可溶性h 2dbigg2b二聚体的构建与表达-construction and expression of soluble h 2 db igg 2b dimer.docxVIP

昆虫杆状病毒双表达载体 pFastBacTMDual 的 2 个启动子 Pp10 和 PpH 的下游，构建获 得 [H-2Db/IgG2b]+pFastBacTMDual+β2m 重组质粒，作为 H-2Db/IgG2b 二聚体的表达载 体。通过 PCR 及限制性内切酶酶切鉴定和测序，证实了重组质粒中 H-2Db/IgG2b 序列 及 β2m 编码基因与预期序列一致。2. 可溶性 H-2Db/IgG2b 二聚体的表达、纯化与鉴定将 [H-2Db/IgG2b]+pFastBacTMDual+β2m 重组质粒转化大肠杆菌 DH10Bac，得到重 组的穿梭质粒 Bacmid+[H-2Db/IgG2b]。应用脂质体转染方法将其转入昆虫细胞系 Sf9 中，收获上清得到成熟杆状病毒颗粒，再用此病毒颗粒大量感染 Sf9 细胞，收集富含 H-2Db/IgG2b 二聚体的病毒感染上清，经 SPA 亲和层析得到浓缩纯化的可溶性H-2Db/IgG2b 二聚体。利用 H-2Db 特异性的 TIB-126 抗体以及抗 IgG2b Fc 抗体进行双 抗夹心 ELISA 鉴定及流式检测，结果显示获得的可溶性 H-2Db/IgG2b 二聚体具有正确 的构象，Western blotting 鉴定结果显示 H-2Db/IgG2b 融合蛋白的分子量为 61kD，与预 期大小一致。关键词：H-2Db/IgG2b 二聚体；昆虫杆状病毒表达系统；亲和层析；EAEConstruction and expression of divalent H-2Db/IgG2b moleculeMaster candidate: Xiaolin Wu Supervisor: Prof. Xiongwen WuAbstractMultiple sclerosis (MS) is the commonest autoimmune disorder of the central nervous system (CNS). Studies of MS and experimental autoimmune encephalomyelitis (EAE), an animal model of MS, focus on the contribution of CD4+ myelin-specific T cells. However, CD8+ T cells specific for myelin antigens have been isolated from peripheral blood of MS patients, and infiltrating CD8+ T cells are present at higher frequency than CD4+ T cells in the CNS, particularly in active demyelinating lesions. Therefore, to invastigate the pathophysiologic mechanism of MS and EAE, studies have become fcous on the role of CD8+ T cells. Blocking autoreactive CD8+ T cells contributes to prevention the occuring and developingof EAE. Previous studies have shown that soluble peptide/major histocompitibility complex class (pMHC) dimer can be used to specific modulate the immune response by interacted with T cell receptors(TCR). In this study, we construct and express of divalent H-2Db/IgG2b molecule on the base of the protophase work. Soluble divalent MOG35-55/H-2Db molecule, which is prepared by the peptide MOG35-55 loading to divalent H-2Db/IgG2b molecule, may help to further develop of inducing Tcells autoreactive tolerance and provide evidence for blocking the