摘要
目的构建两种活性位点相关性CD59突变基因,并重组入真核表达载体。方法选取物种间高度保守W40位点及相邻位置为突变位点,重叠延伸PCR(Overlap extension PCR)法构建两种CD59基因点突变,一种是编码W40的密码子TGG缺失突变,另一种是C39W40K41→W39W40W41的相应基因突变,各设计两条常规引物和两条反向互补突变引物,以已构建CD59 cDNA为模板,三重PCR定点诱变扩增突变基因,重组入克隆载体PMD18-T-Vector,EcoRⅠ单酶切获得目的基因,重组入真核表达载体pIRES。结果通过PCR定点诱变成功获得两种目的CD59突变基因,序列测定及酶切鉴定结果均证实成功构建了pIRES-m1CD59、pIRES-m2CD59重组真核表达载体系统,突变CD59基因长度约500 bp。结论重叠延伸PCR法诱变经济可靠,重组质粒的构建为进一步研究CD59的抗补体活性奠定了基础。
Objective To construct two CD59 active site relative mutagenesis and clone mutants into eukaryotic expression system. Methods The highly conserved W40 and its vicinity were selected for mutant, and site-directed mutagenesis with deleting residue W40 site and changing C39W40K41→W39W40W41 were performed by Overlap extention PCR. Two normal primers and two mutagenesis reverse complemented primers were designed, cDNAs as templates,tri-PCR were used to amplify the mutant genes. Mutant CD59 DNAs were subcloned into the cloning vector PMD18-T-Vector, then cloned into the eukaryotic expression vector pIRES after digested by EcoR Ⅰ . Results Mutants were successfully constructed and confirmed by sequence analysis. Recombinant plasmids of pIRES-mlCD59 and pIRES-m2 CD59 were successfully constructed according to sequence and enzyme digestion analysis. The mutant gene was about 500 bp. Conclusion Overlap extension PCR is economical and in reliable, and the recombinants will be applied for luther study on CD59 anticornplernent activity.
出处
《青岛大学医学院学报》
CAS
2008年第2期95-98,共4页
Acta Academiae Medicinae Qingdao Universitatis
基金
国家自然科学基金资助项目(30170893)
关键词
抗原
CD59
点突变
真核表达
Antigens, CD59
Point mutation
Overlap extension PCR
Eukaryotic expression
