利用乳糖化羧甲基壳聚糖构建小鼠OX40转基因细胞株

目的利用乳糖化羧甲基壳聚糖（Lac-CMCS）构建小鼠OX40转基因细胞株，探讨Lac-CMCS在HepG2细胞定向转染中的可行性。方法从ConA活化的小鼠脾淋巴细胞中提取总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，测序证实后构建pIRLS2-EGFP-OX40重组真核表达载体，利用Lac-CMCS与载体偶联靶向性转染HepG2细胞，G418筛选，RT-PCR法鉴定获得表达OX40分子的阳性细胞株。结果pUCm-T-OX40和pIRFE2-EGFP-OX40经PCR、酶切和测序分析，证实插入的目的片段与GenBank记载的小鼠OX40 cDNA序列完全一致。利用Lac-CMCS能将小鼠OX40靶向转染HepG2细胞并获得表达；经G418筛选后获得稳定表达小鼠OX40的细胞株。结论利用Lac-CMCS能够成功将OX40靶向转染入HepG2细胞中。

Construction of the transfected cell line expressing mouse OX40 gene by lactose-carboxymethyl chitosan

Objection To construct the transfected cell line expressing mouse OX40 gene by using lactose-carboxymethyl chito-(san(Lac-CMCS).) Methods The cDNA fragment encoding mouse OX40 was obtained from the total RNA of ConA-activated lymphocytes of spleen by using RT-PCR and cloned into pUCm-T vector,and then subcloned into the eukaryotic expression vector pIRES2-EGFP.The recombinant vector was transfected into HepG2 cell lines by Lac-CMCS coalesced with pIRES2-EGFP-OX40,and then the positive cell clones were selected and analyzed by G418 and RT-PCR,respectively.Results DNA sequencing indicated that the pUCm-T-OX40 and pIRES2-EGFP-OX40 were consistent with the reported mouse OX40 cDNA in GenBank.The transfected cell line expressing mouse OX40 was established successfully by Lac-CMCS.A stable cell clone was acquired by G418.Conclusion pIRES2-EGFP-OX40 can be transfected into HepG2 through lactose-carboxymethyl chitosam orientationally.