活体电穿孔导入法增强恶性疟原虫核酸疫苗免疫反应研究  

In vivo Electroporation-based Transfer Enhances Immunogenicity of Plasmodium Falciparum DNA Vaccine

作　　者：高宇辉[1] 邓唯唯[1] 张佳佳[1] 李月[1] 

机构地区：[1]中国医学科学院基础医学研究所/北京协和医学院基础学院,100005

出　　处：《医学研究杂志》2016年第6期19-24,共6页Journal of Medical Research

基　　金：国家高技术研究发展计划("863"计划)项目(2012AA02A406);国家科技重大专项基金资助项目(2013ZX09102-043)

摘　　要：目的采用活体电穿孔免疫方法,研究恶性疟原虫DNA核酸疫苗不同免疫剂量对于动物免疫反应的影响,并对载体中Cp G寡脱氧核苷酸基序的免疫刺激活性进行了初步探讨。方法将恶性疟原虫多表位嵌合抗原M.RCAg-1基因克隆于真核表达载体VR1012中,使用电穿孔导入方法免疫小鼠,采用ELISA检测抗体效价、ELISPOT测定细胞因子分泌水平,探索基因疫苗最佳免疫剂量以及考察免疫反应类型。使用计算机分析恶性疟核酸疫苗中特殊CpG基序组成,实验验证基因疫苗中CpG基序的免疫刺激活性。结果相同DNA免疫剂量下,电穿孔免疫法与单纯肌内注射免疫法相比较,小鼠血清IgG抗体效价提高118倍,且能显著诱导体内特异性Th1和Th2型细胞反应。电穿孔免疫小鼠血清可识别天然虫体抗原,抗体效价可达1∶1200。恶性疟原虫核酸疫苗抗原DNA含有24个CpG免疫活性基序,可以有效的刺激人外周血单个核细胞(PBMCs)增殖与IL-6分泌。结论活体电穿孔方法可以显著提高恶性疟原虫DNA疫苗体液免疫和细胞免疫水平。Objective To investigate the protection effect using in vivo electroporation with various amount of malaria DNA vaccine in mammalian.Methods The malaria poly-epitope chimeric antigen M.RCAg-1 gene sequence was cloned in an eukaryotic expression vector VR1012.The DNA vaccine was introduced in BALB/c mice by electroporation method.The total serum IgG antigen titer and cytokines expression were measured by ELISA and ELISPOT respectively,and the optimum DNA vaccine usage was determined as well as the type of immunity.The specific CpG motif sequences were explored by computer analysis.The CpG motifs＇ immune-stimulatory activity was also tested in vitro.Results In vivo electroporation method can induce higher IgG titer up to 118 folds than intramuscular immunization using the malaria DNA vaccine in mice,and enhance both Th1 and Th2 type cellular immunity.Immune serum from mice can recognize native plasmodium falciparum antigen even at titer 1∶ 1200.The CpG motifs buildin malaria vaccine DNA sequence can promote human peripheral blood mononuclear cells proliferation and secretion of IL-6.Conclusion Both humoral and cellular immune response of malaria DNA vaccine are significantly improved by electroporation in vivo.

关 键 词：疟疾 DNA疫苗 电穿孔 VR1012 CPG基序 

分 类 号：R382.9[医药卫生—医学寄生虫学]