hVPS4A 基因克隆及真核表达载体的构建

目的：克隆人细胞空泡蛋白分选因子4A（human vacuolar protein sorting 4A，hVPS4A）基因，构建其真核表达质粒。方法以 Huh7细胞 cDNA 为模板，设计引物扩增全长 hVPS4A 基因，再将目的基因插入到真核载体 pRK5中。重组质粒经PCR、酶切和 DNA 测序确认。结果从 Huh7细胞 cDNA 中扩增得到约1350 bp 大小的目的片段，经回收、纯化、酶切后与载体pRK5连接，转化 DH5α大肠杆菌。选择 PCR 鉴定阳性的重组质粒，经 EcoRⅠ单酶切可见约一条5900 bp 片段；经 EcoRⅠ和HindⅢ双酶切可得到4600 bp 和1350 bp 两个片段，分别与载体 pRK5和目的片段 hVPS4A 预期大小一致；DNA 测序结果显示插入片段与 hVPS4A 参考序列一致。结论成功构建了含 hVPS4A 基因的真核表达载体，为进一步研究 hVPS4A 的生物学功能提供了条件。

Cloning of human vacuolar protein sorting 4A gene and construction of eukaryotic expression vector

Objective To clone human vacuolar protein sorting 4A gene（hVPS4A）and to construct its eukaryotic expressive plasmid.Methods Primers were designed to amplify the full length hVPS4A by PCR using cDNA of Huh7 cell as a template,then the target DNA was inserted into the eukaryotic vector pRK5.The recombinant plasmid was confirmed by PCR,restriction enzyme digestion and DNA sequencing.Results A 1 300 bp fragment was successfully amplified by PCR from the cDNA of Huh7 cells.Af-ter recycled,purified and ligated with the vector pRK5,the recombinant plasmid was transformed into E.coli DH5α.The positive re-combinant plasmid identified by PCR was selectred and digested by EcoRⅠto get a 5 900 bp fragment;and two fragments including 4 600 bp and 1 350 bp were obtained using EcoRⅠand HindⅢ digestion;the size of these two fragments were consistent with the pRK5 target fragment and the inserted hVPS4A as expected.Moreover,DNA sequencing results confirmed that the inserted frag-ment was in accordance with the hVPS4A reference sequence.Conclusion The eukaryotic expression vector containing hVPS4A gene is constructed successfully,which provides the condition for further study on the hVPS4A biological functions.