Williams M N, Freshour G, Darvill A G, Albersheim P, Hahn M G
Complex Carbohydrate Research Center, University of Georgia, Athens 30602, USA.
Plant Cell. 1996 Apr;8(4):673-85. doi: 10.1105/tpc.8.4.673.
Rhamnogalacturonan II (RG-II) is a structurally complex, low molecular weight pectic polysaccharide that is released from primary cell walls of higher plants by treatment with endopolygalacturonase and is chromatographically purified after alkaline deesterification. A recombinant monovalent antibody fragment (Fab) that specifically recognizes RG-II has been obtained by selection from a phage display library of mouse immunoglobulin genes. By itself, RG-II is not immunogenic. Therefore, mice were immunized with a neoglycoprotein prepared by covalent attachment of RG-II to modified BSA. A cDNA library of the mouse IgG1/kappa antibody repertoire was constructed in the phage display vector pComb3. Selection of antigen-binding phage particles resulted in the isolation of an antibody Fab, CCRC-R1, that binds alkali-treated RG-II with high specificity. CCRC-R1 binds an epitope found primarily at sites proximal to the plasma membrane of suspension-cultured sycamore maple cells. In cells deesterified by alkali, CCRC-R1 labels the entire wall, suggesting that the RG-II epitope recognized by CCRC-R1 is masked by esterification in most of the wall and tha such RG-II esterification is absent near the plasma membrane.
鼠李半乳糖醛酸聚糖II（RG-II）是一种结构复杂的低分子量果胶多糖，通过用内切多聚半乳糖醛酸酶处理从高等植物的初生细胞壁中释放出来，并在碱性脱酯后进行色谱纯化。通过从小鼠免疫球蛋白基因的噬菌体展示文库中筛选，获得了一种特异性识别RG-II的重组单价抗体片段（Fab）。RG-II本身没有免疫原性。因此，用通过将RG-II共价连接到修饰的牛血清白蛋白制备的新糖蛋白免疫小鼠。在噬菌体展示载体pComb3中构建了小鼠IgG1/κ抗体库的cDNA文库。对抗原结合噬菌体颗粒的筛选导致分离出一种抗体Fab，CCRC-R1，它以高特异性结合经碱处理的RG-II。CCRC-R1结合主要在悬浮培养的梧桐细胞的质膜附近位点发现的一个表位。在经碱脱酯的细胞中，CCRC-R1标记整个细胞壁，这表明CCRC-R1识别的RG-II表位在大部分细胞壁中被酯化所掩盖，并且在质膜附近不存在这种RG-II酯化。
