CAJ | 학술논문

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AI37 CI20. 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-TQS A. 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-TQS A,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.
목적 사용인면역구단백G( IgG)대금황색포도구균단백A( SPA)단결구역돌변체조합문고진행체외분자진화사선,판단불동돌변체조합적특이결합우세,탐색우세조합분자결구여공능간적관계. 방법 통과 Overlap PCR획득SPA중A화C단결구역돌변체편단조합구성적서균체문고,재사용인IgG대문고진행체외친화사선,기망통과대특정결합분자적정향개조급체외진화획득구유고결합우세적조합분자. 결과 성공구건부합체외사선요구적SPA단결구역A재29、30위안기산정점돌변문고( A1 )、SPA단결구역C재36、37위정점돌변문고( C1 )화SPA단결구역A재37위후삽입3개안기산수궤태( AI37 )、SPA단결구역C재20위후삽입3개수궤련접태( CI20 ) ,장A1、C1수궤조합구건적서균체전시문고명명위고A1C1,장AI37、CI20수궤조합구건적서균체전시문고명명위고AI37 CI20. 량개문고각자경과4、5륜친화사선,문고진화완전.Phage-ELISA고광밀도치적단극륭,측서결과분석위A(Q29K30) A(I29I30)화AI-TQS A. 결론 통과정향개조기술획득료고결합능력적정점돌변분자A(Q29K30) A(I29I30)화삽입돌변분자AI37-TQS A,위Ig결합분자적정향개조급구유신적Ig결합특성적중조Ig결합분자적진일보연구전정료기출.
Objective Using human IgG direct evolutional selection of a combinatorial phage library displaying ran-domly-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 IgG 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 mu-tation 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 li-brary which was made up of a random combination of A1 and C1 named "A1 C1". And the phage display library which was made up of a random combination of AI37 and CI20 named"AI37 CI20". 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-TQS A are obtained with the high combining ability. The study shows the relationship of the structure and function of Ig-bind-ing molecules and lays a foundation for directed improvement of Ig-binding molecules and acquirement of new Ig-binding molecules with new Ig-binding characteristics.