中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
2008年
4期
255-259
,共5页
冯木林%陶爱林%邹泽红%刘绮明%伍秋容%赖荷
馮木林%陶愛林%鄒澤紅%劉綺明%伍鞦容%賴荷
풍목림%도애림%추택홍%류기명%오추용%뢰하
铜绿假单胞菌外毒素A%基因克隆%三级结构%抗原性%氨基酸置换%聚合酶链反应
銅綠假單胞菌外毒素A%基因剋隆%三級結構%抗原性%氨基痠置換%聚閤酶鏈反應
동록가단포균외독소A%기인극륭%삼급결구%항원성%안기산치환%취합매련반응
Psendomonas aernginosa exotoxin A%Gene cloning%Tertiary structure%Allergenicity%Amino acid substitution%Polymerase chain reaction
目的 克隆铜绿假单胞菌外毒素A(PEA)基因及分析不同克隆序列中不同性质氨基酸对抗原性及三级结构的贡献.方法 以铜绿假单胞菌基因组DNA为模板,以PEA编码基因特异区段为引物,采用Touchdown PCR克隆PEA编码基因.阳性克隆经菌落PCR鉴定后进行DNA测序及Clustal X(1.83)比对分析;采用生物信息学在线软件BIMAS及SWISS-Model对所得基因编码蛋白进行抗原性评估及三级结构模拟.人为置换关键部位的氨基酸残基,考察不同性质氨基酸对三级结构的影响.结果 从铜绿假单胞菌DNA中获得3个克隆.抗原性分析及三级结构模拟结果显示,3个克隆与公开发表的序列之间存在少数氨基酸的差异;个别区段上氨基酸的改变可引起相应位点抗原性的改变,但仅有个别位点的改变对空间结构产生影响.结论 基于抗原性评估及三级结构分析,获得了不同性质氨基酸对抗原性及三级结构的贡献等信息,为PEA编码基因用于弱化抗原性靶向性毒素的构建奠定了基础.
目的 剋隆銅綠假單胞菌外毒素A(PEA)基因及分析不同剋隆序列中不同性質氨基痠對抗原性及三級結構的貢獻.方法 以銅綠假單胞菌基因組DNA為模闆,以PEA編碼基因特異區段為引物,採用Touchdown PCR剋隆PEA編碼基因.暘性剋隆經菌落PCR鑒定後進行DNA測序及Clustal X(1.83)比對分析;採用生物信息學在線軟件BIMAS及SWISS-Model對所得基因編碼蛋白進行抗原性評估及三級結構模擬.人為置換關鍵部位的氨基痠殘基,攷察不同性質氨基痠對三級結構的影響.結果 從銅綠假單胞菌DNA中穫得3箇剋隆.抗原性分析及三級結構模擬結果顯示,3箇剋隆與公開髮錶的序列之間存在少數氨基痠的差異;箇彆區段上氨基痠的改變可引起相應位點抗原性的改變,但僅有箇彆位點的改變對空間結構產生影響.結論 基于抗原性評估及三級結構分析,穫得瞭不同性質氨基痠對抗原性及三級結構的貢獻等信息,為PEA編碼基因用于弱化抗原性靶嚮性毒素的構建奠定瞭基礎.
목적 극륭동록가단포균외독소A(PEA)기인급분석불동극륭서렬중불동성질안기산대항원성급삼급결구적공헌.방법 이동록가단포균기인조DNA위모판,이PEA편마기인특이구단위인물,채용Touchdown PCR극륭PEA편마기인.양성극륭경균락PCR감정후진행DNA측서급Clustal X(1.83)비대분석;채용생물신식학재선연건BIMAS급SWISS-Model대소득기인편마단백진행항원성평고급삼급결구모의.인위치환관건부위적안기산잔기,고찰불동성질안기산대삼급결구적영향.결과 종동록가단포균DNA중획득3개극륭.항원성분석급삼급결구모의결과현시,3개극륭여공개발표적서렬지간존재소수안기산적차이;개별구단상안기산적개변가인기상응위점항원성적개변,단부유개별위점적개변대공간결구산생영향.결론 기우항원성평고급삼급결구분석,획득료불동성질안기산대항원성급삼급결구적공헌등신식,위PEA편마기인용우약화항원성파향성독소적구건전정료기출.
Objective To clone the coding sequence of Pseudomonas aeruginosa exotoxin A (PEA) and analyze the contributions of different amino acids to its antigenicity and tertiary structure. Methods Genomic DNA extracted from Pseudomonas aeruginosa ATCC27853 was used as template, and the specific coding region of PEA was used as primer. Touchdown PCR was applied to clone the coding region of immunotoxin-related PEA fragment. The amplified fragment recovered was subcloned into the pGEM-T vector. Positive clones were validated and selected by colony PCR, followed by further confirmation by DNA sequencing and Clustal X (1.83) alignment. The antigenicity and tertiary structure of the encoded peptide was analyzed by bioinformatic softwares BIMAS and SWISS-Model. Amino acid residue of key locus was replaced so as to investigate the effect of different amino acid for tertiary structure. Results Three clones, referred to as PE117, PE215 and PE217, were acquired. Sequence and structure analysis demonstrated that few amino acid sequence disparity existed among the three clones and the sequence registered in GenBank (the accession number is P11439), and thus affecting the antigenicity in the corresponding position. However, slight difference was hardly observed in their tertiary structures. Conclusion Based on sequence analysis, antigenicity evaluation and tertiary structural modeling, some useful information is obtained for antigenicity evaluation, thus would facilitate allergen genetic improvement by amino acid replacement in PEA.
