STUDY OF SIGNAL TRANSDUCTION IN PLASMODIUM DEVELOPMENT A

为了进一步研究恶性疟原虫环磷酸腺苷依赖蛋白激酶(PfPKA)催化亚基的特性，我们克隆并测定了PfPKA基因的序列。PfPKA序列与先前从啮齿动物疟疾寄生虫P.yoelli中发现的PKA基因有70%以上的同源性。PfPKA催化亚基基因在大肠杆菌中以非融合蛋白或硫代还原蛋白融合蛋白的形式表达，但在纯化过程中容易发生快速降解。使用另一种系统将蛋白质表达为内联蛋白融合蛋白并未解决降解问题。我们目前正试图将PfPKA的过度表达引导到包涵体中，以限制宿主中的蛋白分解，并限制其在缺乏蛋白酶的宿主中的表达。我们研制了一种针对PfPKA C端肽的多克隆抗体，该抗体可以检测到PfPKA的表达及其降解副产物。为了研究cAMP及其类似物对恶性疟原虫的影响，我们用cAMP激动剂长期孵育疟疾寄生虫，这些寄生虫没有表现出任何表型变化或性别分化。用PKA抑制剂H89延长孵育时间可降低原虫血症。然而，在低浓度的H89下，血液期寄生虫的裂解液中的PKA活性实际上是升高的。PKA活性升高的机制尚不清楚。为了研究PKA在寄生虫发育中的直接作用，我们采取了两种方法，一种是通过瞬时表达PfPKA来增加其表达，另一种是通过基因敲除来阻断其表达。利用PfPKA序列与恶性疟原虫富组蛋白3(HRP3)的5‘非翻译区和HRP2的3’非翻译区连接，构建PfPKA的表达载体。以刚地弓形虫DHFR基因为选择标记，用改良载体构建基因敲除克隆。

To further characterize thePlasmodium falciparum cyclic AMP-dependent protein kinase (PfPKA) catalytic subunit, we have cloned and sequenced PfPKA gene. PfPKA sequence shows over 70% homolgy to a previously identified PKA gene from the rodent malaria parasite, P. yoelli. The expression of the PKA catalytic subunit gene in E.coli as either a non-fusion protein or a thioreducin fusion protein has generated a transient expression ofPfPKA however, is prone to rapid degradation during purification steps. Using an alternative system to express the protein as an Intein fusion protein has not resolved the problem of degradation. We're currently attempting to direct the overexpression of PfPKA into inclusion bodies to limit proteolysis in host and also its expression in protease-deficient hosts. We have developed a polyclonal antibody against the C-terminal peptide and the antibody could detect the expressed PfPKA and its degraded byproducts. To examine the effect of cAMP and analogs on P. falciparum, we have incubated malaria parasite with cAMP agonists over an extended period, the parasites have not show any phenotypic change or sexual differentiation. Prolong incubation with PKA inhibitor, H89, could reduce the parasitemia in culture. However, under low concentration of H89, blood stage parasites actually showed elevated PKA activity in the lysate. The mechanism of increased PKA activity remains to be established. To investigate direct effect of PKA in parasite development, we have taken two approaches, one to increase its expression by transient expressionof PfPKA or to disrupt its expression by gene knock-out . Expression plasmid of PfPKA was constructed by using PfPKA sequences and flanking them with the 5' untranslated region (UTR) of P. falciparum histine rich protein 3 (HRP3) and 3' UTR of HRP2. The genetic knock-out clone will be constructed with a modified vector using Taxoplasma gondii DHFR gene as the selection marker.