Cao Jun, Kaneko Osamu, Gao Qi, Zhou Hua-Yun, Xia Cao-Ming, Zhuge Hong-Xiang, Tsuboi Takafumi, Torii Motomi
Jiangsu Institute of Parasitic Diseases, Wuxi 214064, China.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2009 Jun;27(3):206-9.
To reveal the transcription profile of PfRON4 gene in Plasmodium falciparum erythrocytic stage.
P. falciparum schizonts were purified by differential centrifugation on a Percoll-sorbitol gradient, after which the released merozoites were allowed to invade uninfected erythrocytes for 4 hours before the clearance of all remaining schizonts using 5% D-sorbitol. The cultured synchronous parasites were harvested for RNA assay immediately, 24 hours later, and then at every 6th hour. PfRON4 and related genes (PfAMA1 and PfRhopH2) were amplified by real-time PCR for establishing standard curves to evaluate the copy number of genes.
P. falciparum parasites were well synchronized. Those quantative analyses were reliable because the R value of standard curves were more than 0.98 and the melting curve showed a single peak. When parasites were in the schizont stage, PfRON4 gene transcription reached a peak in 36-40 hours after invasion.
The transcription of PfRON4 peaks at mature schizont stage, suggesting that the PfRON4 gene may involve in erythrocyte-invasion of P. falciparum.
揭示恶性疟原虫红细胞内期PfRON4基因的转录谱。
通过在Percoll-山梨醇梯度上进行差速离心纯化恶性疟原虫裂殖体,之后让释放的裂殖子侵入未感染的红细胞4小时,然后用5% D-山梨醇清除所有剩余的裂殖体。立即、24小时后以及之后每6小时收集培养的同步化寄生虫用于RNA检测。通过实时PCR扩增PfRON4及相关基因(PfAMA1和PfRhopH2)以建立标准曲线来评估基因拷贝数。
恶性疟原虫寄生虫同步良好。那些定量分析是可靠的,因为标准曲线的R值大于0.98且熔解曲线显示单峰。当寄生虫处于裂殖体阶段时,PfRON4基因转录在侵入后36 - 40小时达到峰值。
PfRON4的转录在成熟裂殖体阶段达到峰值,表明PfRON4基因可能参与恶性疟原虫的红细胞入侵。