Production of a recombinant single-chain variable-fragment (scFv) antibody against sulfoglycolipid
Production of a recombinant single-chain variable-fragment (scFv) antibody against sulfoglycolipid
复制标题
DOI：
10.1093/jb/mvi045
复制
发表时间：
2005-03-01
影响因子：
2.7
通讯作者：
Honke, K
中科院分区：
文献类型：
--
作者：
Cheng, XY;Zhang, YL;Honke, K
哺乳动物磺基糖脂由两大类化合物组成，即硫苷脂（SO3 - 3Gal-神经酰胺）和胆固醇脂（SO3 - 3Gal-烷基酰基甘油）。硫苷脂以相对高的水平存在于髓磷脂中，并且胆固醇脂存在于睾丸中。这些磺基糖脂的硫酸化是由一种常见的酶，糖苷磺基转移酶（CST）催化的。小鼠Cst基因的破坏表明，硫苷脂和胆固醇脂分别对髓鞘形成和精子发生至关重要。本研究描述了一种重组单链可变片段（scFv）抗体的产生，用于在活细胞中的磺基糖脂的功能分析。一个阳性的杂交瘤产生抗磺基糖脂IgG 3，称为DI8，最初获得免疫CST-空小鼠与分离的硫苷脂。DI8单克隆抗体被发现与末端3-O-硫酸化半乳糖结构的磺基糖脂特异性结合，如通过ELISA和薄层色谱免疫染色所证明的。该抗体只与野生型睾丸生精细胞表面的磷脂发生反应，而与CST基因敲除睾丸生精小管中的任何细胞均不发生反应。从该杂交瘤中提取总RNA，并通过RT-PCR获得编码IgG 3重链和轻链可变区的cDNA。这些DNA片段通过编码（Gly（4）Ser）（3）的DNA接头连接，然后将重组scFv片段插入噬菌粒载体pCANTAB 5E中。以g3p融合蛋白形式展示在M13噬菌体尖端的scFv抗体与硫苷脂结合。此外，在ELISA中还发现可溶形式的scFv抗体与磺基糖脂结合。
Mammalian sulfoglycolipids are comprised of two major classes of compounds, sulfatide (SO3-3Gal-ceramide) and seminolipid (SO3-3Gal-alkylacylglycerol). Sulfatide is present in relatively high levels in myelin, and seminolipid is present in testis. The sulfation of these sulfoglycolipids is catalyzed by a common enzyme, cerebroside sulfotransferase (CST). Disruption of the Cst gene in mice revealed that sulfatide and seminolipid are essential for, respectively, myelin formation and spermatogenesis. The present study describes the generation of a recombinant single-chain variable fragment (scFv) antibody against sulfoglycolipid, for use in the functional analysis of sulfoglycolipids in living cells. A positive hybridoma producing anti-sulfoglycolipid IgG3, referred to as DI8, was initially obtained by immunizing CST-null mice with an isolated sulfatide. The DI8 monoclonal antibody was found to bind specifically to sulfoglycolipids with the terminal 3-O-sulfated galactose structure, as evidenced by ELISA and thin-layer chromatogram-immunostaining. The antibody stained seminolipid on the cell surface of spermatogenic cells of wild-type testis, but it did not react with any cells in the seminiferous tubules of CST-null testis. Total RNA was extracted from this hybridoma, and cDNAs that encode the variable regions of the heavy and light chains of IgG3 were obtained by RT-PCR. These DNA fragments were linked through a DNA linker coding (Gly(4)Ser)(3), and the recombinant scFv fragment was then inserted into a phagemid vector pCANTAB 5E. The scFv antibody that was displayed at the tip of the M13 phage in the form of a g3p fusion protein bound to sulfatide. Furthermore, a soluble form of the scFv antibody was also found to bind to the sulfoglycolipids in ELISA.