| 摘要: |
| 为了克隆山羊β-酪蛋白基因的调控区并检测5′调控区的启动子活性,利用Long PCR技术对西农莎能奶山羊β-酪蛋白基因(CSN2)5′和3′调控区进行了克隆(5′调控区分两段进行克隆,3′调控区进行直接克隆)、测序,将克隆的5′调控区的两个片段通过共有的单一性酶切位点HindⅢ融合成一个长为6.7 kb的片段,用其替代表达载体pEGFP-C1上原来的启动子CMV,指导报告基因绿色荧光蛋白(GFP)在山羊乳腺上皮细胞中的瞬时表达。结果表明,克隆的GC 3.3、GC 4.3、GC 6.6 3个片段与GenBank中登录的山羊CSN2基因相应序列相比,同源性均为99%,其包含了TATA box、GC island、CAAT box、SAR、AP1、Oct-1、Sm、Glyco、Pu-1、CAC等复合元件和转录翻译调节因子的作用位点,并且5′调控区可以有效指导报告基因的表达,从而初步验证了所克隆的山羊CSN2基因5′调控区的有效性,表明克隆的调控区可用于乳腺生物反应器的研究。 |
| 关键词: 山羊 β-酪蛋白基因（CSN2） 调控区 PCR GFP基因 |
| DOI： |
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| 基金项目:国家“863”高技术项目 (2004AA213072) |
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| Cloning of control region of goat CSN2 and the detection of the promoter |
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| Abstract: |
| To clone the 5′ and 3′control regio of goat CSN2 gene,and to detect the promoter’s activity of 5′control reqion.5′and 3′control region of Xinong Saanen dairy goat CSN2 were partially cloned by Long PCR and sequenced.The 5′control region was divided into two fragments to be cloned,The first fragment of 5′control region was joined to the second one through the Hind Ⅲ restriction site,so a longer fragment which is 6.7 kb in length was constructed.3′control region was cloned directly.Sequence analysis on NCBI with blast indicated that the three cloned fragments had the homology of 99% respectively with the comparable region of goat CSN2.The three fregments contained many multiple components and factors binding sites,such as TATA box,GC island,CAAT box,SAR,AP1,Oct-1,Sm,Glyco,Pu-1,CAC,and so on.The 5′control region replaced previous promoter CMV cloned in pEGFP C1 expressional vector and transfected the cultured mammary epithelial cell in vivo.Through observation under UV microscope,it showed that GFP had been successfully expressed in mammary epithelial cell.Then,the cloned control region could be served for the study of mammary gland expression vector construction. |
| Key words: goat CSN2 control region PCR GFP gene |
