【摘要】 为筛选能够有效抑制J亚型禽白血病病毒(ALV-J)复制的siRNA,本研究根据ALV的pol基因保守序列设计合成5对shRNA序列,并将其分别克隆至pcDNATM6.2-GW/EmGFP-miR载体中,构建siRNA表达重组质粒,分别为pcDNA-sh-pol711、pcDNA-sh-pol814、pcDNA-sh-pol1016、pcDNA-sh-pol1915和pcDNA-sh-pol2516。将重组质粒分别转染DF-1细胞6h后,以100TCID50的ALV-J感染细胞,并利用IFA、westernblot和real-timePCR方法评价其对ALV-J复制的抑制效果。IFA和westernblot检测结果表明:其中pcDNA-sh-pol711、pcDNA-sh-pol814和pcDNA-sh-pol2516可以有效抑制病毒囊膜蛋白的表达。Real-timePCR结果显示:与阴性对照和空载体对照相比,这3种siRNA在mRNA水平对ALV-J的抑制率达29%～86%。本研究在细胞水平上筛选的siRNAs可作为候选siRNAs,为抗鸡ALV-J的研究奠定基础。更多还原

【Abstract】 To select siRNAs for effectively inhibiting the replication of avian leukosis virus subgroup J (ALV-J). Five short hairpin RNAs (shRNAs) targeting the pol gene were designed, systhessised and cloned into pcDNATM6.2-GW/EmGFP-miR vector, respectively, designated as pcDNA-sh-pol711, pcDNA-sh-pol814, pcDNA-sh-pol1016, pcDNA-sh-pol1915, and pcDNA-sh-pol2516. The recombinant plasmids were transfected into DF-1 cell and infected with 100 TCID50 ALV-J at 6 hour post-transfection. The results of IFA and western blot showed that the expression of ALV-J envelope protein was significantly inhibited by the recombinant plasmids of pcDNA-sh-pol711, pcDNA-sh-pol814, pcDNA-sh-pol2516. Real time PCR detection showed that the expression of pol gene was decreased from 29% to 86% by pcDNA-sh-pol711, pcDNA-sh-pol814 and pcDNA-sh-pol2516 comparing with negative control. The selection of the siRNAs could be served as candidate siRNAs for basic research on ALV-J.更多还原