GST-HDAC4融合蛋白载体的构建及其蛋白表达

Construction of GST-HDAC4 Fusion Protein Vector and Its Protein Expression

目的构建人HDAC4蛋白与谷胱甘肽转移酶(GST)重组的融合蛋白原核表达载体并进行表达和纯化。方法用EcoRI单酶切pcDNA3.1-HDAC4质粒,得到hHDAC4全长编码序列,并将其克隆至原核表达载体pGEX-5X-1构建成新载体GST-HDAC4,经鉴定完全正确后转化大肠埃希菌BL21,通过异丙基硫代-β-D半乳糖苷(IPTG)诱导表达,并纯化获得目的蛋白。Western blot检测重组质粒的表达。结果酶切鉴定和测序结果显示,GST-HDAC4融合蛋白原核表达载体构建成功。考马斯亮蓝染色和Western blot结果显示,成功获得有生物活性的GST-HDAC4融合蛋白。结论成功构建了HDAC4原核表达载体,获得有生物活性的GST-HDAC4蛋白。

Objective To construct the pmkaryofic expression vector of human HDAC4 protein and glutathione-S-transfemse （GST） for protein expression and purification. Methods pcDNA3.1-HDAC4 was digested by EcoR I ,the hHDACA coding sequence was obtained and cloned into prokaryotic expression vector pGEX-5X-1 to generate novel vector GST-HDAC4. After identification of GST-HDAC4, Escbenchia coli BL21 was transformed and induced by isopropyl-β-D-thiogalactoside （IPTG） ,and target protein was obtained after purification. The ex- pression of the recombinant plasmid was proved by Western blot. Results Restriction enzyme digestion and sequencing indicated that prokaryotic expression vector of GST-HDAC4 fusion protein was successfully constructed. Coomassie brilliant blue staining and Westem blot revealed that GST-HDAC4 fusion protein with bioactivity was successfully obtained. Conclusion The prokaryotie expression vector of HDAC4 was successfully constructed. The fusion protein of GST-HDAC4 with bioactivity has been obtained.