CAJ | 학술논문

目的从噬菌体随机肽库中筛选人Rh(D)血型抗原模拟表位,并对其免疫学活性进行鉴定.方法以抗Rh(D)单克隆抗体作为固相筛选分子,对噬菌体随机十二肽库进行3轮"吸附-洗脱-扩增"筛选过程,随机挑选35个克隆,经噬菌体ELISA和交叉反应试验,确定阳性克隆,再对这些克隆进行DNA序列测定和抗体竞争抑制试验,以获取人Rh(D)血型抗原模拟表位.然后采用蛋白质免疫印迹法(Western blot)对所获噬菌体进行鉴定和抗原性分析.结果经过3轮淘洗后,噬菌体得到富集,获得11个阳性克隆.DNA序列测定和竞争抑制实验结果表明,这11个克隆噬菌体所展示的外源多肽中有7个克隆氨基酸序列含有相同的色氨酸(W)、脯氨酸(P)和谷氨酰胺(Q)结构(-WP-Q-),且均具有40%以上的抑制率;其余4个克隆没有共性,抑制率较低,可能为非特异性结合.Western blot分析表明,被选定的噬菌体能被抗Rh(D)血清特异识别,具有类似于Rh(D)蛋白的抗原性.结论利用抗Rh(D)单克隆抗体筛选噬菌体随机肽库,成功获得含有(-WP-Q-)结构的Rh(D)抗原模拟表位,为进一步探讨Rh(D)新生儿溶血病的发病机制和疫苗的研究奠定基础.
목적 종서균체수궤태고중사선인Rh(D)혈형항원모의표위,병대기면역학활성진행감정.방법 이항Rh(D)단극륭항체작위고상사선분자,대서균체수궤십이태고진행3륜"흡부-세탈-확증"사선과정,수궤도선35개극륭,경서균체ELISA화교차반응시험,학정양성극륭,재대저사극륭진행DNA서렬측정화항체경쟁억제시험,이획취인Rh(D)혈형항원모의표위.연후채용단백질면역인적법(Western blot)대소획서균체진행감정화항원성분석.결과 경과3륜도세후,서균체득도부집,획득11개양성극륭.DNA서렬측정화경쟁억제실험결과표명,저11개극륭서균체소전시적외원다태중유7개극륭안기산서렬함유상동적색안산(W)、포안산(P)화곡안선알(Q)결구(-WP-Q-),차균구유40%이상적억제솔;기여4개극륭몰유공성,억제솔교저,가능위비특이성결합.Western blot분석표명,피선정적서균체능피항Rh(D)혈청특이식별,구유유사우Rh(D)단백적항원성.결론 이용항Rh(D)단극륭항체사선서균체수궤태고,성공획득함유(-WP-Q-)결구적Rh(D)항원모의표위,위진일보탐토Rh(D)신생인용혈병적발병궤제화역묘적연구전정기출.
Objective To screen the mimic epitopes of Rh(D)blood group antigens and identify their immunity from phage display peptide library.Methods A twelve mer phage peptide library was biopanned with anti-Rh(D)monoclonal antibody immobilized on plastic surface.After three round panning,thirty-five clones were randomly selected and positive clones were identified by ELISA and cross-reaction,followed by antibody competition inhibition assay and DNA sequencing to obtain the mimic epitopes of Rh (D)blood type antigens.The target phage clones were characterized and the antigenicity was analyzed by Western blot.Results After the third round screening,phages were enriched,and eleven positive clones were obtained.According to sequencing and competition inhibition analysis,the same"-WP-Q-"structure existed in seven of the eleven clones,and they had more than 40%inhibition ratio.The other clones had no same characteristics with low inhibition ratio possibly due to non-specific binding.Western blot analysis indicated that these phage clones could be specifically recognized by the anti-Rh(D)serum and they shared the same antigenicity of Rh(D)protein.Conclusions Rh(D)mimotope of"-WP-Q-"structure is successfully obtained by phage peptide library screening with anti-Rh(D)monoelonal antibody.The results lay the foundation for further exploration of pathogenesis and vaccine development of Rh(D)hemolytic diseases of newborn.