hSp17真核表达载体构建及其在卵巢癌细胞中的表达

摘要

Objective To construct eukaryotic expression vector of pcDNA3.1( + )/hSp17 containing human sperm protein 17 (hSpl7) cDNA, so to provide basis for its clinical application. Methods hSpl7 cDNA was obtained by PCR, and to construct eukaryotic expression vector pcDNA3. 1 ( + )/GNLY by Nhe I , Kpn I digestion and sticky end ligation techniques. The recombinant plasmid was transfected into H08910 cells with lipofectamine 2000. Expression of hSpl7 in the transfected cells was observed by immunocytochemistry. Cells with stable expression of hSp17 were selected by G418. Re-sults Sequencing and restriction enzyme digestion showed pcDNA3. 1 ( + )/GNLY was correctly constructed. The stably hSpl7 gene transfected HO8910 cell clones were obtained through G418 screening and immunocytochemistry assay were fur-ther performed to confirm the expression of hSpl7 in the HO8910 cells. Conclusion An ovarian carcinoma cell model that stably expressed hSpl7 can be successfully established; which can provide basis for immunotherapy of ovarian carcinoma.%目的 构建人精子蛋白17(hSp17)基因的真核表达载体pcDNA3.1(+)/hSp17,为其临床应用奠定基础.方法 用PCR方法获取hSp17基因片段,Nhe Ⅰ、Kpn Ⅰ双酶切、粘端连接的方法构建含有hSp17基因的peDNA3.1(+)/hSp17真核表达载体.将该载体行脂质体介导转染卵巢癌细胞HO8910,免疫细胞化学方法检测转染细胞hSp17蛋白表达,G418筛选稳定表达hSp17蛋白的细胞株.结果 酶切和测序结果均证实pcDNA3.1(+)/hSp17重组质粒的构建完全正确.免疫细胞化学方法证实外源性hSp17基因能够在卵巢癌HO8910细胞瞬时表达.经G418连续筛选,得到阳性克隆细胞株,并证实了外源性hSp17基因在卵巢癌HO8910细胞中稳定表达.结论 成功构建了稳定表达外源性hSp17蛋白的卵巢癌细胞株；其可为卵巢癌的免疫治疗奠定基础.