糖尿病病人突变CD59蛋白在真核细胞中的表达

EXPRESSION OF HUMAN MUTANT CD59 IN CHINESE HAMSTER OVARY CELLS IN PATIENTS WITH DIABETES

目的构建两种含有人CD59蛋白突变体的真核表达载体,获得中国仓鼠卵巢(CHO)细胞真核表达系统,为在基因水平阐明糖尿病血管增殖性病变的发病机制奠定基础。方法将两种含有不同突变体的人CD59全长cDNA序列重组pALTER质粒,应用阳离子脂质体导入法与pcDNA共转染CHO细胞,用G418筛选阳性克隆,应用细胞免疫组化及流式细胞仪检测CD59在CHO细胞表面的表达。结果含有人CD59两种突变体的重组pLATER质粒经酶切和琼脂糖凝胶电泳鉴定,得到长496 bp的电泳条带,与所插入片段完全相符。运用阳离子脂质体导入法将重组pALTER质粒共转染CHO细胞成功,筛选出的阳性克隆经细胞免疫组化检测,证明突变的CD59可在CHO细胞表面表达,流式细胞仪测定表达率分别为68.6%、71.7%。结论成功建立了两种高效表达人CD59突变体的真核表达系统,能够进一步研究人CD59的突变体糖基化及抗补体活性,为阐明糖尿病血管增殖性病变的发病机制奠定了基础。

Objective To construct a eukaryotic expression bearer of two human mutant CD59 and obtain Chinese ham ster ovary （CHO） cell eukaryotic expression system, so as to interpret the mechanism of vascular proliferation in diabetes. Methods pALTER plasmid containing two mutant CD59 cDNAs was transfected into CHO cells together with pcDNA by positive li posome. The positive clones were obtained by G418. The expression of CD59 on the cell membrane was tested by cell immunohis tochemistry and flow eytometry. Results pALTER plasmid containing CD59 was cut with restriction enzymes and a 496 bp flagment obtained by electrophoresis, which was complete conformity with the insert. The pALTER plasmid was successfully trans fected into CHO cells and positive clones were screened and detected immunohistologically. It was proved that CD59 was able to express on the surface of CHO cell, with the expression rates being 68.6% and 71.7%, respectively. Conclusion Two kinds of high performance expressions of eukaryotic system of human mutant CD59 cDNA were successfully established, which makes further study of mutant CD59 glyeation and anti complement activity possible, and thus elucidate the mechanism of blood vessel proliferation in diabetes.