以减毒鼠伤寒沙门菌为递送载体的人呼吸道合胞病毒融合蛋白DNA疫苗滴鼻免疫抗体分析
发布时间：2018-10-09 21:50
【摘要】：目的:人呼吸道合胞病毒(Human respiratory syncytial virus,RSV)广泛分布于世界各地,是导致婴幼儿严重下呼吸道感染的最重要的病毒病原。目前尚无特异性防治方法,世界卫生组织将发展RSV疫苗列为最优先发展的疫苗项目之一。RSV的11种蛋白中,融合蛋白(Fusion glycoprotein,F)是中和抗原,免疫动物后,可以产生具有免疫保护作用的中和抗体。本研究以减毒鼠伤寒沙门菌(Salmonella typhimurium aroA strain SL7207, SL7207)为载体携带可表达RSV密码子优化的F蛋白的真核表达质粒,探讨不同黏膜免疫途径及密码子优化对免疫效果的影响。 方法:通过对RSV野生型F基因(Fwt)进行密码子优化,获得密码子优化的F基因(Fsyn),并构建可表达Fsyn的真核表达质粒pcDNA3.1/Fsyn,通过Western blot分析来检测真核表达质粒pcDNA3.1/Fsyn的体外表达水平。转化SL7207得到SL7207/pcDNA3.1/Fsyn ,构建重组的减毒鼠伤寒沙门菌疫苗SL7207/pcDNA3.1/Fsyn。经重组菌体外稳定性试验后,分别经单次滴鼻和灌胃免疫途径,免疫BALB/c小鼠,通过体液免疫、黏膜免疫和细胞免疫等指标分析免疫后小鼠RSV特异性免疫应答产生情况,以及免疫效果的持续时间。 结果:取已转化pcDNA3.1/Fsyn的SL7207提取质粒,经限制性内切酶分析、测序与预期一致。与野生型相比较,可表达密码子优化型F蛋白的真核表达质粒pcDNA3.1/Fsyn体外表达水平高;重组的减毒鼠伤寒沙门菌疫苗SL7207/pcDNA3.1/Fsyn具有较好的体外传代稳定性(保持在89%以上)。与灌胃组相比,滴鼻组诱导小鼠产生了更高水平的血清IgG和呼吸道黏膜分泌型IgA(Secretary IgA, SIgA),获得了更好的免疫效果(P0.05)。与野生型相比,密码子优化的F蛋白具有更好的免疫原性(P0.05)。 结论:获得的重组减毒鼠伤寒沙门菌疫苗SL7207/pcDNA3.1/Fsyn,能够诱导机体产生有效的体液免疫和局部免疫。经滴鼻途径免疫和密码子优化能够提高以SL7207为载体的RSV DNA疫苗免疫效果。因此我们认为以减毒伤寒沙门菌为载体的滴鼻DNA疫苗兼具黏膜免疫及DNA疫苗的双重优点,是RSV等通过黏膜途径感染的病毒疫苗研究的重要方向,为今后RSV疫苗免疫策略等研究奠定了坚实基础。
[Abstract]:Objective: human respiratory syncytial virus (Human respiratory syncytial virus,RSV) is widely distributed all over the world and is the most important viral pathogen leading to severe lower respiratory tract infection in infants. At present, there is no specific method of prevention and cure. The World Health Organization (WHO) regards the development of RSV vaccine as one of the most priority vaccine projects. Among the 11 proteins, the fusion protein (Fusion glycoprotein,F) is the neutralizing antigen, after immunizing animals. Neutralizing antibodies with immune protection can be produced. In this study, attenuated Salmonella typhimurium (Salmonella typhimurium aroA strain SL7207, SL7207) was used as the vector carrying the eukaryotic expression plasmid of F protein which could express the codon of RSV. The effects of different mucosal immune pathways and codon optimization on the immune response were investigated. Methods: by codon optimization of RSV wild-type F gene (Fwt), the codon optimized F gene (Fsyn), was obtained and the eukaryotic expression plasmid pcDNA3.1/Fsyn, expressing Fsyn was constructed to detect the expression level of eukaryotic expression plasmid pcDNA3.1/Fsyn by Western blot analysis. Transformation of SL7207 to SL7207/pcDNA3.1/Fsyn, construction of recombinant attenuated Salmonella typhimurium vaccine SL7207/pcDNA3.1/Fsyn. After in vitro stability test of recombinant bacteria, BALB/c mice were immunized with single nasal drip and oral administration respectively. The specific immune response of RSV was analyzed by humoral immunity, mucosal immunity and cellular immunity. And the duration of the immune effect. Results: the SL7207 of transformed pcDNA3.1/Fsyn was extracted and analyzed by restriction endonuclease. Compared with wild type, the eukaryotic expression plasmid pcDNA3.1/Fsyn expressing codon optimized F protein had higher expression level in vitro, and the recombinant attenuated Salmonella typhimurium vaccine SL7207/pcDNA3.1/Fsyn had better in vitro subculture stability (above 89%). Compared with the gavage group, the nasal drip group produced a higher level of serum IgG and respiratory mucosal secretory IgA (Secretary IgA, SIgA), (P0.05). Compared with wild type, F protein optimized by codon had better immunogenicity (P0.05). Conclusion: the recombinant attenuated Salmonella typhimurium vaccine SL7207/pcDNA3.1/Fsyn, can induce effective humoral and local immunity. Immunization by nasal drip and codon optimization can improve the immune effect of RSV DNA vaccine with SL7207 as vector. Therefore, we think that the nasal DNA vaccine with attenuated Salmonella typhimurium as the carrier has both the advantages of mucosal immunity and DNA vaccine, which is an important research direction of RSV and other virus vaccines infected by mucosal pathway. For the future RSV vaccine immunization strategy and other research laid a solid foundation.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
