Malaria Vaccines: CSP-rEPA/ALHYDROGEL

该项目的第一步是生产重组蛋白，研究复性蛋白的条件，并开发一种纯化得到复性重组蛋白的工艺。 设计、合成了恶性疟原虫环子孢子蛋白(CS)编码基因，并将其克隆到原核表达载体中。筛选出表达重组CS蛋白的克隆(RCSP)。表达为包涵体的rCSP具有完整的N-末端，并以二硫键为特征。目前的工作集中在寻找合适的缓冲条件来重折叠溶解的rCSP。 我们利用酵母产生的CS蛋白的C-末端结构域作为免疫抗原，制备了几种单抗。6株单抗可识别子孢子和天然CS蛋白。其中3个是构象依赖的单抗，识别至少2个独立的表位。另外3个识别CS重复序列的单抗是构象无关的。我们正在准备测试它们抑制子孢子侵袭肝细胞的能力。

The first step of this project is to produce the recombinant protein, research for a condition to refold the protein, and develop a process to purify to refolded recombinant protein. A gene encoding the Plasmodium falciparum Circumsporozoite (CS) protein has been designed, synthesized, and cloned into an E. coli expression vector. A clone expressing the recombinant CS protein (rCSP) was selected. The rCSP expressed as the inclusion bodies has an intact N-terminus, and is being characterized for disulfide linkages. The current effort is focused on finding a suitable buffer condition to refold the solubilized rCSP. We have generated several monoclonal antibodies (mAbs) using the C-terminal domain of a yeast produced CS protein as immunizing antigen. Six mAbs recognized the sporozoites and the native CS protein. Three of them are conformation-dependent mAbs, recognizing at least 2 independent epitopes. The other 3 mAbs recognized the CS repeats, are thus conformation independent. We are in preparation to test their ability to inhibit sporozoite invasion of hepatocytes.