Milek R L, DeVries A A, Roeffen W F, Stunnenberg H, Rottier P J, Konings R N
Department of Molecular Biology, University of Nijmegen, Toernooiveld 1, Nijmegen, 6525 ED, The Netherlands.
Exp Parasitol. 1998 Oct;90(2):165-74. doi: 10.1006/expr.1998.4315.
With the aim of developing transmission-blocking vaccines based on the sexual stage-specific surface antigen Pfs48/45 of the human malaria parasite Plasmodium falciparum, the gene encoding Pfs48/45 was incorporated into the genome of a recombinant vaccinia virus. In virus-infected mammalian tissue culture cells, recombinant Pfs48/45 antigen (rPfs48/45) is posttranslational modified to produce a highly N-glycosylated polypeptide. The rPfs48/45 protein was radiolabeled with ethanolamine, consisting of a further posttranslational modification in the form of a glycosylphosphatidylinositol anchor at its carboxy-terminal end. The rPfs48/45 was not detected on the surface of the infected cells; instead, it remained within the secretion pathway of mammalian cells irrespective of the duration of infection or culture temperature. Studies with monoclonal antibodies specific for disulfide band-dependent epitopes of Pfs48/45 revealed that recombinant Pfs48/45 is not folded in its authentic conformation even if N-glycosylation was chemically inhibited. Infection of mice and rabbits with recombinant virus elicited Pfs48/45-specific antibodies; however, the antisera failed to block parasite transmission in a standard mosquito membrane-feeding assay.
为了开发基于人类疟原虫恶性疟原虫有性阶段特异性表面抗原Pfs48/45的传播阻断疫苗，将编码Pfs48/45的基因整合到重组痘苗病毒的基因组中。在病毒感染的哺乳动物组织培养细胞中，重组Pfs48/45抗原（rPfs48/45）经过翻译后修饰，产生高度N-糖基化的多肽。rPfs48/45蛋白用乙醇胺进行放射性标记，在其羧基末端以糖基磷脂酰肌醇锚的形式进行进一步的翻译后修饰。在感染细胞的表面未检测到rPfs48/45；相反，无论感染持续时间或培养温度如何，它都保留在哺乳动物细胞的分泌途径中。对Pfs48/45的二硫键依赖性表位具有特异性的单克隆抗体研究表明，即使化学抑制N-糖基化，重组Pfs48/45也不会折叠成其真实构象。用重组病毒感染小鼠和兔子可引发Pfs48/45特异性抗体；然而，在标准的蚊媒膜饲实验中，抗血清未能阻断寄生虫传播。
