转染小鼠可溶性B淋巴细胞刺激因子稳定表达细胞株的筛选与鉴定

用脂质体包裹纯化的小鼠可溶性B淋巴细胞刺激因子 (msBlyS)重组表达质粒 ,体外转染小鼠结肠癌细胞系CT2 6 ,通过Zeosin抗性筛选获得抗性的单克隆肿瘤细胞株。提取细胞总RNA做半定量RT PCR ,鉴定出高表达msBlyS的稳定细胞克隆 ,进一步做Western blotting鉴定和生物活性测定。转染后得到 8个单克隆抗性肿瘤细胞株 ,其中有 5个经RT PCR扩增出msBlyS目的条带。Western blotting显示该克隆株细胞裂解物可与抗msBlyS的抗体结合 ,在杂交膜上形成一条与预期蛋白分子量一致的条带。生物活性测定显示细胞分泌上清可共刺激(Costimulate)体外培养的B淋巴细胞增殖 ,这表明已成功转染并筛选出msBlyS稳定表达小鼠结肠癌细胞株 ,可以进一步用于msBlyS抗肿瘤作用的观察和研究。

Screening and Identification of Stable Transfectants of Mouse Soluble B Lymphocyte Stimulator

Mouse colon cancer cells CT26 were transfected with constructed plasmid expressing mouse soluble B lymphocyte stimulator (msBlyS). Single cell clones were selected with 100 microg/ml Zeosin and subcloned by serial limiting dilution. Eight resistant transfectants were isolated and expanded, and five of them displayed the desirable msBlyS cDNA band amplified by semi-quantitative RT-PCR assay. Western blot analysis showed that only msBlyS molecules of the expected size were detected in the cell lysates from transfectants. The supernatant of transfectants could costimulate B cell proliferation in standard costimulation assay. Thus we have successfully screened the stable transfectants expressing high levels of msBlyS in CT26 cells, which could be used as cancer vaccines for further anti-tumor immunotherapy.