Objective In order to study the different subclasses of mouse IgG Fc and the binding properties with the SpA or SpG, the in vitro molecular evolution of the library was conducted with four mouse IgG subclasses as the baits, respectively. Methods The in vitro molecular evolution of the library by four mouse IgG subclasses respec-tively were used to generate novel evolved immunoglobulin binding molecules ( NEIBMs ) with special binding advantages. The binding activity of the NEIBMs with the four mouse IgG subclasses were compared by phage ELISA. To further compare the binding activity of NEIBMs with the four mouse IgG subclasses, ELISA was conducted with HRP-labeled NEIBMs. Results After five,four,five and five rounds molecular evolution of the phage library di-rected by mouse IgG1, IgG2a, IgG2b and IgG3, the control phages and one inserted the library was all two inserted domains phages, suggesting that the evolution of the library was finished. Sequence analysed by the software showed that DD, DD, AC and DC were obtained by the mouse IgG1, IgG2a, IgG2b and IgG3 respectively. The phage binding assays confirmed that the three molecules possessed binding advantages with the four mouse IgG sub-classes. The results of ELISA with HRP-labeled NEIBMs were not completely consistent with the in vitro molecular evolution of the library by four mouse IgG subclasses, but the binding strength was consistent, all were: IgG3>IgG2a>IgG2b>IgG1. Conclusion In this work, three novel evolved immunoglobulin binding molecules D-D, A-C and D-C are obtained from the in vitro molecular evolution of a combinatorial phage library displaying randomly-rearranged various binding domains, and they have special binding advantages with the four mouse IgG subclasses that don't exist neither in SpA nor SpG. The three molecules provide the new molecules for the purification and de-tection of the four mouse IgG subclasses.%目的判断不同亚类小鼠IgG Fc段构像及与SpA或SpG的结合特点,采用不同亚类小鼠IgG对SpA和SpG单结构域构成的随机组合噬菌体展示文库进行亲和筛选。方法不同亚类小鼠IgG对噬菌体文库进行亲和筛选,获得具有结合优势的单结构域排列组合,噬菌体ELISA法来比较其与不同亚类小鼠IgG的结合特性,优势组合分子经原核表达蛋白,HRP标记后,ELISA法进一步来比较其与小鼠不同亚类IgG的结合特性。结果小鼠IgG1、IgG2a、IgG2b和IgG3分别经过5、4、5和5轮亲和筛选后,其文库的展示片段大小均为2个domain,表明进化完全。测序分析显示：小鼠 IgG1、IgG2a、IgG2b和IgG3筛选得到的具有结合优势组合分子分别是D-D、D-D、A-C和D-C。噬菌体 ELISA验证结合优势组合分子对小鼠不同亚类IgG的结合能力；蛋白经HRP标记后的ELISA结果和筛选不完全一致,但其结合强弱具有一致性,均为：IgG3>IgG2a>IgG2b>IgG1。结论得到3种不存在于天然SpA、SpG分子中,分别与不同亚类小鼠IgG具有较强结合作用的新型组合分子D-D、A-C和D-C,为进一步研究IgG Fc段构像及与SpA或SpG的结合特点提供了新的参考分子。
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