摘要
目的建立屋尘螨主要变应原Derp1蛋白原核表达产物的纯化方法,获得理想的表达产物并鉴定其免疫原性。方法大量诱导表达含pET24a-Derp1质粒的BL21工程菌,表达产物以重组蛋白包涵体的形式存在,包涵体经洗涤与溶解后,使用结合6组氨酸的镍柱进行亲和层析纯化蛋白质,用稀释复性方法进行重组蛋白的复性,再用屋尘螨过敏性哮喘患者阳性血清经Dot-ELISA方法分析Derp1纯化蛋白的抗原活性。结果分步洗涤可有效去除重组蛋白包涵体沉淀中混杂的多数杂蛋白成分,亲和层析分离可获得高纯度重组变应原Derp1蛋白。Dot-ELISA法检测结果表明经复性并纯化的Derp1蛋白呈强阳性反应,最佳血清稀释度为1∶64,而重组蛋白与正常人血清呈阴性反应。结论纯化后的融合蛋白Derp1具有较高的纯度及较强的免疫活性,可望作为有效的屋尘螨变应原诊断试剂和疫苗的候选分子。
Objective To establish a purification procedure for the recombinant mitoehondfia-relatd protein of Dermatophagoides pteronyssinus expressed in Escherichia coll. Method After centrifugation of the bacterial culture, the bacterial pellet was resuspended and lysed by freezing-thawing treatment and ultrasonieation. The inclusion bodies were purified by Gel fdtration ehromatography. Dot-ELISA was used to detect the antibody titer of allergic diseases sets. Result The purity of the fusion protein Der p 1 was verified by using SDS-PAGE and it could reset with the serum of the patients with allergic diseases. Dot-ELISA result showed that the antibody titer in the serum was 1：64. Conclusion The purified fusion protein Der p 1 with high purity and good immunological activity could be used for diagnosis and vaccine development.
出处
《热带医学杂志》
CAS
2006年第6期656-659,共4页
Journal of Tropical Medicine
基金
国家八六三计划(No.2002AA214011)
国家自然科学基金(No.30271226
30471505)
广东省科技计划重大项目(No.2003C104019)
广州市科技计划重点项目(No.2002Z2-E4021)
深圳市科技计划(No.200218)
关键词
屋尘螨
变应原
Der
P
1
融合蛋白
复性
纯化
Dermatophagoides pteronyssinus
allergen
Der p 1
fusion protein
renaturation
purification
