摘要
为了克隆到全长的人促红细胞生成素基因，从而在真核系统中及转基因动物乳腺中进行表达，从一个中国人的血细胞中提取基因组ＤＮＡ，构建了不完全基因组噬菌体文库，文库效价为５×１０４．这种文库的构建方法是用ＢａｍＨⅠ对基因组ＤＮＡ进行完全酶切，回收１０．５ｋｂ左右的酶切片段，插入到λ－噬菌体ＥＭＢＬ３载体中．筛选文库所使用的探针是用ＰＣＲ方法从基因组ＤＮＡ模板中扩增的５５６ｂｐ的ＥＰＯ基因片段．从该文库中筛选到了一个含有完整ＥＰＯ基因的插入片段长度为１２．５ｋｂ的阳性克隆．经全序列分析证实，所克隆的ＥＰＯ基因编码正确，但在５′－侧翼及第一内含子处与国外发表的序列相比较，发现两处核苷酸差异，这两个核苷酸的差异改变了５′－调控区的两个小的开放阅读框——ＯＲＦ１和ＯＲＦ３，其在基因表达调探方面的作用值得进一步探讨．
Using genomic DNA extracted from leucocyte,originated from a Chinese person,a partial gene library was constructed.Digesting the DNA with BamH Ⅰ,recovering about 10 5 kb fragments,inserting the fragment into lambda EMBL3 vector,a 5×10 4 library was obtained,where the erythropoietin gene was enriched.The probe for screening the library was obtained by PCR using genomic DNA as template.A positive clone embodying human erythropoietin(EPO)gene was obtained by in situ hybridization.The cloned EPO gene was 12 5 kb including 5′ and 3′ flanking region.The coding region was the same as published sequence,but in 5′ flanking and 1st intron there were 2 nucleotides differences,which changed the small open reading frame (ORF1 & ORF3)in 5′regulating region.And a Hinc Ⅱ site was found in 3′flanking.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
1998年第6期684-690,共7页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金
