绿色荧光蛋白标记的霍乱毒素B亚基-胰岛素融合蛋白在家蚕中的克隆表达与鉴定

Specific immunological unresponsive state induced by oral administrating autoantigens such as insulin (Ins) in animals or humans is named oral tolerance, and is capable of suppressing autoimmune type 1 diabetes (T1D) except for needing large quantities of insulin. The mucosal adjuvant cholera toxin B subunit (CTB) can be fused with insulin to alleviate the antigen need pressure and enhance the specific oral tolerance. However, the process how the CTB-Ins fusion protein works in vivo for T1D treatment is unclear. Here, we use one green fluorescent protein (GFP) tagged CTB-Ins fusion protein expressed by Bac-to-bac baculovirus expression system (BES) in silkworm to determine the interactions of the fusion protein with intestine in animals. A flexible tetrapeptide was used to ligate CTB, human insulin and GFP for obtaining the fusion protein. We found that the expression level of CTB-Ins-GFP protein in five-instar silkworm larva hemolymph reached high up to 0.58mg/ml. Further study showed that these proteins were produced in a pentameric form retaining the GM1-ganglioside binding affinity, native CTB, insulin antigenicity and GFP activity. Oral administrating microgramme of the fusion protein could induce specific humoral immunoreactions and reduced pancreatic islet inflammation in non-obese diabetic (NOD) mice. The intestinal frozen sections of NOD mice gut mucosa binding assay showed the fusion protein can specifically combine the mucosal epithelial cells. These results demonstrate that this active GPF-tagged fusion protein can be supplied in a large scale by the silkworm bac-to-bac BES and used as a molecular marker to reveal the in vivo work process for this protein in treating T1D.