CTP-HBcAg18-27-Tapasin融合蛋白胞内转导功能的检测

中国临床药理学与治疗学 ›› 2012, Vol. 17 ›› Issue (1): 25-29.

CTP-HBcAg18-27-Tapasin融合蛋白胞内转导功能的检测

刘红红, 陈小华, 周丽芹, 刘雪妮, 余永胜, 臧国庆, 汤正好

上海交通大学附属第六人民医院感染科,上海 200233

收稿日期:2011-09-15 修回日期:2011-12-01 出版日期:2012-01-26 发布日期:2012-02-16
通讯作者: 汤正好,男,硕导,研究方向:病毒性肝炎临床与基础。E-mail: tzhhao@hotmail.com
作者简介:刘红红,女,研究方向:病毒性肝炎的临床与基础。Tel: 13918664643 E-mail: liu.honghong.ok@163.com
基金资助:
国家自然基金项目(31000414)

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

LIU Hong-hong, CHEN Xiao-hua, ZHOU Li-qin, LIU Xue-ni, YU Yong-sheng, ZANG Guo-qing, TANG Zheng-hao

Department of Infectious Diseases, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiaotong University, Shanghai 200233, China

Received:2011-09-15 Revised:2011-12-01 Online:2012-01-26 Published:2012-02-16

摘要/Abstract

摘要： 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 5 d,再加入脂多糖诱导DC成熟。不同剂量的CTP-HBcAg18-27-Tapasin及RPMI-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-HBcAg18-27-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。

关键词: CTP, HBcAg18-27, Tapasin, 转导, 树突状细胞

Abstract: 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.

Key words: CTP, HBcAg18-27, Tapasin, Transduction, Dendritic cells

中图分类号:

R965.2

刘红红, 陈小华, 周丽芹, 刘雪妮, 余永胜, 臧国庆, 汤正好. CTP-HBcAg18-27-Tapasin融合蛋白胞内转导功能的检测[J]. 中国临床药理学与治疗学, 2012, 17(1): 25-29.

LIU Hong-hong, CHEN Xiao-hua, ZHOU Li-qin, LIU Xue-ni, YU Yong-sheng, ZANG Guo-qing, TANG Zheng-hao. Transmission functions of fusion protein CTP-HBcAg18-27-Tapasin in the cells[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2012, 17(1): 25-29.

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