【摘要】 目的 克隆小鼠次级淋巴组织趋化因子(Secondary Lymphoid-tissue Chemokine，SLC)基因，构建真核表达载体。方法 用逆转录聚合酶反应(reverse transcription-polymerase chain reaction，RT-PCR)方法从C57BL/6小鼠脾脏淋巴细胞中克隆出小鼠SLC基因，再连接到pGEM-T easy载体上并转化至JM109感受态菌中构建成pGEM-T easy-mSLC质粒。先用限制性内切酶Nhe I/EcoR V双酶切质粒pCI/GPI-Fc-Sig和pGEM-T easy-mSLC，构建pCI/GPI-Fc-mSLC中间质粒，再用Nhe I/Not I双酶切质粒pCI/GPI-Fc-mSLC和pcDNA3.1，构建成pcDNA3.1-mSLC-GPI-Fc真核表达载体。转化产物通过PCR扩增筛选、双酶切鉴定，将得到的阳性克隆进行序列测定分析。结果 克隆出的mSLC基因经测序完全正确。PCR、双酶切及测序结果均证实构建的真核表达载体中已插入402bp的SLC的基因片断。结论 小鼠次级淋巴组织趋化因子基因成功克隆，真核表达载体pcDNA3.1-mSLC-GPI-Fc构建成功，为进一步转染真核细胞建立稳定的转染表达细胞株和开展免疫基因治疗奠定了基础。

    关键词 趋化因子 次级淋巴组织趋化因子 基因克隆 真核表达载体

    Chen Xing，Yu Jiyun，Xiu Bingshui，et al.

    Beijing Institute of Basic Medical Science，Beijing100850.

    【Abstract】 Objective To clone mouse Secondary Lymphoid-tissue Chemokine(SLC)，then construct the eukaryotic expression vetor of mouse SLC gene.Methods Totol RNA was extracted from the spleen tissue of a C57BL/6mouse by TRIzol reagent，the mouse SLC gene was cloned by reverse transcription-polymerase chain reacˉtion(RT-PCR).Plasmid of pCI/GPI-Fc-Sig and pGEM-T easy-mSLC were digested by Nhe I/EcoR V to construct the intermedia plasmid pCI/GPI-Fc-mSLC for obtaining suitable multiple cloning site.pCI/GPI-Fc-mSLC and pcDNA3.1were further digested with NheI/NotI to construct recombinant pcDNA3.1-mSLC-GPI-Fc.The recombinants were screened by PCR ......