乙肝病毒RNA复制子疫苗、DNA疫苗的构建及其对小鼠免疫效果的比较

发布时间：2018-01-12 18:19

本文关键词：乙肝病毒RNA复制子疫苗、DNA疫苗的构建及其对小鼠免疫效果的比较　出处：《吉林大学》2005年硕士论文　论文类型：学位论文

【摘要】：本研究的目的是构建乙肝病毒的RNA 复制子疫苗,通过与普通疫苗和DNA 疫苗进行比较,探索RNA 复制子疫苗最佳的形式和免疫途径。首先,充分利用生物信息学工具及多条重叠引物PCR 拼接的策略,设计并合成了两个抗原基因:S2-S 和MAG。将两种抗原基因的PCR产物分别与pGEM-T 载体连接,并筛选到序列正确的克隆质粒pGEM-S2S 和pGEM-MAG。本研究利用S2-S、MAG 作为两个抗原基因,以IL-2 为分子佐剂,利用pcDNA3.1+构建DNA疫苗,利用RNA复制子载体pSFV构建RNA复制子疫苗,共构建8个疫苗载体:pSFV-S2S,pSFV-MAG,pcDNA-S2S,pcDNA-MAG,pSFV-S2S-II-IL2,pSFV-MAG-II-IL2,pcDNA-S2S-II-IL2,pcDNA-MAG-II-IL2。其中II 是本研究中从载体pIRES1neo 上扩增IVS和IRES 的片段(本研究中通称为II)。将上述八个疫苗载体分别转染BHK-21 细胞,ELISA 检测其表达情况,结果表明以RNA 复制子疫苗载体的表达效率高于DNA 疫苗。另外,RNA 复制子疫苗载体转染BHK-21 细胞后,细胞呈现凋亡特征。将所构建的疫苗载体以不同的剂量分三次免疫BALB/c 小鼠,ELISA检测体液免疫效果。建立稳定表达S2S 和MAG 的P815 细胞作为CTL检测的靶细胞,通过CTL 方法检测细胞免疫效果。结果表明,RNA 复制子疫苗体液免疫和细胞免疫效果均优于DNA 疫苗,但二者的体液免疫效果不及基因工程亚单位疫苗。
[Abstract]:The purpose of this study is to construct hepatitis B virus RNA replicon vaccine. Through comparing with the common vaccine and DNA vaccine, we explore the best form and immune way of RNA replicon vaccine.
First, make full use of bioinformatics tools and multiple overlapping primers PCR splicing strategy, designed and synthesized two antigen genes: S2-S and MAG. PCR antigen gene products of the two are respectively connected with the pGEM-T vector, and screened the correct sequence of plasmid pGEM-S2S and pGEM-MAG.
This study uses S2-S and MAG as the two antigen gene, using IL-2 as a molecular adjuvant DNA vaccine constructed by pcDNA3.1+, using the RNA replicon vector pSFV to construct the RNA replicon vaccine, we constructed 8 vaccine vectors: pSFV-S2S, pSFV-MAG, pcDNA-S2S, pcDNA-MAG, pSFV-S2S-II-IL2, pSFV-MAG-II-IL2, pcDNA-S2S-II-IL2, pcDNA-MAG-II-IL2. and II are in this study from the vector pIRES1neo amplified IVS and IRES fragments (known as II in this study).
The eight vaccine vector were transfected into BHK-21 cells, detect the expression of ELISA, the results showed that the expression efficiency of replicon vaccine vector with RNA was higher than that of DNA vaccine. In addition, RNA replicon vaccine vector were transfected into BHK-21 cells, the cells exhibited the characteristics of apoptosis. The vaccine carrier with different doses of three immune BALB/c mice, ELISA detection of humoral immune effect. The establishment of stable expression of S2S and MAG of P815 cells as the target cells of CTL detection, by detecting the effect of cellular immunity in CTL method. The results show that the RNA replicon vaccine humoral and cellular immune effects were better than the DNA vaccine, but less than two of the humoral immunity effect of gene engineering subunit the vaccine.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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