弓形虫基因疫苗pBudCE4.1-AMA1-ROP18免疫小鼠的保护作用研究
发布时间：2018-12-23 07:58
【摘要】：弓形虫为寄生于细胞内的原虫,它感染所有的恒温动物,包括人类,是寄生于人体最普遍的寄生虫之一,全球约有三分之一的人口感染弓形虫。弓形虫感染在先天感染和免疫系统受损的个体中有高发病率和高死亡率。先天感染可导致流产、新生儿畸形或胎儿出生后导致其他缺陷,如失明和严重的认知缺陷等。在免疫系统受损的个体中,感染寄生虫可导致脑炎和神经损伤。除此之外,食用感染弓形虫包囊动物的肉,是人体感染弓形虫的一个主要途径。 棒状体蛋白18(ROP18)为弓形虫棒状体蛋白,当弓形虫进入宿主细胞时,会生成一层保护膜包裹自身,使自己受细胞环境影响而不被杀死,ROP18的作用则是让宿主细胞某些蛋白失效而无法破坏这层保护膜。其作用机制是通过和宿主细胞中一类与免疫有关的鸟苷酸酶(GTPase)相结合,使得后者失效,进而无法破坏弓形虫周围的保护膜。AMA1为弓形虫顶端膜抗原,是一种微线体蛋白,为I型膜蛋白,分泌于弓形虫表面,在所有复顶亚门寄生虫中高度保守。有实验表明,AMAl在弓形虫入侵宿主细胞的过程中起重要作用,AMA1缺失将使弓形虫无法进入宿主细胞。 pBudCE4.1表达载体可同时表达哺乳动物细胞系的两个基因,并可同时独立表达两个重组蛋白,可在多种类型哺乳动物细胞中高效稳定的表达。 本研究根据已发表的AMA1mRNA序列和ROP18DNA序列,设计合成引物,构建真核表达质粒pBudCE4.1-AMA1、pBudCE4.1-ROP18,然后构建pBudCE4.1-AMA1-ROP18真核重组质粒。然后转染人包皮成纤维细胞(HFF),48-72小时后,提取细胞RNA,通过SDS-PAGE及逆转录鉴定其表达情况,证实重组质粒在HFF细胞中正常表达。此后,通过肌肉注射重组质粒免疫BALB/c小鼠,设立PBS对照组及空载体对照组,于第15天和第30天加强免疫,并在免疫前取血,收集血清；在第56天时进行攻击实验。将收集的血清进行免疫学检测,检测结果表明重组表达载体诱发宿主的免疫应答反应效果良好。通过攻击感染实验的验证,证明重组DNA疫苗对小鼠均具有一定的免疫保护作用,而且,重组质粒基因疫苗的免疫效果远远比单基因疫苗的免疫效果高,这对今后进一步研制弓形虫基因疫苗奠定了坚实的实验基础。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is one of the most common parasites in human body. It infects all isothermal animals including humans. About 1/3 people worldwide are infected with Toxoplasma gondii. Toxoplasma gondii infection is associated with high morbidity and mortality in individuals with congenital infections and impaired immune systems. Congenital infections can lead to miscarriage, neonatal malformations, or other defects after birth, such as blindness and severe cognitive impairment. In individuals with impaired immune systems, infection with parasites can lead to encephalitis and nerve damage. In addition, eating the meat of Toxoplasma gondii cyst animals is a main way to infect Toxoplasma gondii. Coryloid protein 18 (ROP18) is a Toxoplasma gondii rodlike protein. When Toxoplasma gondii enters the host cell, it forms a protective membrane that wraps itself in such a way that it is not killed by the cellular environment. The effect of ROP18 is to make certain proteins of host cells fail to destroy the protective membrane. The mechanism is that by combining with a class of immune-related guanylidase (GTPase) in host cells, the latter fails to destroy the protective membrane around Toxoplasma gondii. AMA1 is a microline protein, which is the top membrane antigen of Toxoplasma gondii. It is a type I membrane protein secreted on the surface of Toxoplasma gondii and highly conserved in all parasites. Some experiments have shown that AMAl plays an important role in the process of Toxoplasma gondii invading host cells, and AMA1 deletion will prevent Toxoplasma gondii from entering host cells. PBudCE4.1 expression vector can express two genes of mammalian cell line simultaneously and can express two recombinant proteins independently at the same time. It can be expressed efficiently and stably in many kinds of mammalian cells. Based on the published AMA1mRNA sequence and ROP18DNA sequence, primers were designed and synthesized to construct the eukaryotic expression plasmid pBudCE4.1-AMA1,pBudCE4.1-ROP18, and then construct the pBudCE4.1-AMA1-ROP18 eukaryotic recombinant plasmid. After transfection of human prepuce fibroblasts (HFF),) for 48 to 72 hours, RNA, was extracted and its expression was identified by SDS-PAGE and reverse transcription. The normal expression of the recombinant plasmid was confirmed in HFF cells. After that, BALB/c mice were immunized by intramuscular injection of recombinant plasmid, and PBS control group and empty vector control group were set up. The mice were immunized on the 15th and 30th days, blood was collected before immunization, and serum was collected, and the attack test was carried out on the 56th day. The collected serum was detected by immunology, and the results showed that the recombinant expression vector could induce the host immune response. The results showed that the recombinant DNA vaccine had a certain immune protective effect on mice, and the immune effect of recombinant plasmid gene vaccine was much higher than that of single gene vaccine. This laid a solid experimental foundation for the further development of Toxoplasma gondii gene vaccine.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
