人趋化因子ELC的克隆与表达

Gene cloning and expression of human chemokine ELC

目的 克隆人趋化因子ELC基因,表达并初步纯化ELC融合蛋白。方法 从扁桃体中提取总RNA ,再进行RT PCR ,扩增ELC成熟蛋白基因,并在5′和3′分别添加NcoⅠ和EcoRⅠ酶切位点,通过这两个酶切位点重组于pET3 2a(+ )载体上,转化E .coliDH5α,筛选阳性克隆,酶切鉴定,DNA测序检测插入序列的正确性,缺失突变获得ELC天然蛋白表达载体pET3 2a(+ ) ELC ,SDS PAGE分析其表达,Westernblot验证融合蛋白。大量表达并初步纯化ELC融合蛋白。结果 成功克隆了ELC基因,表达并初步纯化得到ELC融合蛋白。结论 构建的ELC硫氧还蛋白融合表达载体以可溶性蛋白的方式表达ELC硫氧还蛋白。

Objective To clone human chemokine ELC and express the ELC fusion protein. Methods Total RNA from human inflammatory tonsil was extracted and the cDNA was generated with reverse transcription. Mature ELC gene was amplified with PCR and NcoⅠand EcoRⅠ sites were added to the 5′ and 3′ terminal respectively, and then cloned into pET32a(+). E.coli DH5α was transformed with the recombinant plasmid, and positive clones were selected. The inserted DNA was verified by enzyme digestion and DNA sequencing. The fusion expression vector of mature ELC was formed with deletion mutation. ELC expression was analyzed by SDS-PAGE and Western blotting and the ELC fusion protein was purified. Results Human chemokine ELC was successfully cloned and the fusion protein was expressed and purified. Conclusion The ELC fusion protein was expressed with solubility.