Expression of erythrocyte binding protein in Plasmodium falciparum merozoites isolated from endemic area

在疟疾流行地区经常观察到表面缺乏某些蛋白质的突变红细胞，其中一些被认为是由于对疟疾感染的选择性优势而产生的。然而，恶性疟原虫有能力入侵多种类型的红细胞，包括这些突变的红细胞。恶性疟原虫这种能力的分子基础可能是参与入侵步骤的多基因家族中编码的红细胞结合蛋白所致。本研究通过对恶性疟原虫多基因家族PfRhop H1各成员的转录和蛋白表达水平的定量检测，探讨恶性疟原虫调控这些多基因家族的表达以克服红细胞多态的可能性。从恶性疟原虫3D7克隆中制备互补DNA，用于构建含有针对每个成员的实时聚合酶链式反应靶标的载体。这些质粒被用来建立标准曲线。在泰国疟疾流行区采集恶性疟原虫田间标本，提取总RNA。同时在玻片上进行血液涂片，进行IFA蛋白表达分析。为了检测蛋白的表达水平，我们成功地制备了抗PfRhop H1_2、3.1、9的特异性血清。经培养适应的恶性疟原虫克隆在蛋白表达上存在差异。

Mutant erythrocyte that lacked certain proteins on the surface were frequently observed in the malaria endemic areas, and some of them were believed to be generated by the selective advantage against malaria infection. However, Plasmodium falciparum have an ability to invade many types of erythrocyte including these mutant erythrocytes. The molecular base of this ability of P.falciparum could be resulted by the erythrocyte-binding proteins encoded in the multi-gene family involved in the invasion steps. In this study, we explored the possibility if P.falciparum regulated the expression of these multi-gene family in order to overcome the erythrocyte polymorphism by quantitate the transcription and protein expression level of each members of the multi-gene family, PfRhopH1.Firstly, we designed a panel of oligonucleotide primers for real-time PCR method with SYBR green. Complementary DNA was made from the 3D7 clone of P.falciparum and used to make plasmids containing the real-time PCR targets for each member. These plasmids were used to create the standard curve. P.falciparum field samples were collected in the malaria endemic area in Thailand and total RNA were extracted. Simultaneously blood smear were made on the glass slides for the IFA analysis for the protein expression. In order to check the protein expression level, we successfully generated anti-PfRhopHl_2, 3.1, 9 specific sera. Difference in the protein expression was observed among the culture-adapted P. falciparum clones.