重组GPI-B7胞外段在中华仓鼠卵巢癌细胞膜上的表达

目的 通过从衰变加速因子(DAF)来源的GPI修饰性信号序列克隆到PCI-dhfr真核表达载体,利用GPI信号序列对B7-1基因胞外段序列进行修饰,实现GPI-B7-1锚定蛋白在中华仓鼠卵巢癌细胞(Chinese hamster ovary cell,CHO)膜表面的表达。方法 应用RT-PCR方法从B淋巴细胞瘤细胞系(Raji细胞系)总RNA中钓取B7-1(CD80)全长基因,扩增B7-1胞外段分子并克隆到已含有GPI修饰性信号序列的真核表达载体pCI-dhfr上,应用脂质体方法转染到CHO-dhfr-细胞中,用氨甲喋呤(Methotrexate,MTX)进行筛选。重组蛋白的表达用细胞免疫荧光进行鉴定。结果 成功地克隆了B7-1全长基因,并扩增了胞外段基因构建到含有GPI修饰性信号序列的真核表达载体pCI-dhfr上,实现了GPI-B7-1锚定蛋白在CHO细胞膜的表达。结论GPI信号序列能够通过其脂质性尾部将GPI-B7-1锚定蛋白整合到细胞膜表面,本研究结果为将来应用GPI-B7-1分子于肿瘤细胞膜表面的修饰做了技术准备,可作为肿瘤免疫治疗的手段。

Cloning of B7-1 cDNA and expression of GPI-B7-1 on Chinese hamster ovary cell membrane

Objective In order to express B7-1 protein on CHO cell membrane by alternative GPI-modification signal sequences modifying, which were cloned into eukaryotic expression vector PCI-dhfr.Methods In this study, B7-1 cDNA was cloned by RT-PCR from human Burkett's B lymphocyte line(Raji) ,B7-1 gene extracellular region was cloned into eukaryotic expression vector PCI-dhfr containing GPI-modifi-cation signal sequences, Using lipofectine-mediated gene transfer technique, pCI-GPI-B7-1 was transfected into CHO-dhfr- cell, and the trans-fectants were selected with methotrexate (MIX). Expression of the recombinant protein was assessed by cell immunofluorescence stain. Results B7-1 cDNA were successfully cloned by RT-PCR, The immunofluorescence stain results showed GPI-modified B7-1 were expressed on CHO cell membrane. Conclusion Glycosyl-phosphatidylinositol (GPI)-anchored B7-1 protein can incorporate into cell membranes with their lipid tail, The protein GPI-anchoring technique can be useful in tumor cell surface modifying as a new type immunotherapy method.