本文编号：2389656
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is one of the most common parasites in human body. It infects all isothermal animals including humans. About 1/3 people worldwide are infected with Toxoplasma gondii. Toxoplasma gondii infection is associated with high morbidity and mortality in individuals with congenital infections and impaired immune systems. Congenital infections can lead to miscarriage, neonatal malformations, or other defects after birth, such as blindness and severe cognitive impairment. In individuals with impaired immune systems, infection with parasites can lead to encephalitis and nerve damage. In addition, eating the meat of Toxoplasma gondii cyst animals is a main way to infect Toxoplasma gondii. Coryloid protein 18 (ROP18) is a Toxoplasma gondii rodlike protein. When Toxoplasma gondii enters the host cell, it forms a protective membrane that wraps itself in such a way that it is not killed by the cellular environment. The effect of ROP18 is to make certain proteins of host cells fail to destroy the protective membrane. The mechanism is that by combining with a class of immune-related guanylidase (GTPase) in host cells, the latter fails to destroy the protective membrane around Toxoplasma gondii. AMA1 is a microline protein, which is the top membrane antigen of Toxoplasma gondii. It is a type I membrane protein secreted on the surface of Toxoplasma gondii and highly conserved in all parasites. Some experiments have shown that AMAl plays an important role in the process of Toxoplasma gondii invading host cells, and AMA1 deletion will prevent Toxoplasma gondii from entering host cells. PBudCE4.1 expression vector can express two genes of mammalian cell line simultaneously and can express two recombinant proteins independently at the same time. It can be expressed efficiently and stably in many kinds of mammalian cells. Based on the published AMA1mRNA sequence and ROP18DNA sequence, primers were designed and synthesized to construct the eukaryotic expression plasmid pBudCE4.1-AMA1,pBudCE4.1-ROP18, and then construct the pBudCE4.1-AMA1-ROP18 eukaryotic recombinant plasmid. After transfection of human prepuce fibroblasts (HFF),) for 48 to 72 hours, RNA, was extracted and its expression was identified by SDS-PAGE and reverse transcription. The normal expression of the recombinant plasmid was confirmed in HFF cells. After that, BALB/c mice were immunized by intramuscular injection of recombinant plasmid, and PBS control group and empty vector control group were set up. The mice were immunized on the 15th and 30th days, blood was collected before immunization, and serum was collected, and the attack test was carried out on the 56th day. The collected serum was detected by immunology, and the results showed that the recombinant expression vector could induce the host immune response. The results showed that the recombinant DNA vaccine had a certain immune protective effect on mice, and the immune effect of recombinant plasmid gene vaccine was much higher than that of single gene vaccine. This laid a solid experimental foundation for the further development of Toxoplasma gondii gene vaccine.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
【参考文献】
相关期刊论文 前2条
1 金春梅;张厚双;薛书江;权根花;崔金伟;王兴贵;;弓形虫AMA1蛋白真核表达载体的构建及免疫活性检测[J];安徽农业科学;2010年29期
2 程相朝,张敏,王建军,王婷;DNA疫苗的主要意义与特点[J];河南科技大学学报(医学版);2005年03期
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