小鼠紧密连接蛋白ZO-2真核表达载体的构建和表达

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

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。

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.