USP22基因克隆及其融合蛋白真核表达载体的构建

目的克隆人类泛素水解酶22（ubiquitin—specific processing enzyme22，USP22）基因，构建与绿色荧光蛋白融合表达的真核载体。方法利用RT—PCR技术以HeLa细胞总RNA为模板分别扩增USP22基因cDNA序列两片段，依次插入真核表达载体pEGFP—N1；重组质粒转染HEK293T细胞，观察融合蛋白表达及绿色荧光的分布。结果测序结果显示USP22序列与GenBank收录数据一致，酶切显示重组质粒pEG—FP—USP22构建无误，质粒转染后有80％左右HEK293T细胞表达绿色荧光，且在胞质与胞核中广泛分布。结论成功克隆USP22基因并构建与GFP融合表达的真核载体，为进一步深入研究USP22基因的生物学功能奠定了基础。

Cloning of USP22 Gene and Construction of Its Eukaryotic Expression Vector of Fusion Gene

Objective To clone human ubiquitin-specific processing enzyme 22 （USP22） gene and construct eukaryotic expression vector containing USP22 and GFP fusion gene. Methods Using the isolated total RNA from HeLa cells as the templates, two fragments of human USP22 cD- NA were amplified by RT-PCR, and then inserted into eukaryotic expression vector pEGFP-N1 one by one. The recombinant plasmids were transfected into HEK293T cells, and the expression and distribution of green fluorescence were observed. Results Sequencing analysis demonstrated that the sequence of USP22 was consistent with the data of NCBI ; Restrictive endonuclease identification confirmed that the recombinant vector pEGFP-USP22 was constructed correctly. After transfeetion, green fluorescence expressed in 80 % approximately HEK293T cells and distributed in cytoplasm and nucleus extensively. Conclusion The USP22 gene is successfully amplified and the eukaryotic expression vector containing USP22 and GFP fusion gene is constructed, which provides a basis for further study of USP22 biology function.