小鼠紧密连接蛋白ZO-2 真核表达载体的构建和表达

目的:克隆小鼠紧密连接蛋白ZO-2(zonula occludens protein2)基因构建其真核表达载体,并验证其在293T细胞系中的表达,为进一步研究其功能奠定基础。方法:从小鼠淋巴结来源的cDNA中分三段分别扩增,通过基因克隆方法获得紧密连接蛋白ZO-2基因全长,连接至pMD-18T载体中,酶切测序正确后,插入pEGFP-C2载体中,构建真核表达载体pEGFP-C2-ZO2。酶切正确后,瞬时转染入293T细胞中,48h后荧光显微镜观察其绿色荧光GFP融合蛋白的表达,并用Western blot检测其在转染细胞中的蛋白水平表达。结果:通过酶切鉴定和测序结果证明成功克隆了ZO-2基因,western blot结果表明成功构建了真核表达载体pEGFP-C2-ZO2。结论:小鼠紧密连接蛋白ZO-2基因的获得和真核表达载体pEGFP-C2-ZO2的成功构建为下一步研究其生物学功能奠定了基础。

Construction of Eukaryotic Vector of ZO-2 and Expression in 293T Cells

Objective: To clone the mouse tight junction protein ZO-2(zonula occludens protein2) gene,to construct its eukaryotic expression vector and to verify its expression in the 293T cell lines,which will establish the foundation for future research.Methods: Three different parts of the tight junction protein ZO-2 gene were clone by polymerase chain reaction(PCR) with the cDNA from mouse lymph node,and to combine all these parts to make the full ZO-2 gene.The amplified fragment was cloned into the pEGFP-C2 vector.The recombinant plasmid was identified and transfected into 293T cells.The expression of ZO-2 protein was verified by fluorescence mi-croscopy and westeren-blot.Result: The gene of full ZO-2 was acquired and the eukaryotic expression vector of pEGFP-C2-ZO2 was constructed.The protein of ZO-2 was successfully expressed.Conclusion: Construction of the mZO-2-eukaryotic expression vector will be beneficial to guiding further study of its biological function.