中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2008年
4期
343-347
,共5页
林旭瑷%潘建平%罗依惠%毛亚飞%李立伟%严杰
林旭璦%潘建平%囉依惠%毛亞飛%李立偉%嚴傑
림욱애%반건평%라의혜%모아비%리립위%엄걸
问号钩端螺旋体%属特异性外膜蛋白%OmpL1/LipL21%抗原表位/预测%噬菌体展示/鉴定
問號鉤耑螺鏇體%屬特異性外膜蛋白%OmpL1/LipL21%抗原錶位/預測%噬菌體展示/鑒定
문호구단라선체%속특이성외막단백%OmpL1/LipL21%항원표위/예측%서균체전시/감정
Leptospira interrogans%Genus-specific envelope proteins%OmpL1/LipL21%Antigenicepitope/prediction%Phage display/identification
目的 筛选问号钩端螺旋体(简称钩体)属特异性外膜蛋白OmpL1和LipL21有效T和B细胞联合抗原表位,为研制多抗原肽(multiple antigenic peptide,MAP)疫苗提供基础.方法 采用生物信息学方法预测OmpL1和LipL21分子中T和B细胞联合抗原表位.采用PCR扩增候选联合抗原表位片段并分别构建其噬菌体展示系统.分别以rOmpL1或rLipL21、黄疸出血群赖株、钩体患者抗血清为一抗,采用Western blot检测各抗血清与目的表位的免疫反应性及其强度.结果 通过抗原表位预测,选择了高分值的4个OmpLl和2个LipL21联合表位.经扩增获得了预期的各抗原表位片段,各目的表位序列均准确插入噬菌体PⅢ蛋白N端并有效表达.各抗血清均能识别上述6个联合表位.其中LipL21的97～112和176-184表位对任一抗血清均显示相似强度的杂交条带.综合4个OmpL1表位对3种抗血清的不同Western blot结果及其实际意义,杂交信号从强到弱依次为173～191、87～98、297～320和59～78表位.结论 所研究的6个联合表位均分别为LipL21和OmpL1的有效抗原表位,其中LipL21的97～112、176～184和OmpL1的87～98、173～191表位可应用于钩体MAP疫苗研制.
目的 篩選問號鉤耑螺鏇體(簡稱鉤體)屬特異性外膜蛋白OmpL1和LipL21有效T和B細胞聯閤抗原錶位,為研製多抗原肽(multiple antigenic peptide,MAP)疫苗提供基礎.方法 採用生物信息學方法預測OmpL1和LipL21分子中T和B細胞聯閤抗原錶位.採用PCR擴增候選聯閤抗原錶位片段併分彆構建其噬菌體展示繫統.分彆以rOmpL1或rLipL21、黃疸齣血群賴株、鉤體患者抗血清為一抗,採用Western blot檢測各抗血清與目的錶位的免疫反應性及其彊度.結果 通過抗原錶位預測,選擇瞭高分值的4箇OmpLl和2箇LipL21聯閤錶位.經擴增穫得瞭預期的各抗原錶位片段,各目的錶位序列均準確插入噬菌體PⅢ蛋白N耑併有效錶達.各抗血清均能識彆上述6箇聯閤錶位.其中LipL21的97～112和176-184錶位對任一抗血清均顯示相似彊度的雜交條帶.綜閤4箇OmpL1錶位對3種抗血清的不同Western blot結果及其實際意義,雜交信號從彊到弱依次為173～191、87～98、297～320和59～78錶位.結論 所研究的6箇聯閤錶位均分彆為LipL21和OmpL1的有效抗原錶位,其中LipL21的97～112、176～184和OmpL1的87～98、173～191錶位可應用于鉤體MAP疫苗研製.
목적 사선문호구단라선체(간칭구체)속특이성외막단백OmpL1화LipL21유효T화B세포연합항원표위,위연제다항원태(multiple antigenic peptide,MAP)역묘제공기출.방법 채용생물신식학방법예측OmpL1화LipL21분자중T화B세포연합항원표위.채용PCR확증후선연합항원표위편단병분별구건기서균체전시계통.분별이rOmpL1혹rLipL21、황달출혈군뢰주、구체환자항혈청위일항,채용Western blot검측각항혈청여목적표위적면역반응성급기강도.결과 통과항원표위예측,선택료고분치적4개OmpLl화2개LipL21연합표위.경확증획득료예기적각항원표위편단,각목적표위서렬균준학삽입서균체PⅢ단백N단병유효표체.각항혈청균능식별상술6개연합표위.기중LipL21적97～112화176-184표위대임일항혈청균현시상사강도적잡교조대.종합4개OmpL1표위대3충항혈청적불동Western blot결과급기실제의의,잡교신호종강도약의차위173～191、87～98、297～320화59～78표위.결론 소연구적6개연합표위균분별위LipL21화OmpL1적유효항원표위,기중LipL21적97～112、176～184화OmpL1적87～98、173～191표위가응용우구체MAP역묘연제.
Objective To screen the efficient antigenic epitopes in genus-specific envelope proteins OmpL1 and LipL21 of Leptospira interrogans for further development of multiple antigenic peptide (MAP)vaccine.Methods Based on bioinformatic technique,the combined epitopes of T and B lymphcytes in OmpL1 and LipL21 molecules were screened.Nucleotide fragments of each epitopes were amplified by PCR and then constructed their phage display systems.Using antisera against rOmpL1,rLipL21,L.interrogans serogroup Icterohaemorrhagiae strain Lai and leptospirosis patients' sera as the first antibodies.respectively,Western blot assays were performed to determine the immunoreaetivity and reactive ability of the epitopes with different antisera.Resuits Four combined epitopes of OmpL1 and two combined epitopes of LipL21 were selected out by the predicting procedure.All the amplified epitope fragments were accurately inserted into the region at N end of phage PⅢ protein and successfully expressed.All of the antisera could recognize each of the epitopes.Based on the results of Western blot,the two LipL21 epitopes at 97-112 and 176-184 showed similar strong hybridization signals with any of the antisera,and the hybridization signals of four OmpL1 epitopes with the three antisera were 173-191,87-98,297-320 and 59-78,from strong to weak.Conclusion The six combined epitopes in this study are efficiently antigenic.And the epitopes at positions 97-112 and 176-184 in LipL21 as well as the epitopes at position 87-98 and 173-191 in OmpL1 have a potential for developing leptospiral MAP vaccine.
