摘要
目的分析恶性疟原虫棒状体颈部蛋白4基因(PfRON4)在红内期不同发育阶段的转录水平。方法用山梨醇结合等渗细胞分离液(Percoll)对实验室体外培养的恶性疟原虫进行均一化处理,收集间隔为6h不同发育阶段的疟原虫,提取RNA。根据PfRON4基因及相关基因(PfAMA1和PfRhopH2)的序列设计特异性引物,构建标准质粒并制作标准曲线,对PfRON4及相关基因的mRNA进行定量检测分析。结果纯化并同步后的疟原虫生长发育较为同步均一,用于定量分析的标准曲线相关性较好,PfRON4、PfAMA1和PfRhopH2的相关系数(r值)分别为-1.00、-0.98和-0.98。产物熔解曲线分析结果均显示为单一波峰。定量分析结果显示,在恶性疟原虫红内期发育过程中,PfRON4基因的转录水平在裂殖子入侵红细胞后36～40h(即成熟裂殖体阶段)达到高峰。结论恶性疟原虫PfRON4基因在成熟裂殖体阶段高表达。
Objective To reveal the transcription profile of PfRON4 gene in Plasmodium falciparum erythrocytic stage. Methods P. falciparum schizonts were purified by differential centrifugation on a Percoll-sorbitol gradient, after which the released merozoites were allowed to invade uninfected erythroeytes for 4 hours before the clearance of all remaining sehizonts 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. Results P. falciparum parasites were well sychronized. Those quantative analyses were reliable because the R value of standard curves were more than 0.98 and the mehing curve showed a single peak. When parasites were in the scbizont stage, PfRON4 gene transcription reached a peak in 36-40 hours after invasion. Conclusion The transcription of PfRON4 peaks at mature schizont stage, suggesting that the PfRON4 gene may involve in erythrocyte-invasion of P.falciparum.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2009年第3期206-209,共4页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家自然科学基金(No.30700695,30872214)
江苏省自然科学基金(No.BK2008109)~~
