CTP-HBcAg18-27-Tapasin融合蛋白胞内转导功能的检测

目的:观察融合蛋白CTP-HBcAg18-27-Tapasin在抗原提呈细胞内的转导功能.方法:体外分离培养近交系BALB/c小鼠髓源性DC,加入重组粒细胞-巨噬细胞集落刺激因子和白细胞介素-4培养5d,再加入脂多糖诱导DC成熟.不同剂量的CTP-HBcAg18-27-Tapasin及RP-MI-1640培养液加入细胞培养介质中,激光共聚焦显微镜下观察免疫荧光在细胞内的分布及定位,并对荧光强度进行定量分析,进一步以Western blot观察不同组细胞中Tapasin表达的差异来检测CTP-HBcAg18-27-Tapasin的转导效率.结果:成功体外诱导培养并鉴定小鼠骨髓源性树突状细胞,免疫荧光法证实CTP-HBcAg18-27-Tapasin能够穿透树突状细胞膜进入细胞质,而不能进入细胞核,且荧光强度50 μg/L CTP-HB-cAg18-27-Tapasin组依次高于10 μg/L CTP-HBcAg18-27-Tapasin组和空白组,Western blot 也证实CTP-HBcAg18-27-Tapasin能够穿透细胞膜进入树突状细胞,结果显示差异有统计学意义.结论:CTP-HBcAg18-27-Tapasin具有穿透树突状细胞膜定位于胞浆的能力.

Transmission functions of fusion protein CTP-HBcAg18-27-Tapasin in the cells

AIM: To observe the function of transduction of fusion protein CTP-HBcAg18-27-Tapasin in antigen presenting cells.METHODS: Bone marrow derived DCs isolated from BALB/c mice were cultured with recombinant granulocyte-macrophage colony-stimulating factor and recombinant interleutin-4 for 5 days followed by LPS added to induce DCs maturation.Then,different dosages of CTP-HBcAg18-27-Tapasin and RPMI-1640 were added to cell culture medium.Subsequently,the distribution and localization of intracellular immunofluorescence were observed by confocal microscopy with fluorescence intensity assaying quantitatively.Futhermore,the transduction efficiency of CTP-HBcAg18-27-Tapasin was evaluated by detecting the differences of expression of Tapasin using western blot in different group.RESULTS:DCs were cultured and identified successfully;Immunofluorescence confirmed the CTP-HBcAg18-27-Tapasin could penetrate the membrane into the cytoplasm of DCs but not into the nucleus.Fluorescence intensity of 50 μg/L CTP-HBcAg18-27-Tapasin group were higher than 10 μg/L CTP-HBcAg18-27-Tapasin group and blank groups.Western blot also confirmed that CTP-HBcAg18-27-Tapasin can penetrate the cell membrane into the DCs,and the data showed the difference was statistical.CONCLUSION: CTP-HBcAg18-27-Tapasin has the ability of penetrating and locating in the cytoplasm of DCs.