摘要
目的 :在大肠杆菌表达人lrg ,制备鉴定人Lrg免疫血清 .方法 :构建pcTAT lrg重组表达质粒 ,并在大肠杆菌中诱导表达相应的融合蛋白 ;对表达的融合蛋白采用镍柱和SDS PAGE纯化 ;以纯化蛋白作为免疫抗原 ,对新西兰白兔实施多次免疫 (间隔时间分别为 1mo,3wk ,2wk) .结果 :获得兔抗人Lrg免疫血清 .经过ELISA和WesternBlot检测 ,效价在 1∶1 0 0 0以上 .真核细胞实验表明 ,6His TAT Lrg蛋白具有良好的穿透性 ,可以在 30min内从培养液进入细胞质 ;免疫组化实验表明Lrg是一种胞质蛋白 ;兔抗人Lrg免疫血清可有效应用于细胞和组织的检测 .结论 :制备得到兔抗人Lrg免疫血清 。
AIM: To express human lrg in E.coli and to obtain an anti-Lrg immune serum. METHODS: lrg cDNA was cloned into pcTAT vector. The fusion proteins were expressed in E.coli and purified by Ni-NTA column and SDS-PAGE. Rabbits were immunized with preliminary purified Lrg protein and reinforced three times. The antiserum was collected. RESULTS: Antiserum against Lrg was obtained in immunized rabbits and its titer was more than 1∶1000 proven by ELISA and Western Blot. The purified Lrg protein could get across membranes of Papilla Dental Cells within 30 min. Immunohistochemical test with this antiserum showed that Lrg was a cytoplasmic protein and rabbit antiserum against human Lrg protein could be used in eukaryotic cell and normal tissues. CONCLUSION: The immune serum specific against Lrg is obtained, which can be used in normal tissues and cells.
出处
《第四军医大学学报》
北大核心
2004年第16期1456-1459,共4页
Journal of the Fourth Military Medical University
基金
国家自然科学基金资助项目 (30 1 70 361 )
