【摘要】 目的使用人免疫球蛋白G(IgG)对金黄色葡萄球菌蛋白A(SPA)单结构域突变体组合文库进行体外分子进化筛选,判断不同突变体组合的特异结合优势,探索优势组合分子结构与功能间的关系。方法通过Overlap PCR获得SPA中A和C单结构域突变体片段组合构成的噬菌体文库,再使用人IgG对文库进行体外亲和筛选,期望通过对特定结合分子的定向改造及体外进化获得具有高结合优势的组合分子。结果成功构建符合体外筛选要求的SPA单结构域A在29、30位氨基酸定点突变文库(A1)、SPA单结构域C在36、37位定点突变文库(C1)和SPA单结构域A在37位后插入3个氨基酸随机肽(AI37)、SPA单结构域C在20位后插入3个随机连接肽(CI20),将A1、C1随机组合构建的噬菌体展示文库命名为库A1C1,将AI37、CI20随机组合构建的噬菌体展示文库命名为库AI37CI20。两个文库各自经过4、5轮亲和筛选,文库进化完全。Phage-ELISA高光密度值的单克隆,测序结果分析为A(Q29K30)A(I29I30)和AI-TQSA。结论通过定向改造技术获得了高结合能力的定点突变分子A(Q29K30)A(I29I30)和插入突变分子AI37-TQSA,为Ig结合分子的定向改造及具有新的Ig结合特性的重组Ig结合分子的进一步研究奠定了基础。更多还原

【Abstract】 Objective Using human Ig G direct evolutional selection of a combinatorial phage library displaying randomly-rearranged mutant binding domains of SPA,judging the advantages of the specific combination of different mutations and exploring the relationship between the structure and the function. Methods With using overlap PCR,A and C single structure domain mutant fragments in the protein A( SPA) staphylococcus could be obtained.And then used human Ig G to affinity screening the phage library,expecting to obtain the integrate molecule with a high combination advantage,through directional transform the particular combinational molecular and evolving it in vitro. Results Two libraries could meet the requirement for the in vitro molecular evolution. SPA single domain structure A in 29,30 amino acids site-directed mutation was library( A1),structure C in 36,37 site-directed mutation was library( C1),SPA single domain structure A insert 3 amino acids after 37 random peptide was library( AI37),SPA single domain structure C after 20 insert 3 random peptide was library( CI20). The phage display library which was made up of a random combination of A1 and C1 named "A1C1". And the phage display library which was made up of a random combination of AI37 and CI20named "AI37CI20". The capacity of these two libraries were respectively: 3. 0 × 106 and 2. 0 × 106. The titers were respectively: 2. 3 × 1012 and 2. 1 × 1012( TU/ml).These two libraries had evolved completely through four or five times of affinity screening. Besides,Phage-Elisa monoclonal obtained a monoclonal with high OD,and the testing analytical result was A( Q29K30)A( I29I30)and AI37-TQS A. Conclusion A fixed point mutation molecules A( Q29K30)A( I29I30)and insertion mutation molecular AI37-TQSA are obtained with the high combining ability. The study shows the relationship of the structure and function of Ig-binding molecules and lays a foundation for directed improvement of Ig-binding molecules and acquirement of new Igbinding molecules with new Ig-binding characteristics.更多还原