-頂X313T经过分泌途径时不被蛋白酶降解
[0172] 通过用含有頂X313T的质粒的DNA免疫获得的结果强烈地表明当所述分子经过分 泌途径（此处富含蛋白酶）时该分子的尾部不被蛋白酶切割。为了更直接地检查此问题， 使用用来体内表达NPm-頂X313T的pcDNA3质粒进行CHO K1细胞的转染。该转染如别处所 述（Krammer)进行。
[0173] 十八至二十四小时后，转染的细胞的上清通过离心回收，并过滤然后加载于肝 素琼脂糖凝胶（Heparin Sepharose)柱上，如2013年12月11日提交的专利申请PCT/ EP2013/076289 中所述。
[0174] 可见小的"峰C"，这在SDS-PAGE和Western印迹上证明含有蛋白NPm-頂X313T。
[0175] 实施例10-产生重组NPm-頂X313P蛋白
[0176] 为了表达NPm-頂X313P蛋白，将表达NPm-頂X313T的质粒通过用頂X313P基因 取代頂X313T基因进行修饰，这通过用来自编码頂X313P的质粒的限制片段（Pml I-Hind III)交换代替编码NPM-IMX313T蛋白的质粒中相应的片段来进行。然后如实施例7中表 达和纯化融合蛋白。图19显示纯化的蛋白；主要条带是单体，但是寡聚的形式也是可见的 (在过量加载的凝胶上）在主要条带上方。
[0177] 实施例11_产生对于頂X313P的超免疫抗血清
[0178] 将一组五只雌性BALB/C小鼠用頂X313P蛋白肌内免疫六次，间隔14天，每次注射 使用50 y g。
[0179] 通过使用修饰的ELISA方法测试血清中IgG抗体。将X级硫酸鱼精蛋白 (Sigma)、IMX313或IMX313P用于涂覆微孔板的孔以捕获抗体。检测抗体为羊-抗-小鼠 IgG-HRP(Sigma)，其与过氧化氢反应以产生405nm的吸光度读数。
[0180] 用頂X313P免疫的小鼠的所有血清呈现对于頂X313P的高效价的IgG抗体，和一 些与頂X313交叉反应的抗体；但均不与鱼精蛋白交叉反应（图20)。
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