摘要
目的观察柯萨奇B组病毒3型衣壳蛋白1(CVB3-VP1)特征性小干扰RNA(smallinterferingRNA,siRNA)对CVB3复制及VP1蛋白表达的抑制作用。方法根据CVB3VP1基因的序列及其结构特征,设计并用化学方法合成小干扰RNA(siRNA)。以CVB3感染HeLa细胞,通过脂质体介导以siRNA转染HeLa细胞。48h后,观察细胞病变的程度,用TCID50法测定病毒滴度,免疫荧光法测定CVB3-VP1蛋白的表达,RT-PCR法检测CVB3-RNA的水平。结果化学合成的4对针对CVB3-VP1的siRNA,发现其中有两对(VP1-1、VP1-2)对CVB3的复制具有明显的抑制作用,同时VP1蛋白的表达明显减少;细胞病变的程度也明显减轻。结论CVB3-VP1siRNA可有效地抑制CVB3在HeLa细胞中的复制及表达VP1。
AIM： To inhibit the expression of CVB3 VP1 protein and the replication of CVB3 with synthesized siRNAs. METHODS： According to the sequence and secondary structure of CVB3 VP1 protein, four pieces of siRNAs were designed following the requirement from Journal of Nature Cell Biology were synthesized in Shanghai GeneChem Company, Then they were transfected into infected HeLa cells by liposome （Lipofectamine^TM 2000 ）, but the non-transfected cells and non-specific siRNAs were taken as control, 48 hours later, the patho-morphous changes were observed, virus titer changes were examined by TCID50, CVB3-VP1 protein expression were detected by immunofluorescence with FITC dyeing, and CVB：RNA level was tested by semi-quantitative RT-PCR. RESULTS： Two pieces of the four specific synthesized siRNAs （ VP1-1 and VP1-2 ） were found to have obvious inhibitory effect on CVB3 replication and VP1 protein were reduce greatly. Besides, the changes of pathological cells were obviously mitigated. CONCLUSION： Specific siRNAs can effectively inhibit the expression of CVB3 VP1 protein and the replication of CVB3 in HeLa cells.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第3期306-309,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(No.30271665)
