医学分子生物学杂志2013，Vol.10Issue(2)：63-68,6.DOI:10.3870/j.issn.1672-8009.2013.02.001
attB位点核心区定点突变对ΦC31整合酶效率的影响
Effect of Mutations in attB Core Area on the Efficiency of ΦC31 Integrase
摘要
Abstract
Objective To investigate the effect of base mutations in attB site core area on the efficiency of ΦC31 integrase in eukaryotes. Methods Some bases in attB site core area , which may influence the efficiency of ΦC31 integrase , were chosen as target bases for mutation. The primers that contain mutations of target bases were designed , and used in two rounds of PCR amplification to obtain attB segments , which carried the mutated bases. Then, the attB segments were linked to pEGFP-N1 to form pEGFP-N1-attBm. pEGFP-N1-attB, which carries a wild attB site , served as control. The aforementioned plasmids along with the plasmid expressing ΦC31 integrase were trans-fected into HeLa cells. After G418 selection, cell dyeing and colony counting, the integration rate was compared among groups. Results Nine plasmids containing one or two mutations in attB site core area were constructed and identified by restriction enzyme digestion and sequencing . Among them, six plasmids contained the target base mutations , while the other three contained unexpected mutations due to the PCR amplification. Cell experiment showed that the integration rate of the plas -mids that carried the three mutations had no obvious difference from that of the wild type attB -contained plasmid , while the plasmids that harbored the other six mutations had significantly lower inte -gration rate than the wild type attB -contained plasmid ( P < 0. 05 ) . Two attB sites each with two mutations had an extremely lower integration rate than the control plasmid (P <0. 001 ) . Conclusion attB site core area is a conserved area for integration mediated by ΦC31 integrase, and mutations of some bases in attB site can decrease the integration rate of ΦC31 integrase significantly.关键词
attB位点/点突变/大引物/整合率Key words
attB site/ mutation / integration rate / long primer分类
医药卫生引用本文复制引用
李振海,谢飞,马晴雯..attB位点核心区定点突变对ΦC31整合酶效率的影响[J].医学分子生物学杂志,2013,10(2):63-68,6.基金项目
国家重点基础研究发展计划(973计划)(No.2010CB529902) （973计划）
