以減毒鼠傷寒沙門菌為遞送載體的人呼吸道合胞病毒融合蛋白DNA疫苗滴鼻免疫抗體分析
發(fā)布時間：2018-10-09 21:50
【摘要】：目的:人呼吸道合胞病毒(Human respiratory syncytial virus,RSV)廣泛分布于世界各地,是導致嬰幼兒嚴重下呼吸道感染的最重要的病毒病原。目前尚無特異性防治方法,世界衛(wèi)生組織將發(fā)展RSV疫苗列為最優(yōu)先發(fā)展的疫苗項目之一。RSV的11種蛋白中,融合蛋白(Fusion glycoprotein,F)是中和抗原,免疫動物后,可以產(chǎn)生具有免疫保護作用的中和抗體。本研究以減毒鼠傷寒沙門菌(Salmonella typhimurium aroA strain SL7207, SL7207)為載體攜帶可表達RSV密碼子優(yōu)化的F蛋白的真核表達質(zhì)粒,探討不同黏膜免疫途徑及密碼子優(yōu)化對免疫效果的影響。 方法:通過對RSV野生型F基因(Fwt)進行密碼子優(yōu)化,獲得密碼子優(yōu)化的F基因(Fsyn),并構建可表達Fsyn的真核表達質(zhì)粒pcDNA3.1/Fsyn,通過Western blot分析來檢測真核表達質(zhì)粒pcDNA3.1/Fsyn的體外表達水平。轉(zhuǎn)化SL7207得到SL7207/pcDNA3.1/Fsyn ,構建重組的減毒鼠傷寒沙門菌疫苗SL7207/pcDNA3.1/Fsyn。經(jīng)重組菌體外穩(wěn)定性試驗后,分別經(jīng)單次滴鼻和灌胃免疫途徑,免疫BALB/c小鼠,通過體液免疫、黏膜免疫和細胞免疫等指標分析免疫后小鼠RSV特異性免疫應答產(chǎn)生情況,以及免疫效果的持續(xù)時間。 結果:取已轉(zhuǎn)化pcDNA3.1/Fsyn的SL7207提取質(zhì)粒,經(jīng)限制性內(nèi)切酶分析、測序與預期一致。與野生型相比較,可表達密碼子優(yōu)化型F蛋白的真核表達質(zhì)粒pcDNA3.1/Fsyn體外表達水平高;重組的減毒鼠傷寒沙門菌疫苗SL7207/pcDNA3.1/Fsyn具有較好的體外傳代穩(wěn)定性(保持在89%以上)。與灌胃組相比,滴鼻組誘導小鼠產(chǎn)生了更高水平的血清IgG和呼吸道黏膜分泌型IgA(Secretary IgA, SIgA),獲得了更好的免疫效果(P0.05)。與野生型相比,密碼子優(yōu)化的F蛋白具有更好的免疫原性(P0.05)。 結論:獲得的重組減毒鼠傷寒沙門菌疫苗SL7207/pcDNA3.1/Fsyn,能夠誘導機體產(chǎn)生有效的體液免疫和局部免疫。經(jīng)滴鼻途徑免疫和密碼子優(yōu)化能夠提高以SL7207為載體的RSV DNA疫苗免疫效果。因此我們認為以減毒傷寒沙門菌為載體的滴鼻DNA疫苗兼具黏膜免疫及DNA疫苗的雙重優(yōu)點,是RSV等通過黏膜途徑感染的病毒疫苗研究的重要方向,為今后RSV疫苗免疫策略等研究奠定了堅實基礎。
[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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392
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