Bifunctional VSG,as a signal and a receptor

非洲锥虫的血流保护并保持其种群连续地将变异型(VAT)特异性表面糖蛋白(VSG)的表达从一个切换到另一个。VSG基因的表达方式已经得到了较好的解释，然而，这种转换的机制或调控系统本身还不清楚。我们一直在研究威康菌株对布氏锥虫冈比亚的调节作用。为此，我们制备了抗KUTAT-1及其衍生的KUTAT-2和KUTAT-3的单抗。克隆的KUTAT-3型小体在体外培养3个月后，被表达KUTAT-1或原始VAT的锥体所取代，而克隆的KUTAT-1锥体保留了6个月以上的VAT。为了分析异型锥体与同型锥体比值的变化，我们尝试应用免疫荧光技术和免疫毒素。流式细胞仪的分析倍数可达1：103。蓖麻毒素A链标记的单抗对血细胞有致死作用，但杀死时间长于24小时。结果显示了实时测定比值的局限性。抗体亲和层析纯化锥体表面VSG的效果最好，应用VSG的抗交叉活性决定簇是非常有用的，因为它是可用的，而不受VAT的影响。生物素标记方法显示，除VSG外，血流表面还存在几种蛋白质，其中几种蛋白质显示出磷酸化的模式，但不受抗VSG抗体的刺激。我们既可以克隆后循环型的VSG基因，也可以克隆血流型的VSG基因。亚循环转化后的血流形成的第一个增加的种群持续表达亚环VSG，这些VSG基因可能是主要的表达群。

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.