Development of transmission blocking malaria vaccine using recombinant groteins.

为了开发一种模型传播阻断免疫原，我们研究了伯氏疟原虫主要的卵母细胞表面蛋白Pbs21。在小鼠中，在大肠杆菌表达系统（EcrPbs21）中表达的重组Pbs21在诱导传输阻断抗体方面不如天然Pbs21 （nPbs21）有效，这可能是因为EcrPbs21和nPbs21在翻译后加工方面存在差异。为了提高重组分子的有效性，我们在此描述了在家蚕中使用杆状病毒表达载体系统。注射含有Pbs21 cDNA的重组杆状病毒后，B。morilarvae合成了分子量与nPbs21相近的重组Pbs21 （BmrPbs21）。用亲和层析法从一只感染幼虫的血淋巴中纯化了50微克的BmrPbs21。与nPbs21还原敏感表位反应的单克隆抗体在Western blotting分析中检测到BmrPbs21，但未检测到EcrPbs21。经纯化BmrPbs21免疫后感染伯氏单胞杆菌的Balb/c小鼠为食的斯氏按蚊，其卵囊形成的阻断率为99.1 ~ 100%。这些结果表明，杆状病毒-家蚕系统产生了大量的重组Pbs21，在有限的研究中，重组Pbs21在结构和免疫原性上与天然分子没有区别。

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.