【作者】 王崇龙；

【导师】 贡成良；

【作者基本信息】 苏州大学 ， 特种经济动物饲养， 2006， 硕士

【摘要】 RNA干扰(RNAi, RNA interference)是dsRNA介导的特异性基因表达沉默现象,是生物界一种古老而且进化上高度保守的基因表达调节机制。RNAi在生物体中普遍存在,原虫、线虫、真菌、昆虫以及哺乳类动物中都已发现RNAi现象。本研究将IE-1基因+18~+446区以反向串联方式连接,构建在细胞内能利用IE-1基因启动子转录形成发夹状dsRNA的RNAi表达载体pSK-IE,和转基因RNAi载体pigA3-IE-Neo。将pSK-IE转染细胞,注射蚕蛹均表现出对BmNPV抑制作用。表明IE-1启动子可以用作构建RNAi DNA载体的启动子,转染效率的高低直接影响到RNAi的检测。用pigA3-IE-Neo转染细胞,G418筛选4个月,获得了转pigA3-IE-Neo细胞,阳性率可达到80%。感染BmNPV后,转基因细胞相比正常细胞表现出对BmNPV更强的抑制作用。另外,克隆了BmNPV的alk-exo基因,并构建了基于alk-exo基因的系统进化树;用原核表达系统和昆虫表达系统成功实现了SARS-CoV S与M基因的融合表达。更多还原

【Abstract】 RNA interference (RNAi) was the process by which a gene was suppressed using double-stranded RNA (dsRNA). The phenomenon has been observed as gene expression mediated mechanism in protozoan, elegans, fungi, insect, and mammal etc., which was very old and highly conserved in evlution.In this reseach, IE-1 gene was inserted into plasmid pBluescripⅡSK(+)(pSK-IE) in a series-opposing form that could transcript into hairpin dsRNA drivered by IE-1 promoter. The IE dsRNA expression element was cloned into piggyBacA3 plasmid, and achieved the transgene RNAi plasmid pigA3-IE-Neo. Both transfecting cell and injecting silkworm pupa using pSK-IE showed inhibiting BmNPV effection. The results indicated it feasible to construct RNAi DNA vector using IE-1 promoter, and RNAi detectability was determined by transfection efficiency. Selected by G418 for 4 months, cells induced pigA3-IE-Neo, whose positive rate reach out of 80%, were obtained. Transgene cells showed stronger inhibiting BmNPV effection than ordinary cells after infection. In addition, alk-exo gene of BmNPV was cloned and Phylogenetic tree of alk-exo gene was constructed. And fused expression of S and M gene of SARS-CoV was fulfilled in prokaryotic expression system and insect expression system.更多还原