A型流感病毒NS1基因密码子去优化改造引起病毒毒力减弱的研究

发布时间：2018-12-11 17:51

【摘要】： 疫苗接种是预防流感最有效的方法,目前投入使用的流感疫苗可分为灭活苗和减毒活苗两大类:灭活苗通过肌肉注射途径免疫,能够诱发机体产生针对血凝素的中和抗体,但无法刺激鼻腔黏膜产生分泌型抗体,也无法诱导产生细胞免疫应答;减毒活苗(冷适应减毒活苗)通过鼻吸入的方式免疫,其免疫的方式更接近自然感染的过程,可充分诱导体液免疫及细胞免疫应答。减毒活苗无论从免疫途径还是免疫效果上都优于传统灭活苗。自反向遗传操作技术建立以来,研究者们尝试过多种方法开发减毒活苗,降低流感病毒毒力,提高其免疫原性,研究出安全有效的减毒活苗已成为新型流感疫苗的发展方向。在本研究中,我们根据A型流感病毒密码子使用偏嗜性的统计规律,在保留NS片段包装信号和不影响NS1/NS2剪切位点的前提下,选取稀有密码子对A/Puerto Rico/8/34(H1N1)病毒NS1基因内部110个氨基酸区域进行密码子同义突变改造,将其替换为病毒使用频率最低的密码子,全基因合成NS基因,利用12质粒反向遗传操作技术拯救出含有密码子去优化NS1基因的重组病毒(deoptimized NS, deoNS)。MDCK细胞噬斑形成实验表明,病毒在细胞上的成斑能力大大减弱;病毒在MDCK细胞上的生长曲线表明,其在细胞内的复制能力比野生型病毒低约1000倍。BALB/c小鼠体内致病力实验证明,deoNS病毒不能引起小鼠发病和死亡,感染第3天病毒在小鼠肺内的复制滴度比野生型病毒低100倍,感染第5天其在小鼠肺内的滴度比野生型病毒低1000倍。本研究初步探索了通过基因组密码子去优化改造途径降低A型流感病毒毒力的可行性,首次证明流感病毒NS1基因密码子去优化同义突变可以降低病毒毒力,为流感减毒活疫苗的研究提供了新的思路。
[Abstract]:Vaccination is the most effective way to prevent influenza. The influenza vaccine currently in use can be divided into two categories: inactivated vaccine and live attenuated vaccine: the inactivated vaccine is immunized by intramuscular injection, which can induce the body to produce neutralizing antibodies against hemagglutinin. However, it can not stimulate nasal mucosa to produce secretory antibody, nor can it induce cellular immune response. Attenuated live vaccine (cold acclimated attenuated vaccine) is immunized by nasal inhalation, which is closer to the process of natural infection and can fully induce humoral and cellular immune responses. The live attenuated vaccine is superior to the traditional inactivated vaccine in both the immune pathway and the immune effect. Since the establishment of reverse genetic operation technology, researchers have tried many methods to develop live attenuated vaccine, reduce the virulence of influenza virus, improve its immunogenicity, and find out that the safe and effective live attenuated vaccine has become the development direction of new influenza vaccine. In this study, according to the statistical rule of codon usage bias of influenza A virus, we kept the NS fragment packaging signal and did not affect the NS1/NS2 splicing site. A rare codon was selected to modify the 110 amino acid region of the NS1 gene of A/Puerto Rico/8/34 (H1N1) virus. The codon was replaced by the codon with the lowest frequency of the virus, and the whole gene was synthesized into the NS gene. Using 12 plasmid reverse genetic manipulation technique to save the recombinant virus (deoptimized NS, deoNS). MDCK cell plaque formation with codon to optimize the NS1 gene, the results showed that the ability of the virus to form plaque on the cell was greatly weakened. The growth curve of the virus on MDCK cells showed that the replication ability of the virus in the cells was about 1000 times lower than that of the wild type virus. The virulence test in BALB/c mice showed that deoNS virus could not cause the disease and death of the mice. On the 3rd day of infection, the replication titer of the virus in the lung of mice was 100 times lower than that of the wild type virus, and the titer of the virus in the lung of the mice on the 5th day was 1000 times lower than that of the wild type virus. This study preliminarily explored the feasibility of reducing the virulence of influenza A virus by optimizing the route of genome codon modification. It is the first time that the codon optimization of NS1 gene codon of influenza virus can reduce the virulence of influenza virus. It provides a new idea for the study of live attenuated influenza vaccine.
【学位授予单位】：中国科学技术大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【共引文献】