真核表达载体pIRES2-EGFP-KAI1的构建及其在胆管癌细胞的表达

Construction of the eukaryotic expression vector pIRES2-EGFP-KAI1 and its expression in human biliary carcinoma cells line

目的构建真核表达载体pIRES2-EGFP-KAI1,并在胆管癌细胞系QBC939中进行表达。方法采用RT-PCR法从人胆管组织中克隆KAI1基因,连接T载体并测序正确后与真核表达载体pIRES2-EGFP连接,经EcoRⅠ和SalⅠ双酶切鉴定后通过脂质体法转染QBC939细胞中,用荧光显微镜检测细胞中增强型绿色荧光蛋白(EGFP)的表达,用免疫组化染色的方法检测细胞中KAI1的表达。结果成功构建真核表达载体pIRES2-EGFP-KAI1,用脂质体法转染胆管癌细胞QBC939后,经荧光显微镜和免疫组化染色法检测可见细胞内有EGFP及KAI1的表达。结论真核表达载体pIRES2-EGFP-KAI1构建成功并在胆管癌细胞QBC939中得到稳定表达,为研究KAI1对肿瘤的生物学作用以及KAI1在肿瘤基因治疗中的应用奠定了基础。

Objective To construct the eukaryotic expression vector pIRES2-EGFP-KAI1,and to express KAI1 in human biliary carcinoma cell line.Methods KAI1 gene was cloned from human biliary tissue by RT-PCR and inserted into T-vector.After DNA sequencing verification,it was then sub-cloned into eukaryotic expression vector pIRES2-EGFP.The plasmid was transfected into the QBC939 cells using lipofectamine.The expressed EGFP was observed under fluorescent microscope and the KAI1 protein expression was detected by immunostaining using anti-KAI1 antibody.Results The eukaryotic expression vector pIRES2-EGFP-KAI1 was constructed and transfected successfully into QBC939 cells.The green fluorescence of EGFP was observed in the plasma and nuclei of transfected cells,and KAI1 protein was only found in the plasma.Conclusion The recombinant expression vector pIRES2-EGFP-KAI1 is constructed,and the EGFP and KAI1 gene can be co-expressed in the QBC939 cells.This study leads a foundation for the further research of the function of KAI1 in cell differentiation,growth and tumorigenesis.