RKIP蛋白在多个人肺癌细胞系中的表达下调

Downregulation of Raf kinase inhibitory protein in human lung cancer cell lines

目的检测肺癌细胞系中RKIP的表达情况,探讨其与肺癌细胞侵袭、转移的关系。方法RT-PCR法检测多个人肺癌细胞系中RKIP的表达情况,构建表达人RKIP的融合蛋白,纯化后免疫新西兰白兔制备多克隆抗体。ELISA鉴定血清特异抗体效价,Western blot鉴定血清特异抗体并检测肺癌细胞系中RKIP表达。结果成功构建表达人RKIP的融合蛋白,制备的多抗效价达到106,可特异检测细胞内RKIP表达;有转移性的3个肺癌细胞系中RKIP表达明显下调,且在转移性较高的A549细胞中下调更明显。结论获得了效价和特异性都良好的RKIP多抗,适合应用于RKIP的检测。RKIP表达下调与肺癌细胞侵袭、转移有关,有可能成为肺癌治疗的靶点。

Objective To detect the expression level of RKIP in human lung cancer cell lines and to investigate the relationships between RKIP and cell metastasis. Methods The expression level of RKIP in human lung cancer cell lines was analyzed by RT-PCR. To construct and express human RKIP fusion protein and to immunize New Zealand rabbit with purified fusion protein to prepare polyclonal antiserum. The antiserum was titered by ELISA. The specifcity of antiserum and expression level of RKIP in human lung carcinoma cell lines was analyzed by westeru blotting. Results The fusion protein was successfully expressed. The prepared polyclonal antibody reacted specifically with RKIP in human cells. Downregulation of RKIP was detected in three lung cancer cell lines with metastasis, and it is more significant in A549 with higher metastasis. Conclusion The RKIP antiserum with high titer and good specificity could meet the requirement for studying on RKIP. Downregulation of RKIP has correlation with human lung carcinoma cell metastasis.