摘要
目的: 构建抗菌肽Aurein1 2基因多拷贝串联体,并将目的基因串联产物克隆到载体pUC18上.方法: 分别合成含相同黏性末端的Aurein1 2单拷贝基因、EcoRⅠ前接头及SalⅠ后接头.单拷贝基因分别与前、后接头连接,通过控制基因和接头加入的量及时间,可得到两侧有EcoRⅠ和SalⅠ酶切位点的同向串联的多拷贝基因,选取合适拷贝数的基因,将其克隆到载体pUC18上.结果: PCR鉴定、酶切鉴定和DNA测序证明多拷贝基因重组质粒构建成功.结论: 该方法能方便高效地获得所需的多拷贝基因,为进一步克隆到表达载体并进行高效表达打下基础.
AIM: To construct a tandem repeated gene recombinant plasmid pUC18-nAurein1.2.METHODS: We designed and synthesized a DNA fragment encoding Aurein1.2 and two adapters which had the same cohesive end (AAC/TTG).The Aurein1.2 gene was ligated with the two adapters respectively.The gene was added every six hours.At proper time,the contents in the two Eppendorfs were mixed and kept ligating until multi-copies in the same direction formed.The produced multi-copy was then cloned into pUC18 vector.RESULTS: PCR amplification and DNA sequencing analysis confirmed that 5-copy gene was correctly inserted into the vector.CONCLUSION: With this method we can conveniently and efficiently obtain the desired multi-copy gene,which lays the basis for the cost-effective expression.
出处
《第四军医大学学报》
北大核心
2005年第10期875-877,共3页
Journal of the Fourth Military Medical University
基金
甘肃省自然科学基金(3ZS041 A25 055)
关键词
抗菌肽
Aurein1.2基因
同向串联
多拷贝
克隆
分子
antibacterial peptide
Aurein1.2
tandem in the same direction
multi-copy
cloneing,molecular
