【摘要】 本文扩增了包含卵清蛋白5’侧翼上游约3.4kb片段及其第一外显子、第一内含子与第二外显子的一部分,并分别删除5’端的一个DNA酶超敏感位点(DHSs)和第一内含子,得到四个不同长度的调控序列.经测序后,将四个片段分别连接在带有绿色荧光蛋白基因的pcDH-EGFP载体上,并同时为了消除载体上的CMV启动子的影响而将之切除,从而得到OV1.1k-EGFP,OV3.4k-EGFP,OV1.1k-intron-EGFP和OV3.4k-intron-EGFP四种慢病毒载体.经酶切鉴定这四种表达载体构建正确,并且尝试用OV1.1k-EGFP慢病毒载体转染培养家鸡原代输卵管细胞后,标记基因(EGFP)成功表达.这些结果为后续深入探究家鸡卵清蛋白基因调控序列功能奠定基础.更多还原

【Abstract】 To identify the regulatory region driving specific expression,a fragment containing ovabumin5’upstream regions(~3.4kb)and the first exon,first intron,part of the second exon of ovalbumin gene was cloned.After a DNase I hypersensitive sites(DHSs)located in the 5’terminal and the first intron were respectively deleted,resulting four regulatory sequences in different length.After sequencing,these four fragment were subcloned to vector pcDH-EGFP which containing the sequence of EGFP.And the promoter of CMV in the vector was been deleted by enzyme digestion,to avoid influence from it.At last,we obtained four retroviral vectors,named OV1.1k-EGFP,OV3.4k-EGFP,OV1.1k-intron-EGFP and OV3.4k-intron-EGFP.Each vector was constructed correctly by restriction enzymes,OV1.1k-EGFP was used to transfect chicken primary oviduct epithelial cells,and the reporter gene(EGFP)was expressed.These vectors can be used for further functional analyses.更多还原