中文摘要：

目的：克隆与表达针对人CD133分子两个不同胞外段的单链抗体。方法：提取杂交瘤细胞总mRNA，利用简并引物PCR获得针对人CD133分子两个胞外段抗体可变区基因，再通过重叠延伸PCR的方法，用一段柔性linker （GGGGS）3将其相连，得到CD133 Ex-1 scFv和CD133 Ex-2 scFv基因。随后，将其克隆至携带6×His标签的原核表达载体pRSET-B中，然后对其进行了表达和鉴定研究。结果：获得分别针对CD133不同胞外段的单链抗体基因，并成功获得针对CD133两个胞外段的单链抗体，但该单链抗体主要以不溶性包涵体形式存在于胞内。包涵体蛋白经复性和镍柱亲和层析纯化后，得到纯度较高的单链抗体，通过蛋白质印迹实验证实，融合蛋白确实为单链抗体。结论：成功制备出针对CD133两个胞外段的单链抗体。该研究结果为其功能研究及其在肿瘤靶向治疗研究中奠定了物质基础。

英文摘要：

Objective：To clone and express the single chain variable fragment antibodies （scFv） against two ectodomains of human CD133. Methods：Total mRNA was extracted from the hybridoma ceils that can secrete monoclonal antibodies recognizing different ectodomains of human CD133 molecule. The encoding sequences of the heavy chain （V,） and light chain （VL） were successfully obtained using degenerate PCR. Then we joined the Vs and Vr genes with a flexible linker （ GGGGS ） 3 by overlap extension PCR,after that we respectively cloned the CDl33 Ex - 1 scFv or CD133 Ex -2 scFv into the pRSET - B vector that with a 6 x His tag on N - terminus. The expression and identification of CD133 Ex - 1 scFv and CD133 Ex - 2 scFv were examined after construction. Results： We successfully cloned and linked the VH and VL genes to construct two single chain variable fragment antibodies that respectively against the two ectodomains of human CD133. After expressed in E. coli, the production of the two scFvs was identified by Western blot assay using His tag mAb. Conclusion：The single chain variable fragment antibodies were expressed in E. coli in the form of inclusion - body in cytoplasm. Then the inclusion - body was renatured and purified by Ni - NTA resin to obtain the scFv antibody with high purity. We believe that construction and application of these scFvs could shed light on the investigation of the function of human CD133 molecule and, more importantly, develop novel tumor targeting therapy.