| 朱冰柯1 秦鸿雁2 付伟1 梁世倩2 曹秀丽2 韩骅2△ 梁英民1.小鼠紧密连接蛋白ZO-2 真核表达载体的构建和表达[J].,2012,12(11):2001-2004 |
| 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 |
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| DOI： |
| 中文关键词: 紧密连接蛋白ZO-2 293T 细胞 真核表达 |
| 英文关键词: Zonula occludns protain2 293T cells Eukaryotic expression |
| 基金项目:国家自然科学基金面上项目（81071874）；国家自然科学基金青年基金项目（100354）；
陕西省自然科学基础研究计划项目（2010 JQ 4002） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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 microscopy
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. |
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