自身免疫性肝炎新型抗原分子高迁移率族蛋白B1的克隆表达鉴定及蛋白纯化

目的获得高纯度的人高迁移率族蛋白B1(high mobility group box-1 protein,HMGB1),为制备单克隆抗体及研究其与自身免疫性肝炎(autoimmune hepatitis,AIH)的关系打基础。方法应用PCR从乳腺文库中扩增得到HMGB1基因片段,并将其克隆到pET28a( )载体上,对获得的pET28a( )-HMGB1重组质粒扩增后进行PCR、酶切和测序鉴定。将鉴定完全正确的质粒转化大肠杆菌BL21(DE3),IPTG诱导重组蛋白表达,表达产物经SDS-PAGE电泳分析和Western blotting鉴定。最后进行大量诱导并纯化,得到带His标签的HMGB1蛋白。结果成功构建pET28a( )-HMGB1重组质粒,并经诱导表达,得到了纯化的His-HMGB1蛋白。结论构建pET28a( )-HMGB1重组质粒并得到其表达蛋白,为今后深入研究HMGB1在AIH中的作用机理提供有力的实验工具。

Construction, Expression and Purification of His- HMGB1 and Novel Antigen in Autoimmune Hepatitis

Objective To obtain purified human high mobility group box-1 protein (HMGB1) used in further research of the pathophysiolgical role of HMGB1 in autoimmune hepatitis (AIH). Methods Full length HMGB1 cDNA cloned by PCR from mammary library was inserted into pET28a( ) vectors to construct pET28a( )-HMGB1 plasmid. After confirmation, the positive pET28a( )-HMGB1 recombinant plasmid was transformed into E.coli BL21(DE3), and the protein induced by IPTG was examined by Western blotting analysis. Finally, the recombinant His-HMGB1 protein was purified. Results pET28a( )-HMGB1 recombinant plasmid was constructed correctly, and purified His-HMGB1 protein was prepared. Conclusion Correct construction of pET28a( )-HMGB1 recombinant plasmid and purification of His-HMGB1 protein provide effective tools for further research of AIH.