中文摘要：

目的：检测ClC-2是否能被促分裂原活化蛋白激酶（MAPK）磷酸化，为进一步研究其在细胞增殖、分化过程中的调控机制提供基础。方法：从含有家兔ClC-2cDNA的质粒pSPORT1中PCR扩增ClC-2胞浆内的羧基端DNA编码序列，构建重组载体pGEX-4T-1／ClC-2CT；重组质粒经酶切、测序鉴定正确后转化大肠杆菌BL21菌株；经异丙基β-D-硫代半乳糖（IPTG）诱导后，通过Gluthathion Sepharose4B亲和层析柱纯化融合蛋白；并行融合蛋白的体外磷酸化实验。结果：酶切、测序鉴定重组载体pGEX-4T-1／ClC-2CT构建正确，并纯化得到GST／ClC-2CT融合蛋白。而且该融合蛋白能被MAPK磷酸化，而GST不能被MAPK磷酸化。结论：氯通道ClC-2能被MAPK磷酸化。

英文摘要：

Objective：To investigate if chloride channel ClC-2 could be phosphorylated by mitogen activated protein kinase （MAPK） for further study of its regulation mechanism in proliferation and differentiation of cells. Methods：The coding sequence containing the cytosolic C-termlnus of ClC-2 was amplified from pSPORT1/ClC-2 plasmid, including rabbit ClC-2 eDNA, by polymerase chain reaction（ PCR）, the fragment was cloned into pGEX4T-1 plasmid for the construction of GST-tagged fusion protein expressing vector, pGEX4T-1/ClC-2CT. After being identified by enzyme digestion and sequencing, the recombinant vector was transformed into a strain of E. coli BI21. The expression of GST-tagged fusion protein was induced with IPTG and purified with Gluthathion Sepharose 4B affinity chromatography. Then the phosphorylation of ClC-2 by MAPK was examined by using phosphorylation assays in vitro. Results：The construction of pGEX4T-1/ClC-2CT recombinant vector was proved by enzyme digestion and sequencing. The purified fu- sion protein GST/ClC-2CT could be phosphorylated by MAPK, however the GST could not. Conclusion：Chloride channel ClC-2 can be phosphorylated by MAPK.