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目的应用噬菌体展示免疫球蛋白(Ig)结合分子组合文库,进化筛选获得与兔IgG结合的代表性序列,测定其与兔IgG及人IgG结合活性的差异,以判断筛选获得的代表性序列对不同种属Ig的结合是否存在倾向性的差别。方法应用包含Ig效应子结合蛋白Protein A的D结构域(PA-D)、A结构域(PA-A),Protein G的B2结构域(PG-B2)及protein L的B3结构域(PL-B3)的单结构域随机组合文库,以兔IgG对该组合文库进行进化筛选,序列比较分析筛选获得的代表性序列与人IgG进化筛选文库所得代表性序列的异同性;用ELISA和竞争抑制试验分别测定阳性噬菌体克隆与兔IgG及人IgG的结合活性。结果以兔IgG经4轮分子进化筛选,获得在天然细菌蛋白中不存在的新的结构形式PA-A—PA-A,该结果与人IgG进化筛选所得代表性序列PA-A—PG有显著差异;兔IgG筛选序列PA-A—PA-A和人IgG筛选序列PA-A—PG分别显示出与兔Ig和人Ig结合力更强的特性。结论该研究首次应用噬菌体展示技术研究不同种属抗体效应子构象的不同,证实兔IgG和人IgG两者效应子构象之间确实存在差异。这一结论有助于阐明抗体效应子构象在不同种属之间的差异性。
OBJECTIVE: To use phage display immunoglobulin (Ig) binding molecule combinatorial library to obtain the representative sequence of binding to rabbit IgG by evolution screening and determine the binding activity to rabbit IgG and human IgG, so as to judge whether the representative sequences obtained by screening are different There is a predisposition to the binding of species Ig. METHODS: The D domain (PA-D), the A domain (PA-A), the B2 domain of Protein G (PG-B2) and the B3 domain of protein L (PL- B3) single-domain random combinatorial library, and the combinatorial library was screened by rabbit IgG. The similarity and similarity of the representative sequences obtained from screening and comparison with the representative sequences obtained from the human IgG evolution screening library were compared and analyzed by ELISA and competitive inhibition test The binding activity of the positive phage clones to rabbit IgG and human IgG was measured respectively. Results A new structural form PA-A-PA-A, which was not found in native bacterial proteins, was obtained by four rounds of molecular evolutionary screening using rabbit IgG. This result was significantly different from PA-A-PG, a representative sequence obtained from human IgG evolutionary screening PA-A-PA-A and PA-A-PG of rabbit IgG screening sequence showed the stronger binding ability to rabbit Ig and human Ig, respectively. Conclusions This study, for the first time, uses phage display technology to study the differences in the effector conformations of different species of antibodies, demonstrating that there is indeed a difference between the effector conformations of rabbit IgG and human IgG. This conclusion helps to elucidate the differences between antibody species in different species.