小鼠OX40真核表达载体的构建及表达

目的构建小鼠OX40的真核表达载体。方法从ConA活化的小鼠牌淋巴细胞中提取总RNA，应用RT-PCR方法，扩增获得编码小鼠OX40分子的cDNA，并将其克隆到PUCm-T载体，经PCR、酶切和测序分析证实，进而脂质体法转染HUVECS，G418筛选，RT-PCR法鉴定获得表达OX40分子的阳性细胞株。结果构建的pIRES2-EGFP-OX40重组真核表达载体经PCR、酶切和测序分析，证实该片段与GeneBank记载的小鼠OX40cDNA序列完全一致。筛选获得能表达小鼠OX40蛋白的HUVECs转基因细胞。结论成功构建小鼠OX40转基因细胞，该克隆细胞为进一步研究OX40分子的功能奠定了基础。

Construction of the Transfected Cell Line Expressing the Mouse OX40 Gene

Objective To construct HUVECs cell line expressing mouse OX40. Methods The cDNA fragment encoding the mouse OX40 was obtained from the total RNA of ConA-activated lymphocytes of spleen by using RT-TCR and cloned into PUCm-T vector and then was inserted into the eukarotic expression vector pIRES2-EGFP. The recombinant vector was transfected into HUVECS lines with lipofectine, and the positive cellular clones were selected by G418, a stable cell line expressing the mouse OX40 was established. Results The recombinant plasmid was conformed by DNA sequencing, PCR and enzyme digestion, which was consistent with the reported mouse OX40 cDNA in GeneBank, which was successfully cloned, and the stable HUVECs clone expressing the mouse OX40 was acquired. Conclusion Transfected cell line expressing the mouse OX40 gene has been constructed successfully.