关键词: 前列腺特异抗原;单域抗体;克隆;表达

    摘 要:目的 扩增出抗前列腺特异抗原(PSA)单克隆抗体(MAb)的重链可变区(VH)单域抗体基因，并在大肠杆菌中表达. 方法 从分泌抗PSA mAb的杂交瘤细胞系E4 B7 中提取总RNA，经RT-PCR扩增VH 单域抗体基因，将其克隆到融合蛋白表达载体pGEX-4T-1中进行表达. 结果 VH 单域抗体基因全长363bp，含起始码和终止码.将其克隆到pGEX-4T-1内，转化大肠杆菌DH5α，获得高效表达，表达量占全菌总蛋白质的38%，以包涵体形式存在.经初步纯化和复性后，用谷胱甘肽(GST)亲和色谱纯化，再经凝血酶水解获得抗PSA VH 单域抗体.竞争结合抑制实验证明，该表达产物具有前列腺癌细胞亲和活性. 结论 构建成功抗PSA的VH 单域抗体，为进一步临床应用奠定基础.

    Construction and expression of anti┐prostate specif┐ic antigen VH single domain antibody gene

    YUAN Jian-Lin WANG Zhi WU Guo-Jun HAO Xiao-Ke

    1 Department of Urology Surgery，Xijing Hospital，Fourth Military Medical University，Xi'an710033，

    2 Department of Biochemistry，Faculty of Preclinical Medicine，Fourth Military Medical University，

    3 Central Laboratory，Tangdu Hospital，Fourth Mil-itary Medical University

    Keywords:prostate-specific antigen;single domain antibody;cloning;expression

    Abstract:AIM To amplify the VH single domain gene of an-ti-prostate specific antigen(PSA)monoclonal antibody(mAb)，and express it in E.coli.METHODS Total RNA was extracted from the hybridoma cell line E4 B7 ......