摘要
目的 制备人假想镁离子转运蛋白(HPT)抗血清并检测其在胃癌耐药细胞中的表达 ,以观察其在胃癌耐药发生中的作用。方法 克隆编码HPT蛋白的cDNA并测序鉴定 ;构建原核表达载体pRSETB HPT ,诱导表达 6×His与HPT的融合蛋白 ;用含融合蛋白的聚丙烯酰胺凝胶颗粒免疫小鼠 ,以Westernblot检测小鼠血清的抗体活性 ;以所制备的抗血清检测HPT蛋白在胃癌各细胞系中的表达。结果 DNA测序结果证实所克隆的基因片段与预计相符 ;利用原核系统表达出 1条约 5 5kD的新生蛋白带 ,Westernblot证实其为与6×His融合的蛋白 ;该融合蛋白免疫血清仅结合SGC790 1细胞中约 5 0kD的单一蛋白带 ;利用该血清检测发现SGC790 1 /ADR中HPT蛋白表达水平高于SGC790 1。结论 成功地制备了鼠抗人HPT蛋白抗血清 。
Objective To prepare an antiserum of human putative magnesium transporter protein (HPT protein) to study its biological effects on the drug resistance of gastric cancer cell by observing its expression level. Methods The cDNA encoding HPT protein was cloned and sequenced. Prokaryotic expression vector was constructed and HPT protein fused with histidine tag was expressed. Mice were immunized with the polyacrylamide gel particles containing the fusion protein, and the antisera of them were detected for antibody activity by Western blot. Using the antiserum, HPT protein expression level was assessed in different gastric cancer cell line. Results The sequence of cloned cDNA fragment was consistent with that of HPT. After BL21/pRSETB bacterium was induced with IPTG, a new protein band with a relative molecular weight of 55kD was showed on SDS PAGE profile, and further proved to be fusion protein with histidine tag by Western blot. The antiserum of immunized mice only recognized a unique protein band with a relative molecular weight of 50kD. HPT protein expression level in SGC7901/ADR was higher than in SGC7901. Conclusion The antiserum of HPT protein was successfully prepared, and HPT was found to probably take part in the gastric cancer cell MDR
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2004年第6期530-533,共4页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金重点项目 (编号 30 0 30 1 4 0 )
创新研究群体基金项目(编号 30 0 2 4 0 0 2 )资助课题
