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IgGI、gE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IgGI、gE浓度。结果琼脂糖凝胶电泳分离PCR产物,于750 bp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。
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 notexist.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.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2011年第2期155-160,174,共7页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
国家自然科学基金资助项目(No.30772010)~~
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
bronchial asthma
humulus scandens grass pollen allergen
DNA vaccine
eukaryotic expression
immunogenicity
