节点文献
FMDV抗原表位多肽及分子佐剂对小鼠上呼吸道免疫的影响
The Immune Response of Mouse upper Respiratory Tract Vaccinated by FMDV Epitopes Peptide with Molecular Adjuvants
【作者】 李勐；
【导师】 王君伟；
【作者基本信息】 东北农业大学 ， 预防兽医学， 2009， 博士
【摘要】 上呼吸道接种免疫作为新型的FMDV接种途径,正在被越来越多的预防医学研究工作者所重视。很大一部分流行病都是由呼吸道感染机体,因此是否能够建立起上呼吸道黏膜免疫保护是机体能否抵御病毒入侵的关键,上呼吸道粘膜免疫保护机制也为预防医学提供了研究防控疾病的有效途径。粘膜佐剂作为黏膜免疫研究的一个重要组成部分能够显著增强粘膜免疫保护效果。很多免疫增强剂都被应用到黏膜免疫的研究中来,但绝大多数佐剂都是经验总结的产物,作用机理难以透彻研究。FMDV经济上具有重大的影响,其每次大规模爆发流行都会给人类带来了数以亿计的经济损失,研究防控各亚型大流行的新型高通量疫苗迫在眉睫。因此研究上呼吸道黏膜免疫对防控该病流行是行之有效的手段之一。本研究结合了上呼吸道黏膜免疫、FMDV抗原表位多肽、CTB佐剂、核酸佐剂和细胞因子佐剂等研究内容在小鼠上进行了对比试验。利用分子生物学技术、基因工程技术、生物信息学技术和DNA逆向工程技术构建Asia 1 FMDV抗原表位多肽基因经Linker蛋白基因与CTB多肽基因串联构建了抗原表位多肽基因31c。反转录法克隆了成年牛干扰素cDNA即BoIFN-γ基因,将表达纯化得到的BoIFN-γ成熟多肽蛋白作为了辅助31C多肽的佐剂。在建立的多肽与全病毒对照组、细胞因子核酸佐剂与活性多肽佐剂对照组、上呼吸道免疫与皮下接种免疫对照组和CTB佐剂研究组等一系列研究中,通过间接ELISA检测分泌型IgA抗体水平,应用微量血清中和试验检测体液免疫水平,淋巴细胞增殖实验检测细胞免疫水平以及应用反转录荧光定量PCR检测IL-2、IL-4、IL-10和IFN-γ在颈部淋巴、肺脏及脾脏内的变化。通过以上数据统计分析得出黏膜免疫保护水平、全身体液免疫水平、全身细胞免疫水平和黏膜免疫诱导应答调节各个器官的细胞因子调节变化规律。实验数据表明人工合成的抗原表位级联多肽31C的免疫活性显著高于商品化多肽,证明了抗原表位多肽31C可以诱导小鼠产生中和性抗体和分泌型抗体。证明了鼻腔免疫多肽抗原不仅可以诱导口腔及鼻腔内产生分泌型抗体,其还可以诱导远端生殖道内产生分泌型抗体。鼻腔免疫具有较皮下免疫更早诱导免疫记忆性细胞的能力。验证了CTB佐剂可以辅助融合表达多肽显著提高后者的免疫原性。且证明了CTB具有提高口鼻分泌液中sIgA抗体水平的功效,通过鼻腔免疫小鼠,CTB可以显著增加结合在其表面抗原表位的体液免疫抗体水平。研究了mBoIFN-γ多肽pBoIFN-γ基因在粘膜佐剂中的作用,mBoIFN-γ作为效应细胞因子可以显著增强注入部位的IFN-γ表达水平和sIgA表达水平,并能增强全身的细胞免疫水平。pBoIFN-γ基因在作为粘膜佐剂注入机体后能持续发挥免疫增强效应,但是对于免疫部位而言,由于其连续持续的免疫使得局部免疫细胞对IFN-γ反应产生耐受并显著降低免疫位点的局部免疫水平。
【Abstract】 Upper respiratory tract immunity as a novel way of immunization, more workers in immunology research got into it. A lot of infectious disease infected body from the respiratory tract, which is the best research strategy to research how to control the virus provides. Mucosal adjuvant as an important component of mucosal immunity, a lot of scholars are researching immune enhancer for mucosal immune, but the mechanism of majority of adjuvants are unknown. FMDV as an enormous economic and political impact of the disease, has brought hundreds of millions of human beings in economic losses, Investigating the prevention and controling of the popular new subtype of large high-throughput vaccine is imminent.In this study, the upper respiratory tract mucosal immunity, FMDV peptide epitopes, CTB adjuvant, gene adjuvant and cytokine adjuvants were studied in mice. Molecular biology techniques, genetic engineering technology, bio-informatics technology and methods of reverse engineering DNA technology were used to build the Asia 1 FMDV peptide epitopes with CTB adjuvant by Linker Protein, we got a peptide 31C. Reverse transcription from the adult cow total RNA,we cloned BoIFN-γgene and purified mature BoIFN-γprotein peptide 31C as a adjuvant polypeptide. Comparing the peptide and the virus group, cytokine gene adjuvant and peptide adjuvant group, upper respiratory tract immunity and subcutaneous injection immunization group, CTB adjuvant group, we got the level of sIgA and serum antibody levels of humoral immunity by indirect ELISA detection, the level of cell-mediated immunity by lymphocyte proliferation assay, detectde the Change of IL-2, IL-4, IL-10 and IFN-γin the neck lymphatic, lung and spleen by Realtime RT-PCR. Analysing the data obtained through the above-mentioned we got the changes of protection of mucosal immunity, humoral immunity of the whole body, systemic cellular immunity and cytokines in various organs of the regulation changes.Experimental data show that the immunological activity of 31C was significantly higher than the commercialization of peptide epitopes, 31C polypeptide could make the mice get sIga and humoral antibody. Nasal immunization induced the reproductive tract get sIgA. Nasal immunization got memory cells more earlier than the other group. The CTB adjuvant can help significantly enhance epitope peptide immunogenicity. CTB not only enhance the level of sIgA by nasal immunization, It could significant increase the activity of the epitope protein immunogenicity on it. The mBoIFN-γcan significantly enhance IFN-γexpression and sIgA levels in the injection site, and to enhance the cellular immunity level of body . The pBoIFN-γgene injected in the body as a mucosal adjuvant had The immune-enhancing effects for a long time, at the inject sites, got the tolerance to second vaccination.Researching about the mucosal immunity and immune peptides stiff job, this experiment got a lot of breakthrough in it.