Bifunctional VSG,as a signal and a receptor

非洲锥虫的血流形式保护并保持它们的种群连续地将变异型（VAT）特异性表面糖蛋白（VSG）的表达从一种切换到另一种。VSG基因的表达方式已经得到了很好的解释，但其转换机制和调控系统尚不清楚。我们一直在研究锥虫B的调控。Wellcome菌株的冈比亚布氏杆菌。为此目的，我们提出了针对KUTat 1和KUTat 2和KUTat 3的单克隆抗体，其衍生自KUTat 1。当克隆的KUTat 3型体在体外培养3个月时，它们被表达KUTat 1或原始VAT的锥虫所取代。另一方面，克隆的KUTat 1锥虫保持其VAT超过6个月。为分析异型与同型锥虫比例的变化，我们尝试应用免疫荧光技术和免疫毒素。流式细胞仪可分析高达1：10^3的比例。蓖麻毒素A链标记的单克隆抗体对血吸虫有致死作用，但致死时间超过24小时。结果表明，实时测定比值存在局限性。用抗体亲和层析法从血液锥虫表面纯化VSG效果最好，VSG的抗交叉活性决定簇的应用非常有用，因为它是可用的，而不受VAT的影响。生物素标记法显示，在血液锥虫表面除VSG外还存在几种蛋白质，这些蛋白质呈磷酸化模式，尽管它不受抗VSG抗体刺激的影响。我们可以克隆亚循环型和血流型的VSG基因。由亚循环型转化后的第一个增加的血流型群体保持持续表达亚循环型VSG，这些VSG基因可能是一个优势表达群。

Bloodstream from of African trypanosomes protect and keep their population serially switching expression of the variant type (VAT)-specific surface glycoprotein (VSG) from one to another. The way of the VSG gene expression is fairly explained, however, the mechanism or regulatory system of the switching itself is unknown. We have been studying on the regulation of Trypanosoma b. brucei gambiense of Wellcome strain. For that purpose we raised monoclonal antibody against KUTat 1, and KUTat 2 and KUTat 3, derived from KUTat 1. When cloned KUTat 3 typanosomes were kept in in vitro culture for 3 months they were replaced by trypanosomes expressing VAT of KUTat 1 or original VAT.On the other hand, the cloned KUTat 1 trypanosomes kept their VAT for more than 6 months. To analyze the change of the ratio of the heterotype to homotype trypanosomes, we tried to apply immunofluorescence technique and immunotoxin. FACS could analyze up to the ratio of 1 : 10^3. Ricin A chain conjugated monoclonal antibody could give lethal effect on bloodstream froms however killed them taking longer times than 24 hrs. The results showed the limitation for determination of the ratio at realtime. The antibody affinity chromatography gave the best result to purify VSG from the surface of bloodstream trypanosomes and it was very useful to apply anti-cross active determinant of VSG,because it is available, regardless of VAT.The biotin labeling method revealed there were several proteins on the surface of bloodstream froms, besides VSG.The several proteins showed a phosphorylated pattern, though it was not affected by the stimulation with anti-VSG antibody. We could clone the VSG genes of metacyclic forms as well as bloodstream forms. The first increased population of bloodstream forms after transformation from metacyclics kept continuously to express metacyclic VSG.Those VSG genes might be a dominant group to be expressed fraquently.