Bifunctional VSG,as a signal and a receptor

非洲锥虫的血液保护和保持其种群在不同类型（VAT）特异性表面糖蛋白（VSG）表达上的连续切换。VSG基因的表达方式已得到较好的解释，但其开关本身的机制或调控系统尚不清楚。我们一直在研究布氏锥虫冈比亚型威康菌株的调控作用。为此，我们制备了抗KUTat 1的单克隆抗体，以及从KUTat 1衍生的KUTat 2和KUTat 3。克隆的KUTat 3型锥虫体在体外培养3个月后，用表达KUTat 1型增值酶或原增值酶的锥虫体代替。另一方面，克隆的KUTat 1锥虫保持了6个月以上的VAT。为了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技术。FACS可以分析到1:10 ^3的比率。蓖麻毒素A链偶联单克隆抗体对血液有致死性作用，但杀灭时间超过24小时。结果表明，实时测定该比值存在一定的局限性。抗体亲和层析法从血流锥虫表面纯化VSG的效果最好，并且由于VSG的抗交叉活性决定因子是可获得的，因此对VSG的抗交叉活性决定因子的应用非常有用。生物素标记法显示，除VSG外，血流表面还存在多种蛋白质。这几种蛋白表现出磷酸化模式，但不受抗vsg抗体刺激的影响。我们可以克隆元环型和血流型的VSG基因。从元循环转化后的第一批增加的血流形态持续表达元循环VSG。这些VSG基因可能是频繁表达的优势群体。

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.