pET28a-Streptag I-SbpA-Ag85B重组质粒的表达与鉴定

目的 应用分子克隆技术获得纳米级Ag85B蛋白并对其进行鉴定和免疫学特性检测.方法 利用S-layer/Streptag I这一具有自我组装能力的纳米模式嵌合结核杆菌分泌的Ag85B蛋白基因,将其克隆到原核表达载体pET28a（＋）中,并在大肠杆菌中表达Ag85B蛋白,表达后的蛋白经SDS-PAGE和Western blotting鉴定.结果 原核表达重组质粒pET28a-Streptag I-SbpA-Ag85B成功构建,并可在大肠杆菌中诱导表达,得到的Streptag I-Sbpa-Ag85B蛋白经SDS-PAGE和Western blotting鉴定正确,重组蛋白能被结核患者血清识别.结论 重组后的Ag85B蛋白具有较强的免疫反应性.所建立的原核表达体系为新型结核疫苗研制提供了一个新的思路.

The expression and identification of recombinant pET28a-Streptag I-SbpA-Ag85B Plasmid

Objective To obtain nanometer Ag85B protein by the molecular cloning techniques and identify its immunological characteristics.Methods The gene sllB coding surface layer protein （S-layer）was truncated and linked with the Mycobacterium tuberculosis secreted protein Ag85B gene.The expression plasmid pET28a-Streptag I-SbpA-Ag85B was transformed into E.coli,the protein was identified by SDS-PAGE and Western blotting.Results The expression system was developed successfully for recombinant nanometer-scale antigen and the fusion protein was identified by SDS-PAGE with correct molecular weight and can be recognized by the serum of tuberculosis patients.Conclusion The recombinant Ag85 B protein has strong immune reaction and small size on nanometer level,this expression system provides a new way for the tuberculosis vaccine design.