HIV-1治疗性重组DNA疫苗的构建、纯化及实验免疫研究

发布时间：2018-01-05 19:30

本文关键词：HIV-1治疗性重组DNA疫苗的构建、纯化及实验免疫研究　出处：《吉林大学》2005年博士论文　论文类型：学位论文

【摘要】：本论文利用分子生物学、免疫学、分子病毒学、生物化学、生物信息学等技术和手段进行了新型治疗性HIV-1 重组核酸疫苗的构建、纯化及实验免疫学研究,内容概述如下: 本实验以选择HIV-1 优势抗原表位为原则,提出以HIV-1 表位基因为基础,以HIV-1 巨分子颗粒p24 为支架载体分子,对抗原基因进行人工分子设计,并辅以计算机模拟来研制治疗性HIV 基因工程疫苗的新思路。该种HIV DNA 疫苗的设计在国内外尚未见报道。将经分子设计的疫苗抗原的核苷酸序列进行人工合成,获得新型HIV-1 治疗性抗原基因(MEG);以PCR 法获得HIV-1 衣壳蛋白基因p24,将其插入到MEG 基因中,获得嵌合基因MEGp24。利用新型安全性真核表达载体质粒pVAXI,构建了新型HIV-1 治疗性重组DNA 疫苗(pVAXI-MEG-p24),经过哺乳动物细胞转染结果表明,pVAXI-MEG-p24 成功地表达了MEGp24 抗原蛋白。将已构建好的治疗性HIV-1 重组DNA 疫苗的质粒(pVAXI-MEG-p24),经发酵罐建立发酵工艺,通过阴离子交换层析方法(Q Sepharose XL)进行纯化,随后将得到的质粒DNA 通过排阻层析方法进行进一步的纯化及脱盐,浓缩。经过质量鉴定结果表明,该纯化质粒疫苗符合FDA 推荐的临床用质粒DNA 的质量标准,表明所建立的工艺是可行。纯化后的重组DNA 疫苗质粒pVAX1-MEG-p24 进行实验免疫研究表明,纯化后HIV-1 重组DNA 疫苗使小鼠和恒河猴,产生了特异性体液和细胞免疫应答;治疗性HIV-1 重组DNA 疫苗(pVAXI-MEG-p24)与HIV 全结构蛋白基因疫苗(pVAXI-gag-gp120)比较研究表明,pVAXI-MEG-p24 能够诱发比全结构蛋白基因疫苗更强的表位特异性CTL 反应和抗体反应。总之,本研究以新的研究思路,对HIV 治疗性重组核酸疫苗的构建进行了
[Abstract]:In this paper, a novel therapeutic HIV-1 recombinant nucleic acid vaccine was constructed using molecular biology, immunology, molecular virology, biochemistry, bioinformatics and other techniques and methods. Purification and experimental immunological studies are summarized as follows: Based on the selection of HIV-1 epitopes, this study proposed that HIV-1 giant particle p24 should be used as scaffold carrier molecule based on HIV-1 epitope gene. Artificial molecular design of antigen gene. A new idea of developing therapeutic HIV gene engineering vaccine with computer simulation was also presented. The HIV DNA. The design of vaccine has not been reported at home and abroad. The nucleotide sequence of vaccine antigen designed by molecule was synthesized. A novel HIV-1 therapeutic antigen gene was obtained. HIV-1 capsid protein gene p24 was obtained by PCR and inserted into MEG gene. The chimeric gene MEGp24. using a novel safe eukaryotic expression vector plasmid pVAXI was obtained. A novel HIV-1 therapeutic recombinant DNA vaccine pVAXI-MEG-p24 was constructed and transfected into mammalian cells. PVAXI-MEG-p24 successfully expressed MEGp24 antigen protein. The plasmid pVAXI-MEG-p24 of therapeutic HIV-1 recombinant DNA vaccine was constructed and the fermentation process was established. The plasmid DNA was purified by anion exchange chromatography and then further purified and desalted by exclusion chromatography. The results of quality identification show that the purified plasmid vaccine meets the quality standard of clinical plasmid DNA recommended by FDA, and the established technology is feasible. The purified recombinant DNA vaccine plasmid pVAX1-MEG-p24 was used for experimental immunological study. The results showed that the purified HIV-1 recombinant DNA vaccine made mice and rhesus monkeys. Produced specific humoral and cellular immune responses; The comparative study of therapeutic HIV-1 recombinant DNA vaccine pVAXI-MEG-p24 and pVAXI-gag-gp120) showed that pVAXI-MEG-p24 and pVAXI-gag-gp120). PVAXI-MEG-p24 can induce a stronger epitope specific CTL reaction and antibody response than the whole structure protein gene vaccine. In conclusion, the construction of HIV therapeutic recombinant nucleic acid vaccine was carried out with new research ideas.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392.1

【引证文献】