人突变CD59基因在卵巢癌细胞表达及其生物活性检测

EXPRESSION OF MUTANT CD59 GENE IN OVARIAN CANCER CELLS AND ITS ACTIVITY DETECTION

目的研究野生型CD59与突变型CD59在卵巢癌细胞A2780表面的抗补体活性。方法取突变型CD59质粒、野生型CD59质粒分别转染A2780细胞,G418筛选稳定表达细胞克隆;酶免疫法及RT-PCR方法检测细胞表面CD59蛋白及mRNA的表达;制备抗CD59多克隆抗体,MTT法观察CD59蛋白的抗补体活性。结果野生型及突变型CD59质粒扩增成功,并成功建立了稳定转染野生型和突变型CD59的A2780细胞株;与野生型CD59相比,突变型CD59失去对补体的抑制功能,与未转染A2780表面CD59表达无显著差异。结论CD59的W40位点对其功能具有重要作用,封闭该位点能够提高补体溶细胞作用,有望应用于肿瘤治疗。

Objective To study the antieomplementary activity （ACA） of wide-type CD59 and mutant CD59 On ovarian cancer A2780 cells. Methods The pires-WTCD59 plasmid and the pires M1CD59 plasmid were transfected into A2780 cells. Stable expression clones were selected by the addition of G418. Immunohistochemistry method and RT PCR were used to analyze the quantity of protein CD59. The ACA of CD59 was tested by MTT method, Results The pires-WTCD59 plasmid and the pires-MICD59 plasmid were successfully amplified. A2780 cells with pires-WTCD59 and cells with M1CD59 were selected. The re suits suggested that mutant CD59 lost its protection of being against human complement compared with WTCD59. There was no significant difference compared with CD59 on A2780 cells. Conclusion The W40 of human CD59 is important to its activity, prohibition of this site may be a potential way to raise complement activity and treat tumor.