摘要
目的:构建抗乙型肝炎病毒(HBV)的治疗性DNA疫苗并进行体外、体内鉴定。方法:采用PCR技术扩增含有HBV包膜中蛋白(preS2.S)抗原基因和hIL-2/hIFN-γ融合蛋白的基因,将目的基因分别亚克隆于pVAX1真核表达载体,并进行酶切和测序分析;转染双质粒于COS-7细胞检测基因及蛋白表达情况;利用Combinatotial Extension(CE)软件模建分析佐剂质粒表达融合蛋白的空间结构;双质粒联合在体电脉冲技术(EP)注射BALB/c小鼠以检测特异性的体液和细胞免疫应答。结果:经酶谱和测序分析表明,构建的双质粒pVAX1/S2.S(pS2.S)和pVAX1/IL-2IFN-γ(pIIF)的基因片段的方向、序列完全正确;ELISA法定量检测双质粒转染COS-7细胞后48小时目的基因(preS2.S和hIL-2/hIFN-γ)的蛋白表达水平较高,HBsAg、IL-2、IFN-γ的含量分别为45.1、10.03、11.5ng/ml;模拟空间结构分析表明佐剂质粒的融合蛋白中两细胞因子的活性部位均裸露在外;小鼠免疫试验结果表明,疫苗pS2.S能诱导健康小鼠的保护性抗-HBs的抗体产生,且具有剂量相关性;佐剂pIIF可显著增强疫苗pS2.S质粒的免疫效果;在EP辅助作用下,质粒pS2.S和pIIF共肌注BALB/c小鼠,低剂量就能诱导较强特异性的细胞和体液免疫。结论:成功构建了抗HBV的DNA疫苗pS2.S及佐剂pIIF质粒,并具有较特异的体内外活性。
Objective：To develop an effective therapeutic double HBV DNA vaccine with two eukaryotic expressing motif,namely pS2.S and pIIF,a fusion ORF of hIL-2/hIFN-γ.Methods：Two genes were amplified,which separately encoded HBV preS2.S middle envelope protein as a vaccine and human IL-2/IFN-γ fusion protein as an adjuvant by PCR technique from plasmids pcDNA3.1/preS2.S and pcDNA3.1/hIL-2/IFN-γ.Then the genes subcloned into eukaryotic vector pVAX1.The new plasmid was analysed by restriction endonuclease and DNA sequencing.The constructed plasmids were transfected into COS-7 cells in vitro with LipofectamineTM 2000.The expressing products in supernatants were quantified by ELISA.Space structure of fusion hIL-2/IFN-γ expressed by the adjuvant plasmid was simulated by CE software.The double plasmid association were injected into BALB/c mice by in situ electroporation method,and specific humoral and cellular immune responses were examined.Results：The gene segments and inserting direction of pVAX1-S2.S（pS2.S）and pVAX1-IL-2IFN-γ（pIIF）were both correct by genetic analysises.At 48 h after transfection,HBsAg and the cytokines of IL-2 and IFN-γ were respectively 45.1 ng/ml,10.03 ng/ml and 11.5 ng/ml by ELISA.The activity domains of the two cycokines in fusion protein were entirely exposed on surface by CE software.Experiments were pefrormed in mice by injection of plasmid pS2.S.Protective antibodies of anti-HBs were produced in terms of dose-dependment pattern for the efficacy.Co-injection of plasmid pS2.S together with adjuvant pIIF resulted in more evident immune responses and dose of the plasmid pS2.S needed was diminished.There was strongly specific humoral and cell immunity by pS2.S and pIIF co-injection with EP technique.Conclusion：The double plasmids pS2.S and pIIF of anti-HBV are constructed succeedly and induce strongly specific immune activity in vitro and vivo.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2008年第12期1121-1124,1132,共5页
Chinese Journal of Immunology
基金
国家"863"课题基金资助项目(2002AA2Z3317)
关键词
DNA疫苗
构建
转染
基因表达与免疫
HBV DNA vaccine
Construction
Transfection
Gene expression and immunity
