Development of transmission blocking malaria vaccine using recombinant groteins.

我们已经研究了Pbs 21，主要动合子表面蛋白的伯氏疟原虫，为模型的传播阻断免疫原的发展。在小鼠中，在大肠杆菌表达系统中表达的重组Pbs 21（EcrPbs 21）在诱导传递阻断抗体方面不如天然Pbs 21（nPbs 21）有效，这可能是因为EcrPbs 21和nPbs 21之间的翻译后加工的差异。为了提高重组分子的功效，我们在此描述了杆状病毒表达载体系统在家蚕中的应用。在注射含有Pbs 21 cDNA的重组杆状病毒后，B. morilaravirus产生了分子量与nPbs 21无法区分的重组Pbs 21（BmrPbs 21）。使用亲和色谱法从一个感染的幼虫的血淋巴中纯化50微克的BmrPbs 21。与nPbs 21的还原敏感表位反应的单克隆抗体在Western印迹分析中检测到BmrPbs 21而不是EcrPbs 21。在斯氏按蚊中，以用纯化的BmrPbs 21免疫并感染伯氏疟原虫的Balb/c小鼠为食的卵囊形成被99.1- 100%阻断。这些结果表明，杆状病毒-家蚕系统产生有用数量的重组Pbs 21，其在有限的研究中在结构上和免疫原性上与天然分子无法区分。

We have studied Pbs21, a major ookinete surface protein of Plasmodium berghei, for the development of a model transmission blocking immunogen. In the mouse, recombinant Pbs21 expressed in the Escherichia coli expression system (EcrPbs21) is not as effective in inducing transmission blocking antibodies as native Pbs21 (nPbs21), possibly because of differences in post-translational processing between EcrPbs21 and nPbs21. In an attempt to improve the efficacy of the recombinant molecule, we describe here the use of a baculovirus expression vector system in the silkworm Bombyx mori. Following an injection of recombinant baculovirus containing Pbs21 cDNA,B.morilarvae produced recombinant Pbs21 (BmrPbs21) with a molecular weight indistinguishable from nPbs21. Fifty micrograms of BmrPbs21 were purified from the hemolymph of one infected larva using affinity chromatography. Monoclonal antibodies reacting with reduction -sensitive epitopes of nPbs21 detected BmrPbs21 but not EcrPbs21 in Western blotting analyzes. Oocyst formation in Anopheles stephensi mosquitoes, which fed on Balb/c mice immuinized with purified BmrPbs21 and infected with P.berghei, wqs blocked by 99.1-100%. These results suggest that the baculovirus-silkworm system produces useful quantities of recombinant Pbs21 which in limited studies is structurally and immunogenically indistinguishable from the native molecule.