人趋化因子巨噬细胞炎性蛋白3α的克隆与原核表达

Gene cloning and expression of human chemokine MIP-3 alpha

目的克隆人趋化因子MIP-3 alpha基因,表达并初步纯化MIP-3 alpha融合蛋白。方法从人扁桃体中提取总RNA,再进行RT-PCR,扩增MIP-3 alpha成熟蛋白基因,并在5′和3′端分别添加NcoI和EcoRI酶切位点,通过与之对应的存在于pET32a(+)质粒上的酶切位点重组于pET32a(+)载体上,转化E.coli BL21trxB(DE3),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MIP-3 alpha序列前端多余碱基,获得MIP-3 alpha天然蛋白表达载体pET32a(+)/MIP-3 al-pha,SDS-PAGE分析其表达,Western Blot验证融合蛋白。大量表达并初步纯化MIP-3 alpha融合蛋白。结果成功克隆了MIP-3 alpha基因,表达并初步纯化得到MIP-3 alpha融合蛋白。结论在大肠杆菌中成功构建的MIP-3 alpha硫氧还蛋白融合表达载体,以可溶性蛋白的方式表达MIP-3 alpha硫氧还蛋白。

Objective To clone human chemokine MIP-3 alpha and to express the MIP-3 alpha fusion protein. Methods Total RNA from human inflammatory tonsil was extracted and its eDNA was generated with reverse transcription. Mature MIP-3 alpha gene were amplificated with PCR and NcoI and EcoRI sites were added to the 5＇and 3＇end respectively. PCR product was cloned into pET32a（＋） with the two sites. E. coli BL21trxB（DE3） was transfected with the recombinant plasmid, and positive clones were selected. The insert DNA was verified by enzymes digesting and DNA sequencing. The fusion expession vector of mature MIP-3 alpha was formed with deletion mutation. Expression of MIP-3 alpha was analysed by SDS-PAGE and Western Blot. And the MIP-3 alpha fusion protein was purified. Results Human chemokine MIP-3 alpha was successfully cloned, and the fusion protein is expressed and purified. Conclusion The MIP-3 alpha fusion protein is expressed with solubility.