The analysis of role of calcium-dependent protein kinase in the mechanism of parasitism of malarial parasites

1994年，根据多种生物钙依赖蛋白激酶(蛋白激酶C和钙调素依赖性蛋白激酶)的高度保守区设计了一对寡核苷酸引物，对约氏疟原虫基因组DNA进行了聚合酶链式反应(PCR)，得到了一条聚合酶链式反应产物。以该聚合酶链式反应产物为引物，对约氏假丝酵母基因组DNA文库进行了筛选。对阳性克隆的插入片段进行测序表明，它含有一个编码蛋白激酶的基因。1995年：将该寄生虫蛋白激酶的一级结构与其他生物的各种蛋白激酶的一级结构进行了比较，表明该寄生虫蛋白属于cAMP依赖性蛋白激酶催化亚单位家族成员。约氏疟原虫蛋白的原位凝胶分析表明，约氏疟原虫至少存在8种蛋白激酶。在滋养体晚期至裂殖体阶段，钙依赖蛋白激酶活性最高。从裂殖体到滋养体早期，在EGTA存在下更具活性的蛋白激酶活性最高。不同蛋白激酶抑制剂对恶性疟原虫体外生长影响的研究表明，钙依赖蛋白激酶抑制剂(蛋白激酶C和钙调素依赖性蛋白激酶)对恶性疟原虫生长有抑制作用。

In 1994 : Polymerase chain reaction (PCR) was carried out against P.yoelii genomic DNA with oligonucleotide primers which were designed from highly conserved regions among calcium-dependent protein kinases (protein kinase C and Ca^<2+>/calmodulin-dependent protein kinase) of a wide range of organisms and a PCR product was yielded. P.yoelii genomic DNA library was screened using this PCR product as a primer. Sequencing of the insert of a positive clone revealed that it contained a gene which codes a protein kinase.In 1995 : The comparison of the primary structure of the parasite protein kinase with those of various kinds of protein kinases of other organisms showed that the parasite protein belongs to cAMP-dependent protein kinase catalytic subunit family members. In situ gel assay of P.yoelii proteins showed that there are at least 8 kinds of protein kinases of P.yoelii. And the calcium-dependent protein kinase showed the highest activity from late trophozoite stage to schizont stage. The protein kinase which is more activated in the presence of EGTA showed the highest activity from schizont stage to early trophozoite stage. The study of the effect of various inhibitors of protein kinases on the growth of P.falciparum in vitro demonstrated that inhibitors of calcium-dependent protein kinases (protein kinase C and Ca^<2+>/calmodulin-dependent protein kinase) inhibit the growth of P.falciparum.