that seminolipid is included in lipid rafts of the plasma membranes of spermatogenic cells. Monoclonal antibodies were then generated by immunizing syngenic mice with the DIM fraction of spermatogenic cells. Three clones were selected on the basis that they strongly reacted to DIM, but weakly reacted to seminolipid. All of them were IgM. Two of the three clones reacted to specific protein bands on Western blotting analysis. The other clone (#1) did not react to any bands, suggesting that it may recognize a three-dimensional structure. The #1 Ab -recognizing molecular complex was found to bind to sulfatide but not to galactosylceramide. Identification of the #1 Ab -recognizing molecular complex is underway.3)Production of an animal model of autoimmune disease against sulfoglycolipids : After immunizing glycolipid sulfotransferase-knockout mouse with sulfatide, and confirming that antiserum activity against sulfatide was raised, spreen cells were isolated from the immunized mouse and transplanted into syngenic wild-type mice. As a result there was no increase of the titer of anti-sulfatide antibody in the recipient mice, and they showed no abnormality. Less