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目的 免疫球蛋白类别转换在生发中心 B细胞中进行 ,从而 B细胞由产生 Ig M转换到产生其他类别的免疫球蛋白如 Ig G,Ig A,Ig E.目前导致类别转换的机制还不清楚 .我们试图克隆参与类别转换的基因 .方法 原发性免疫缺陷患者 B细胞不能进行类别转换 ,而正常细胞则可以 .据此 ,我们将患者与正常人的 B细胞在同一条件下分别培养以诱导类别转换 .从培养的细胞中分别提取 RNA,并反转录成 c DNA,由此制备扩增物 (amplicon) .最后将来源于患者 B细胞的扩增物与正常人 B细胞的扩增物混合在一起 ,在一定条件下进行剔除杂交 .经 5轮剔除杂交以后 ,将剔除共同基因以后的剩余物进行克隆 .经筛选以后将阳性克隆物进行测序及序列分析 .结果 在所有克隆中 ,我们发现一个新基因 ,命名为整合酶样基因 - 1.后证实 ,该基因来源于实验室细菌株 TOP10 F’序列分析表明 ,IL G1具有原核基因特性 ,但缺乏真核细胞的基因特性 .在 DNA水平 ,IL G1与任何基因都不具有同源性 ,但在蛋白水平 ,它与来源于溶原性 p4噬菌体的整合酶具有高达 5 8%的同源性 ,我们认为 IL G1定居于一个新的溶原性 p4原噬菌体 Φ- TOP10 F.IL G1的催化活性区具有高度保守特性 ,因为活性区的关键氨基酸残基是保守的 ,这包括高度保守的 R- H- R- Y结构 .结论
The objective immunoglobulin class switching takes place in germinal center B cells so that B cells are converted from producing Ig M to other classes of immunoglobulins such as Ig G, Ig A, Ig E. The mechanisms by which class switching now leads are unclear. We tried to clone genes involved in class switching.Methods B cells from patients with primary immunodeficiency were unable to switch between classes but normal cells were able to do so.We cultured the patients and normal B cells under the same conditions to induce categories The RNA was extracted from the cultured cells and reverse transcribed into c DNA to prepare an amplicon Finally, the amplicons derived from the patient’s B cells were mixed with the amplicons of normal human B cells Together, under certain conditions for the removal of hybridization.After 5 rounds of hybridization, will remove the common gene residues after cloning.After screening the positive clones were sequenced and sequence analysis.ResultsIn all the clones, we found A new gene, named the integrase-like gene, was identified as 1. This gene was derived from the laboratory bacterial strain TOP10 F ’Sequence analysis showed that IL G1 has the original But lacking the genetic characteristics of eukaryotic cells.In the DNA level, IL G1 has no homology to any of the genes, but at the protein level it has as high as 58% of the integrase from lysogenic p4 phage , We believe that IL G1 resides in a novel, protolytic p4 prophage, Φ-TOP10 F. The highly active catalytic domain of IL G1 is conserved because of the conserved key amino acid residues in the active domain, including Highly conserved R-H-R-Y structure. Conclusion