安徽医科大学学报
安徽醫科大學學報
안휘의과대학학보
ACTA UNIVERSITY MEDICINALIS ANHUI
2014年
9期
1227-1232,1233
,共7页
徐文竹%丁莹莹%吴莉莉%张婧%冯娇娇%王锦红%潘卫%邓松华
徐文竹%丁瑩瑩%吳莉莉%張婧%馮嬌嬌%王錦紅%潘衛%鄧鬆華
서문죽%정형형%오리리%장청%풍교교%왕금홍%반위%산송화
噬菌体文库%定向进化%亚类%新型免疫球蛋白结合分子
噬菌體文庫%定嚮進化%亞類%新型免疫毬蛋白結閤分子
서균체문고%정향진화%아류%신형면역구단백결합분자
phage library%directed molecular evolution%subclass%NEIBM
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。
目的判斷不同亞類小鼠IgG Fc段構像及與SpA或SpG的結閤特點,採用不同亞類小鼠IgG對SpA和SpG單結構域構成的隨機組閤噬菌體展示文庫進行親和篩選。方法不同亞類小鼠IgG對噬菌體文庫進行親和篩選,穫得具有結閤優勢的單結構域排列組閤,噬菌體ELISA法來比較其與不同亞類小鼠IgG的結閤特性,優勢組閤分子經原覈錶達蛋白,HRP標記後,ELISA法進一步來比較其與小鼠不同亞類IgG的結閤特性。結果小鼠IgG1、IgG2a、IgG2b和IgG3分彆經過5、4、5和5輪親和篩選後,其文庫的展示片段大小均為2箇domain,錶明進化完全。測序分析顯示：小鼠 IgG1、IgG2a、IgG2b和IgG3篩選得到的具有結閤優勢組閤分子分彆是D-D、D-D、A-C和D-C。噬菌體 ELISA驗證結閤優勢組閤分子對小鼠不同亞類IgG的結閤能力；蛋白經HRP標記後的ELISA結果和篩選不完全一緻,但其結閤彊弱具有一緻性,均為：IgG3>IgG2a>IgG2b>IgG1。結論得到3種不存在于天然SpA、SpG分子中,分彆與不同亞類小鼠IgG具有較彊結閤作用的新型組閤分子D-D、A-C和D-C,為進一步研究IgG Fc段構像及與SpA或SpG的結閤特點提供瞭新的參攷分子。
목적판단불동아류소서IgG Fc단구상급여SpA혹SpG적결합특점,채용불동아류소서IgG대SpA화SpG단결구역구성적수궤조합서균체전시문고진행친화사선。방법불동아류소서IgG대서균체문고진행친화사선,획득구유결합우세적단결구역배렬조합,서균체ELISA법래비교기여불동아류소서IgG적결합특성,우세조합분자경원핵표체단백,HRP표기후,ELISA법진일보래비교기여소서불동아류IgG적결합특성。결과소서IgG1、IgG2a、IgG2b화IgG3분별경과5、4、5화5륜친화사선후,기문고적전시편단대소균위2개domain,표명진화완전。측서분석현시：소서 IgG1、IgG2a、IgG2b화IgG3사선득도적구유결합우세조합분자분별시D-D、D-D、A-C화D-C。서균체 ELISA험증결합우세조합분자대소서불동아류IgG적결합능력；단백경HRP표기후적ELISA결과화사선불완전일치,단기결합강약구유일치성,균위：IgG3>IgG2a>IgG2b>IgG1。결론득도3충불존재우천연SpA、SpG분자중,분별여불동아류소서IgG구유교강결합작용적신형조합분자D-D、A-C화D-C,위진일보연구IgG Fc단구상급여SpA혹SpG적결합특점제공료신적삼고분자。
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.
