中华实验和临床病毒学杂志
中華實驗和臨床病毒學雜誌
중화실험화림상병독학잡지
CHINESE JOURNAL OF EXPERIMENTAL AND CLINICAL VIROLOGY
2009年
2期
146-148
,共3页
陈操%王桂荣%石琦%张宝云%梅国勇%李媛%王新%周瑞敏%韩俊%赵玉军%董小平
陳操%王桂榮%石琦%張寶雲%梅國勇%李媛%王新%週瑞敏%韓俊%趙玉軍%董小平
진조%왕계영%석기%장보운%매국용%리원%왕신%주서민%한준%조옥군%동소평
蛋白质类%外显子%抗体,病毒%朊病毒病
蛋白質類%外顯子%抗體,病毒%朊病毒病
단백질류%외현자%항체,병독%원병독병
Proteins%Exons%Antibodies,viral%Prion diseases
目的 制备人微管相关蛋白tau N端的外显子2和外显子3特异性多克隆抗体.方法 从人外周血DNA中获得tau蛋白外显子2及外显子3序列并连接至原核表达载体pGEX-2T,表达纯化重组融合蛋白GST-E2、GST-E3;以此融合蛋白免疫家兔及小鼠制备抗血清,通过ProteinG/A、偶联GST蛋白的溴化氰(CNBr)活化柱对抗血清进行亲和纯化;用Western Blot及ELISA方法确定所制备抗体的特异性和敏感性.结果 在大肠埃希菌中表达纯化出人tau外显子2和外显子3重组融合蛋白GST-E2和GST-E3,相对分子质量为29×103.免疫实验动物后获得抗血清,经系列纯化后Western Blot和ELISA检测显示,制备的抗人tau蛋白外显子2和外显子3抗体具有良好的特异性和免疫反应效价.结论 成功制备了4种人tau外显子2和外显子3特异性抗体,为进行tau蛋白在神经退行性疾病中的作用研究提供了基础.
目的 製備人微管相關蛋白tau N耑的外顯子2和外顯子3特異性多剋隆抗體.方法 從人外週血DNA中穫得tau蛋白外顯子2及外顯子3序列併連接至原覈錶達載體pGEX-2T,錶達純化重組融閤蛋白GST-E2、GST-E3;以此融閤蛋白免疫傢兔及小鼠製備抗血清,通過ProteinG/A、偶聯GST蛋白的溴化氰(CNBr)活化柱對抗血清進行親和純化;用Western Blot及ELISA方法確定所製備抗體的特異性和敏感性.結果 在大腸埃希菌中錶達純化齣人tau外顯子2和外顯子3重組融閤蛋白GST-E2和GST-E3,相對分子質量為29×103.免疫實驗動物後穫得抗血清,經繫列純化後Western Blot和ELISA檢測顯示,製備的抗人tau蛋白外顯子2和外顯子3抗體具有良好的特異性和免疫反應效價.結論 成功製備瞭4種人tau外顯子2和外顯子3特異性抗體,為進行tau蛋白在神經退行性疾病中的作用研究提供瞭基礎.
목적 제비인미관상관단백tau N단적외현자2화외현자3특이성다극륭항체.방법 종인외주혈DNA중획득tau단백외현자2급외현자3서렬병련접지원핵표체재체pGEX-2T,표체순화중조융합단백GST-E2、GST-E3;이차융합단백면역가토급소서제비항혈청,통과ProteinG/A、우련GST단백적추화청(CNBr)활화주대항혈청진행친화순화;용Western Blot급ELISA방법학정소제비항체적특이성화민감성.결과 재대장애희균중표체순화출인tau외현자2화외현자3중조융합단백GST-E2화GST-E3,상대분자질량위29×103.면역실험동물후획득항혈청,경계렬순화후Western Blot화ELISA검측현시,제비적항인tau단백외현자2화외현자3항체구유량호적특이성화면역반응효개.결론 성공제비료4충인tau외현자2화외현자3특이성항체,위진행tau단백재신경퇴행성질병중적작용연구제공료기출.
Objective To prepare the specific antibodies against exon 2 and exon 3 of human tan protein. Methods Sequences encoding exon 2 and exon 3 of human tau protein were amplified from human peripheral blood DNA and cloned into a prokaryotic expression vector pGEX-2T. Fusion proteins GST-E2 and GST-E3 were expressed and purified from E. coli system. The antisera were elicited by immunization of the purified fusion proteins to rabbits and mice. The specific antibodies were purified by Protein G/A and CNBr-aetivated sepharose 4B coupled with GST protein. The specificity and sensitivity of the purified antibodies were evaluated by Western blotting and ELISA.Results Recombinant fusion proteins GST-E2 and GST-E3 were expressed and purified from E. coli, which showed Mr. 29 x 103 . Various antisera were collected from the immunized experimental animals. Reliable immunoreactive specificity and titers of the purified antibodies against exon 2 and exon 3 of human tau protein were confirmed by Western blotting and ELISA after serial purification processes. Conclusion Four specific antibodies against exon 2 and exon 3 of human tau protein have been successfully prepared, which provides basis for analyzing the role of tau in neurodegenerative diseases.
