重组人突变CD59真核表达质粒的构建和转染

CONSTRUCTION AND TRANSFECTION OF HUMAN MUTANT CD59 EUKARYOTIC EXPRESSION SYSTEM

目的获取人突变CD59基因(HM3)并构建HM3真核表达体系,转染中国仓鼠卵巢细胞(CHO),以获得稳定转染的细胞克隆。方法应用重组PCR定点诱变技术获得突变的CD59 DNA,进一步构建重组质粒pALTER-HM3;与pcDNA3共转染CHO细胞,转染细胞按一定比例稀释后加入G418压力筛选稳定转染细胞克隆,传代扩增培养并及时冻存备用。结果酶切及序列测定均证实成功构建了突变的pALTER-HM3重组质粒;转染细胞培养约2周后套取出G418抗性细胞克隆,获得生长较好的细胞。结论重组PCR定点诱变技术成功获得突变DNA,合适的细胞稀释有利于转染细胞克隆的筛选纯化。

Objective To construct a human mutant CD59 （HM3） eukaryotic expression system and transfect it into CHO so as to get a stable-transfection cell clone. Methods HM3 DNA was performed by recombinant PCR using human CD59 eDNA template and designed mutagenic primers. HM3 DNA was inserted into the mammalian expression vector pALTER and transfected into CHO together with the selection marker pcDNA3, which conferred resistance to G418. The mutant clones were propagated in great amoums for further research, Results Recombinant plasmids of pALTER- HM3 were successfully constructed according to analysis of sequence and enzyme digestion. The mutant gene was about ,500 bp. Stable transfectants were selected by the addition of G418. Conclusion HM3 DNA is successfully constructed. Appropriate cell dilution is useful to get purified transfected cell clones,