自身抗原核点蛋白Sp100基因克隆和真核表达

目的:克隆人核点蛋白自身抗原Sp100基因, 构建重组表达质粒,获得具有免疫学活性的纯化重组蛋白．方法:从人类肝脏cDNA文库中扩增出Sp100 的基因片段,克隆至PEGH表达载体进行诱导表达,并对表达产物进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(Western blot)鉴定．结果:经重组质粒测序结果证实,Sp100目的基因已正确插入真核表达载体中,基因序列正确,符合表达框架;SDS-PAGE检测表达产物分别在53 ku,55 ku,52 ku,37 ku,42 ku,47 ku 处有一明显的蛋白表达条带,Western blot分析表明重组蛋白2,3,5,6具有人sp100抗原反应性．结论:本研究成功克隆人核点蛋白自身抗原 Sp100基因,并将其在酵母菌中成功表达．

Gene cloning and fusion expression of human autoantigen Sp100 in yeasts

AIM: To clone and construct the plasmid containing human autoantigene Sp100 gene, and then to identify the immunoreactivity of the purified recombinant protein. METHODS: The Sp100 gene was amplified from human liver cDNA library, and then was cloned into PEGH vector to induce the Sp100 expression. The obtained products were identified and analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. RESULTS: The sequence of Sp100 autoantigene gene was confirmed to be the same as the sequence reported in GenBank. The fusion proteins were found at 53-, 55-, 52-, 37-, 42-, and 47-ku strip on SDS-PAGE gel. Western blot analysis showed that the fusion protein with 55, 52, 42 and 47 ku had the same immunoreactivity as human Sp100 autoantigene. CONCLUSION: Human plasmid containing SplOO gene is successfully cloned and expressed in yeast Y258.