CAJ | 학술논문

目的：预测转化生长因子-βⅡ型受体（T RβⅡ）胞外段蛋白与靶向适配子S58的结合位点，并在体外进行验证，证实S58的结构稳定性。方法通过适配子ssDNA序列建立其三维结构，在蛋白质数据库搜索受体蛋白 TRβⅡ胞外段的晶体结构，运用计算机辅助技术对两个分子进行对接实验，根据结果指导剪切优化适配子结构，分别用生物传感器技术及蛋白免疫印迹法验证其亲和力。结果核酸适配子ssDNA S58与 TRβⅡ胞外段蛋白结合的可信位点包括位点Ⅰ（T4、T5、G6、C7）、位点Ⅱ（G13、A14、T15、C16、G17、C18）、位点Ⅲ（T31、G32、T33、C34）及位点Ⅳ（G40、A41、T42、T43、T44、G45、G46）。S58与TRβⅡ胞外段蛋白的亲和力高，S58的α-平滑肌肌动蛋白（α-SMA）表达明显较DMEM 对照降低（P＜0．05），根据结果剪切适配子S58后得到优化后的ssDNA亲和力、α-SMA表达均不如S58。结论核酸适配子S58为受体蛋白TβRⅡ的高特异性分子，具有一定稳定性，任何结构的改变均会降低与TβRⅡ的亲和力。计算机辅助的分子对接技术成为一项探索分子间作用的重要手段，为医学基础研究提供了良好的理论依据。
목적：예측전화생장인자-βⅡ형수체（T RβⅡ）포외단단백여파향괄배자S58적결합위점，병재체외진행험증，증실S58적결구은정성。방법통과괄배자ssDNA서렬건립기삼유결구，재단백질수거고수색수체단백 TRβⅡ포외단적정체결구，운용계산궤보조기술대량개분자진행대접실험，근거결과지도전절우화괄배자결구，분별용생물전감기기술급단백면역인적법험증기친화력。결과핵산괄배자ssDNA S58여 TRβⅡ포외단단백결합적가신위점포괄위점Ⅰ（T4、T5、G6、C7）、위점Ⅱ（G13、A14、T15、C16、G17、C18）、위점Ⅲ（T31、G32、T33、C34）급위점Ⅳ（G40、A41、T42、T43、T44、G45、G46）。S58여TRβⅡ포외단단백적친화력고，S58적α-평활기기동단백（α-SMA）표체명현교DMEM 대조강저（P＜0．05），근거결과전절괄배자S58후득도우화후적ssDNA친화력、α-SMA표체균불여S58。결론핵산괄배자S58위수체단백TβRⅡ적고특이성분자，구유일정은정성，임하결구적개변균회강저여TβRⅡ적친화력。계산궤보조적분자대접기술성위일항탐색분자간작용적중요수단，위의학기출연구제공료량호적이론의거。
Objective To predict the binding sites of transforming growth factor-βreceptor Ⅱ (TβRⅡ ) ectodomain and the aptamer S58 specifically targeted TβRⅡ ,and to confirm the structure stability of the aptamer S 58 in vitro .Methods We created three-dimensional structure by utilizing ssDNA aptamer sequences ,the crystal structure of the TβRⅡ was searched by protein data bank database .According to the results of the molecular docking experiments on aptamer S 58 and TβRⅡ ectodomain ,we sheared the aptamer sequences ,then verified its affinity respectively by biosensor technology and Western blot .Results Binding sites of aptamers S58 and TRβⅡ ectodomain included site Ⅰ(T4 ,T5 ,G6 ,C7) ,site Ⅱ(G13 ,A14 ,T15 ,C16 ,G17 ,C18 ) ,site Ⅲ (T31 ,G32 , T33 ,C34) and site Ⅳ(G40 ,A41 ,T42 ,T43 ,T44 ,G45 ,G46) .We validated the high affinity between aptamer S58 and TRβⅡ ectodo-main .The expression of α-smooth muscle actin(α-SMA) protein in the human tenon′s capsule fibroblasts was descended obviously after the experiment of the aptamer S58 in comparing with the control of DMEM (P< 0 .05) .But the new ssDNA by shear the aptamer ssDNA S58 according to the results were poor than aptamers S58 .Conclusion The aptamer S58 targeted TβRⅡ was high-ly specific with a certain stability ,any changing of structure will reduce the affinity of TβRⅡ .Computer-aided molecular docking technology has become an important means of an exploratory intermolecular interaction ,and can provides a good theoretical basis on medical research .