Abstract: [Objective] To evaluate the potential of camel-derived antibody cAbBCII10 as a non-antibody affinity transfer skeleton, the FSHR binding peptide was grafted to the antigen-binding region of cAbBCII10 using affinity transfer to rapidly obtain anti-FSHR antibody.[Method]The FSH binding motif FSH 33-53 coding sequence was inserted into the CDR1 and CDR3 regions of the nanobody cAbBCII10, respectively, and named VHH-hFSH1 and VHH-hFSH3.These DNA sequences were cloned into pET22b vector and transformed into E.coli BL21 (DE3), and purified single-domain antibody was obtained by IPTG induction and Ni-affinity affinity chromatography.[Result]Finally binding capacity and specificity of purified cAbBCII10, VHH-hFSH1 and VHH-hFSH3 with FSHR were identified by ELISA.The VHH-hFSH1, VHH-hFSH3 and cAbBCII10 proteins obtained by the framework transplantation were expressed in the intercellular space of bacteria.ELISA experiments showed that the VHH-hFSH3 obtained by grafting the FSHR binding peptide FSH33-53 to the CDR3 of cAbBCII10 had a specific binding to FSHR activity.[Conclusion]CAbBCII10 can be used as a graft framework, FSH and FSHR binding peptide grafted into the CDR1 and CDR3 regions of cAbBCII10 can obtain higher affinity anti-FSHR antibody.

摘要： [目的]评价骆驼来源的纳米抗体cAbBCII10作为非抗体亲和力转移骨架的潜能,采用亲和力转移的方法,将FSHR结合肽段移植到cAbBCII10的抗原结合区以快速获得抗FSHR抗体.[方法]将FSHR结合肽FSH33-53编码序列,分别移植入纳米抗体cAbBCII10的CDR1和CDR3区域中,命名为VHH-hFSH1 和VHH-hFSH3.采用DNA合成方法获得VHH-hFSH1,VHH-hFSH3和cAbBCII10的DNA编码序列,把这些DNA序列克隆到pET22b载体上,将其转化到BL21(DE3)感受态细胞中.经过诱导和表达再用Ni离子亲和纯化获得纯的单域抗体.采取ELISA方法鉴定纯化的cAbBCII10,VHH-hFSH1、VHH-hFSH3与FSHR的结合能力及特异性.[结果]通过框架移植获得的VHH-hFSH1 ,VHH-hFSH3和cAbBCII10蛋白均在细菌胞间质可溶性表达.将FSHR结合肽FSH33-53移植到cAbBCII10的CDR3获得的VHH-hFSH3具有特异结合FSHR活性.[结论]cAbBCII10可以作为移植的框架,FSH和FSHR结合肽段移植到cAbBCII10的CDR1和CDR3区可以获得亲和性较高的抗FSHR抗体.