Malaria vaccine development of Plasmodium vivax, by using ookinete surface proteins as vaccine antigens

在非洲以外的许多疟疾流行地区，开发传播阻断疫苗将需要同时针对恶性疟原虫和间日疟原虫的活性。间日疟原虫传播阻断疫苗的研究工作由于无法从这种寄生虫克隆疫苗候选基因而受到阻碍。为了寻找编码间日疟原虫卵母细胞表面蛋白的基因，我们对P25亚家族（Pfs25、Pgs25、Pys25、Pbs25）和P21/28亚家族（Pfs28、Pgs28、Pys2l、Pbs2l）中已知的8个蛋白的基因序列进行了比对。利用最高同源性的区域设计简并PCR寡核苷酸。间日疟原虫Sail基因组DNA、基因组文库和splinkerette DNA文库作为PCR模板。对Pvs28的氨基酸序列进行分析，发现其具有分泌信号序列、4个egf样结构域、6个七肽重复序列（GSGGE/D）和一个短疏水区。Pvs28被认为是P21/28亚家族成员的同源物，因为第四个egf样结构域有四个而不是六个半胱氨酸。Pvs25的氨基酸序列与Pvs28的结构相似。在第4个egf样结构域存在6个而不是4个半胱氨酸，这表明Pvs25是P25亚家族成员的同源物。因此，我们成功地从间日疟原虫中克隆了新的卵母细胞表面蛋白基因Pvs28和Pvs25作为传播阻断痘苗候选抗原。

In many malarious regions outside of Africa, development of transmission-blocking vaccine will require activity against both Plasimodiun falciparum and P.vivax. Work on P.vivax transmission-blocking vaccines have been hampered by the inability to clone the vaccine candidate genes from this parasite. To scarch for genes encoding the ookinete surface proteins from P.vivax, the gene sequences of tile eight known proteins in P25 subfamily (Pfs25, Pgs25, Pys25, Pbs25) and in P21/28 subfamily (Pfs28, Pgs28, Pys2l, Pbs2l) were aligned. Regions of highest identity were used to design degenerate PCR oligonucleotides. P.vivax Sail genomic DNA and genomic and splinkerette DNA libraries were used as PCR templates. Analysis of tile deduced amino acid sequence of Pvs28 revealed a secretory signal sequence, four EGF-like domains, six copies of the heptad amino acid repeat (GSGGE/D) and then a short hydrophobic region. Pvs28 is the presumed homologue of P21/28 subfamily member because the fourth EGF-like domain has four rather than six cysteines. Analysis of the deduced amino acid sequence of Pvs25 revealed a similar structure to Pvs28. Tile presence of six rather than four cysteines in tile fourth EGF-like domain suggested that Pvs25 is the homologue of P25 subfamily member. Accordingly, we have successfully cloned novel ookinete surface protein genes, Pvs28 and Pvs25, as the transmission-blocking vaccille candidate antigens, from P.vivax.