[摘 要] 目的 克隆人趋化因子MIP-3α基因，表达并初步纯化MIP-3α融合蛋白。 方法 从扁桃体中提取总RNA，再进行RT-PCR，扩增MIP-3α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pET32a(+)载体上，转化E.coli.DH5α，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MIP-3α天然蛋白表达载体pET32a(+).MIP-3α，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-3α融合蛋白。 结果 成功克隆了MIP-3α基因，表达并初步纯化得到MIP-3α融合蛋白。 结论 构建的MIP-3α硫氧还蛋白融合表达载体以可溶性蛋白的方式表达MIP-3α硫氧还蛋白。

    [关键词] MIP-3α;融合表达;载体;Western blot;蛋白纯化

    Gene cloning and expression of human chemokine MIP-3α

    LUO Fu-kang，ZHENG Hong，SHEN Da-bin

    (Laboratory Research Center，Southwest Hospital，Third Military Medical University，Chongqing400038，China)

    Abstract:Objective To clone human chemokine MIP-3αand to express and purify macrophage inflammatory protein3α(MIP-3α)fusion pro-tein.Methods Total RNAwas extracted from human inflammatory tonsil and its cDNAwas generated by reverse transcription.Mature MIP-3αgene was amplified by PCR and NcoⅠand EcoRⅠsites were added to the5'and3'end，respectively.PCR product was cloned into pET32a(+)with the two sites.E.coli DH5αwas transfected with the recombinant plasmid，and positive clones were selected.The insert DNAwas verified by enzyme digestion and DNA sequencing.The fusion expression vector of mature MIP-3αwas formed with deletion mutation.Expression ofMIP-3αwas analyzed by SDS-PAGE and Western blotting.MIP-3αfusion protein was purified.Results Human chemokine MIP-3αwas successfully cloned ......