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| 卢家美，孙秀珍，李满祥，刘 昀，徐 晶，张永红，吴媛媛 葎草花粉主要变应原核酸疫苗pcDNA3.1-LC2的真核细胞表达和免疫分析 |
| 论文编辑部-新丝路理论网 2011-03-31 15:20:47 作者:中华医学之家：http://www.xinxi85.com 来源: 文字大小:[大][中][小] |
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Title:
Eukaryotic expression and immunization analysis of the DNA vaccine of the major grass pollen allergen of humulus scandens
- 文章编号:
- 1671-8259(2011)02-0155-06
- 作者:
- 卢家美; 孙秀珍; 李满祥; 刘 昀; 徐 晶; 张永红; 吴媛媛
- 西安交通大学医学院第二附属医院呼吸内科，陕西西安 710004
- Author(s):
- LU Jia-mei; SUN Xiu-zhen; LI Man-xiang; LIU Yun; XU Jing; ZHANG Yong-hong; WU Yuan-yuan
- Department of Respiratory Medicine, the Second Affiliated Hospital, Medical School of Xi’an Jiaotong University, Xi’an 710004, China
- 关键词:
- ; 支气管哮喘; 葎草花粉变应原; 核酸疫苗; 真核表达; 免疫原性
- Keywords:
- ; bronchial asthma; humulus scandens grass pollen allergen; DNA vaccine; eukaryotic expression; immunogenicity
- 分类号:
- R562.1
- DOI:
- -
- 文献标识码:
- A
- 摘要:
- 摘要：目的 验证葎草花粉主要变应原核酸疫苗(pcDNA3.1-LC2)能否在真核细胞内表达，并进行免疫原性、免疫保护作用、安全性等分析。方法 应用磷酸钙沉淀法将pcDNA3.1-LC2转染至HEK293细胞，提取RNA并行RT-PCR，利用琼脂糖凝胶电泳分离PCR产物，验证所分离出的条带与目的片段是否一致；应用pcDNA3.1-LC2免疫正常小鼠，ELISA方法测定血清中IgG、IgE浓度和脾组织匀浆中IL-4、IFN-γ浓度；制备葎草花粉变应性哮喘小鼠模型，应用pcDNA3.1-LC2对葎草花粉变应性哮喘模型小鼠进行免疫治疗，行肺泡灌洗液沉渣细胞计数，肺泡灌洗液沉渣滴片瑞士染色并嗜酸性粒细胞分类计数；取左肺上叶行肺脏病理HE染色，ELISA方法测定肺泡灌洗液中IL-4、IFN-γ浓度，并测定血清中IgG、IgE浓度。结果 琼脂糖凝胶电泳分离PCR产物，于750bp处分离出一明显的条带，与目的片段一致；采用pcDNA3.1-LC2免疫正常小鼠后，血清IgG浓度升高，IgE浓度无明显变化，脾组织匀浆中IL-4浓度降低，IFN-γ浓度升高；免疫过程中未引起过敏反应、免疫损伤及急性肺损伤；应用该核酸疫苗免疫葎草花粉变应性哮喘小鼠模型，可明显降低气道炎症级别、减轻炎症细胞浸润、降低嗜酸性粒细胞百分比，引起血清中IgG浓度升高和IgE浓度降低，肺泡灌洗液中IL-4浓度降低和IFN-γ浓度升高，再次激发时哮喘症状明显减轻。结论 pcDNA3.1-LC2可在真核细胞内表达，诱导TH1/TH2平衡向TH1方向转化，具有免疫原性及免疫保护作用，安全性较好。
- Abstract:
- ABSTRACT: Objective To test whether the DNA vaccine of the major grass pollen allergen of humulus scandens (pcDNA3.1-LC2) can be expressed in eukaryatic cells and to further analyze its immunogenicity, immune protection and safety. Methods HEK293 cells were transfected with the DNA vaccine of the major grass pollen allergen of humulus scandens using Ca3 (PO4)2. RNA was extracted and used for RT-PCR. Products of PCR were separated on agarose gel electrophoresis. The normal BALB/C mice were immunized with the DNA vaccine. The concentration of IgG, IgE, IL-4 and IFN-γ was measured. BALB/C mice of asthma model were immunized with the DNA vaccine. Lung pathology and BALF cell sediment drop films were advanced; cell count and differential count of eosinophils were conducted, too. The concentration of IL-4 and IFN-γ in the BALF was measured. The concentration of IgG and IgE in the serum was measured at the same time. Results Our study showed a clear strip at 750bp and confirmed the expression of the DNA vaccine of the major grass pollen allergen of humulus scandens in eukaryotic cells using agarose gel electrophoresis. The normal mice could produce IgG rather than IgE after being immunized with the DNA vaccine, which suggested the immunogenicity of it. At the same time, IFN-γ concentration increased and IL-4 concentration decreased. Allergic reactions, immune injury and acute lung injury were not caused in the immune process, which suggested that the potential injury risk of the DNA vaccine did not exist. It caused the following changes after the model of asthma of BALB/C mice being immunized with the DNA vaccine: Levels of lung inflammation and infiltration of inflammatory cells and the percentage of eosinophils were significantly reduced. The generation of IL-4 and IgE was inhibited and the generation of IgG and IFN-γ was evoked significantly. The symptoms were relieved greatly if the mice were excited again after being immunized with the DNA vaccine. Conclusion The DNA vaccine of the major grass pollen allergen of humulus scandens can be successfully expressed in eukaryotic cells. It can induce the TH1/TH2 balance to the transformation of TH1 direction. It has proved to have immunogenicity, immune protection and safety.
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基金项目：国家自然科学基金资助项目(No.30772010)
Supported by the National Natural Science Foundation of China (No.30772010)
通讯作者：孙秀珍，教授. E-mail: doc-ly@sohu.com
作者简介：卢家美，(1980-)，女(汉族)，博士. 研究方向：哮喘的特异性治疗与免疫机制研究. E-mail: lu-jia1980@163.com
中华医学之家：http://www.xinxi85.com
投稿信箱：zhyxzj85@163.com
联系电话：029--85327298 主编QQ：693891972 |
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