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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 zonula occludens protein2 gene and construct its eukaryotic expression vector and verify its expression in 293T cell line, and lay a foundation for further study of its function. METHODS: The full length cDNA of ZO-2 was amplified from mouse lymph node cDNA by gene cloning. The full-length ZO-2 gene was ligated into pMD-18T vector. The correct sequence was inserted into pEGFP-C2 vector The eukaryotic expression vector pEGFP-C2-ZO2 was constructed. The recombinant plasmid was transiently transfected into 293T cells and the expression of green fluorescent GFP fusion protein was observed by fluorescence microscope 48h later. The protein expression level in transfected cells was detected by Western blot. Results: The ZO-2 gene was successfully cloned by restriction enzyme digestion and sequencing. Western blot results showed that the eukaryotic expression vector pEGFP-C2-ZO2 was successfully constructed. CONCLUSION: The construction of mouse ZO-2 tight junction protein and the successful construction of eukaryotic expression vector pEGFP-C2-ZO2 lay the foundation for the further study of its biological functions.