pCMV-DKK1/IL10双表达核酸疫苗的构建及其免疫保护效果研究

中国临床药理学与治疗学 ›› 2014, Vol. 19 ›› Issue (11): 1211-1217.

pCMV-DKK1/IL10双表达核酸疫苗的构建及其免疫保护效果研究

张晓清, 刘思博, 窦云鹏, 杜雨轩, 袁慧慧, 赵文明

首都医科大学基础医学院免疫学系,北京 100069

收稿日期:2014-09-05 修回日期:2014-11-07 出版日期:2014-11-26 发布日期:2014-12-09
通讯作者: 赵文明,男,教授,博士生导师,研究方向:类风湿关节炎的免疫调节治疗。 Tel: 010-83911824 E-mail: zhaowenming@163.com
作者简介:张晓清,女,硕士研究生,研究方向:类风湿关节炎的免疫调节治疗。Tel: 18302442086 E-mail: zhangxiaoqing2014@126.com
基金资助:
国家自然科学基金(31400767)资助

Construction and protection effects of the dual expression DNA vaccine pCMV-DKK1/IL10

ZHANG Xiao-qing, LIU Si-bo, DOU Yun-peng, DU Yu-xuan, YUAN Hui-hui, ZHAO Wen-ming

Department of Immunology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069,China

Received:2014-09-05 Revised:2014-11-07 Online:2014-11-26 Published:2014-12-09

摘要/Abstract

摘要： 目的构建双表达人Dickkopf 1(DKK1)表位序列和IL-10功能序列的核酸疫苗载体,检测其对胶原诱导关节炎小鼠的保护作用。方法选择4段免疫原性较高的片段并与T细胞表位片段连接,将此基因片段与IL-10的功能片段克隆至pCMV6-XL5载体中,得到pCMV-DKK1/IL10双表达核酸疫苗载体;用此质粒DNA转染COS7细胞并肌肉注射BALB/c小鼠,分别检测核酸疫苗在细胞和小鼠体内的表达;用核酸疫苗免疫BALB/c小鼠,检测小鼠体内产生的特异性抗体滴度;制备胶原诱导关节炎小鼠模型,评估核酸疫苗对胶原诱导关节炎小鼠的保护作用。结果成功构建了pCMV-DKK1/IL10双表达核酸疫苗载体,DKK1重组蛋白和IL-10能够在COS7细胞和小鼠肌肉组织中成功表达;免疫后4周即可检测到高滴度的DKK1抗体;核酸疫苗可以减轻胶原诱导关节炎小鼠的炎症症状和骨破坏的程度。结论 pCMV-DKK1/IL10双表达核酸疫苗对胶原诱导关节炎小鼠具有保护作用,该研究结果为类风湿关节炎的治疗提供了新的思路和方向。

关键词: 类风湿关节炎, 核酸疫苗, DKK1, IL-10

Abstract: AIM: The eukaryotic expression vector of human Dickkopf 1(DKK1) and interleukin-10 (IL-10) DNA vaccine was constructed to evaluate its protection effects in collagen-induced arthritis (CIA) mice. METHODS: Four DNA fragments of human DKK1 with high immunogenicity were selected and then connected with T-cell epitopes. The recombinant sequences of DKK1 and IL-10 were cloned into eukaryotic vector pCMV6-XL5, resulting in the dual expression vector pCMV-DKK1/IL10. The plasmid was transfected into COS7 cells and administrated into the skeletal muscles of BALB/c mice. The expression of the target proteins in COS7 cells and the injection site were detected using Western blotting and immunohistochemistry. The specific antibodies of DNA vaccine was determined using indirect enzyme-linked immunosorbent assay (ELISA). The arthritis incidence, clinical score, radiography and histopathology of the collagen-induced arthritis mice were used as indexes to evaluate the protective effects of the DNA vaccine. RESULTS: The recombinant plasmids pCMV-DKK1/IL10 was successfully constructed and expressed the target proteins both in COS7 cells and in the skeletal muscles of mice. The high expression of anti-human DKK1 antibodies were elicited at the fourth week after immunization with pCMV-DKK1/IL10. In addition, the DNA vaccine attenuated the inflammation and bone erosion in CIA mice.CONCLUSION: The results showed that the immunization of the mice with pCMV-DKK1/IL10 ameliorated the symptoms of CIA mice. This study may provide a potential therapeutic strategy in rheumatology arthritis (RA).

Key words: rheumatoid arthritis(RA), DNA vaccine, DKK1, IL-10

中图分类号:

R965.2

张晓清, 刘思博, 窦云鹏, 杜雨轩, 袁慧慧, 赵文明. pCMV-DKK1/IL10双表达核酸疫苗的构建及其免疫保护效果研究[J]. 中国临床药理学与治疗学, 2014, 19(11): 1211-1217.

ZHANG Xiao-qing, LIU Si-bo, DOU Yun-peng, DU Yu-xuan, YUAN Hui-hui, ZHAO Wen-ming. Construction and protection effects of the dual expression DNA vaccine pCMV-DKK1/IL10[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2014, 19(11): 1211-1217.

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