采用CDR3亲和力转移方法快速获得抗FSHR抗体  

Rapid Generation of Anti-FSHR Nanobody by CDR3 Affinity Transfer Approach

作　　者：席欧彦 邱玲玲[1] 马晓玲[1] 秦瑞坪 赵婷[1] 李江伟[1] 

机构地区：[1]新疆大学生命科学与技术学院/新疆生物资源基因工程省部共建重点实验室,乌鲁木齐830046

出　　处：《新疆农业科学》2017年第7期1298-1304,共7页Xinjiang Agricultural Sciences

基　　金：国家自然科学基金项目"骆驼单域抗体框架区作为小分子抗体通用移植骨架的研究"(31370933);国家自然科学基金项目"基于高通量测序对骆驼抗体库的多样性分析和数据挖掘及新纳米抗体的发现"(31570935)~~

摘　　要：【目的】评价骆驼来源的纳米抗体c Ab BCII10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c Ab BCII10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c Ab BCII10的CDR1和CDR3区域中,命名为VHH-h FSH1和VHH-h FSH3。采用DNA合成方法获得VHH-h FSH1,VHH-h FSH3和c Ab BCII10的DNA编码序列,把这些DNA序列克隆到p ET22b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c Ab BCII10,VHH-h FSH1、VHH-h FSH3与FSHR的结合能力及特异性。【结果】通过框架移植获得的VHH-h FSH1,VHH-h FSH3和c Ab BCII10蛋白均在细菌胞间质可溶性表达。将FSHR结合肽FSH33-53移植到c Ab BCII10的CDR3获得的VHH-h FSH3具有特异结合FSHR活性。【结论】c Ab BCII10可以作为移植的框架,FSH和FSHR结合肽段移植到c Ab BCII10的CDR1和CDR3区可以获得亲和性较高的抗FSHR抗体。【 Objective] To evaluate the potential of camel - derived antibody cAbBCIIlO as a non - anti-body affinity transfer skeleton, the FSHR binding peptide was grafted to the antigen - binding region of cAbB- CII10 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 cAbBCIIlO, respective-ly , and named VHH - hFSHl 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 cAbB- CII10, VHH - hFSHl and VHH - hFSH3 with FSHR were identified by ELISA. The VHH - hFSHl, VHH - hFSH3 and cAbBCIIlO 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 cAbBCIIlO had a specific binding to FSHR activity. [ Conclusion] CAbB- CII10 can be used as a graft framework, FSH and FSHR binding peptide grafted into the CDR1 and CDR3 re-gions of cAbBCIIlO can obtain higher affinity anti - FSHR antibody.

关 键 词：亲和力转移 FSHR FSH33-53 c Ab BCII10 纳米抗体 

分 类 号：S188[农业科学—农业基础科学]