[Abstract]:Objective: human respiratory syncytial virus (Human respiratory syncytial virus,RSV) is widely distributed all over the world and is the most important viral pathogen leading to severe lower respiratory tract infection in infants. At present, there is no specific method of prevention and cure. The World Health Organization (WHO) regards the development of RSV vaccine as one of the most priority vaccine projects. Among the 11 proteins, the fusion protein (Fusion glycoprotein,F) is the neutralizing antigen, after immunizing animals. Neutralizing antibodies with immune protection can be produced. In this study, attenuated Salmonella typhimurium (Salmonella typhimurium aroA strain SL7207, SL7207) was used as the vector carrying the eukaryotic expression plasmid of F protein which could express the codon of RSV. The effects of different mucosal immune pathways and codon optimization on the immune response were investigated. Methods: by codon optimization of RSV wild-type F gene (Fwt), the codon optimized F gene (Fsyn), was obtained and the eukaryotic expression plasmid pcDNA3.1/Fsyn, expressing Fsyn was constructed to detect the expression level of eukaryotic expression plasmid pcDNA3.1/Fsyn by Western blot analysis. Transformation of SL7207 to SL7207/pcDNA3.1/Fsyn, construction of recombinant attenuated Salmonella typhimurium vaccine SL7207/pcDNA3.1/Fsyn. After in vitro stability test of recombinant bacteria, BALB/c mice were immunized with single nasal drip and oral administration respectively. The specific immune response of RSV was analyzed by humoral immunity, mucosal immunity and cellular immunity. And the duration of the immune effect. Results: the SL7207 of transformed pcDNA3.1/Fsyn was extracted and analyzed by restriction endonuclease. Compared with wild type, the eukaryotic expression plasmid pcDNA3.1/Fsyn expressing codon optimized F protein had higher expression level in vitro, and the recombinant attenuated Salmonella typhimurium vaccine SL7207/pcDNA3.1/Fsyn had better in vitro subculture stability (above 89%). Compared with the gavage group, the nasal drip group produced a higher level of serum IgG and respiratory mucosal secretory IgA (Secretary IgA, SIgA), (P0.05). Compared with wild type, F protein optimized by codon had better immunogenicity (P0.05). Conclusion: the recombinant attenuated Salmonella typhimurium vaccine SL7207/pcDNA3.1/Fsyn, can induce effective humoral and local immunity. Immunization by nasal drip and codon optimization can improve the immune effect of RSV DNA vaccine with SL7207 as vector. Therefore, we think that the nasal DNA vaccine with attenuated Salmonella typhimurium as the carrier has both the advantages of mucosal immunity and DNA vaccine, which is an important research direction of RSV and other virus vaccines infected by mucosal pathway. For the future RSV vaccine immunization strategy and other research laid a solid foundation.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
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