摘要
根据siRNA设计原则,设计并合成5对靶向禽流感病毒(AIV)PB2基因的siRNA(PB2374,PB2665,PB2936,PB21031和PB21233),将其转染MDCK细胞,分别于转染前和转染后感染H5N1禽流感病毒A/Goose/Guang dong1/96株;在转染后24h、48h和72h分别收集上清液;通过血凝试验(HA)和Real-time PCR检测siRNA抑制AIV增殖的效果。血凝试验结果表明:所设计的5对siRNA中,PB2374抑制AIV增殖的效率为68%～71%,PB2963抑制AIV增殖效率为78%～81%;Real-time PCR检测到PB2374转染组PB2基因的转录水平与对照组比较降低3～3.3倍,PB2963转染组PB2基因的转录水平降低3.9～4.2倍。无论siRNA是先于还是迟于AIV感染,这两对siRNA都能明显地抑制AIV的增殖;本研究结果为开发抗AIV治疗制剂和动物机体抗AIV研究奠定了基础。
To investigate the effectiveness of RNA interference in suppressing avian influenza virus replication in vitro, five siRNA （PB2 374, PB2 665, PB2 936, PB2 1031 and PB2 1233） targeting PB2 gene of AIV were synthesized and transfected into MDCK cells before or after H5NI AIV （A/Goose/Guangdongl/96） infection. Cells culture supernatant was collected at 24 h, 48 h, 72 h respectively, and tested by hemagglutination assay and Real-time PCR. Two siRNA exhibited strong suppressing effect on virus replication. PB2 374 inhibited virus growth by 67 %-71% and PB2 963 by 74 %-8 1%; Real-time PCR showed that the virus PB2 transcription level in the PB2 374 transfected cells was reduced by 3 to 3.3 fold compared to the mock cells, while the PB2 963 transfection resulted in 3.9 to 4.2 fold decrease in PB2 gene expression. Similar inhibitory efficiency was detected for PB2 374 and PB2 963 when administered before or after virus infection.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2008年第4期245-249,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金项目(30300258)
