FMDV抗原表位多肽及分子佐剂对小鼠上呼吸道免疫的影响

发布时间：2018-08-27 18:55

【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 immunization, as a new way of FMDV inoculation, is being paid more and more attention by preventive medicine researchers. Most of the epidemics are caused by respiratory tract infection, so whether the upper respiratory tract mucosal immune protection can be established is the key to the body's ability to resist the invasion of the virus, and the upper respiratory tract mucosal immune protection. Mucosal adjuvants, as an important part of mucosal immunity research, can significantly enhance the protective effect of mucosal immunity. Many immunopotentiators have been applied to the study of mucosal immunity, but most of them are the product of experience and mechanism. It is difficult to make a thorough study. FMDV has a great economic impact. Every outbreak of FMDV will bring hundreds of millions of economic losses to human beings. It is imminent to study new high-throughput vaccines to prevent and control pandemics of various subtypes.
In this study, a comparative study was carried out in mice by combining the research contents of upper respiratory mucosal immunity, FMDV epitope polypeptide, CTB adjuvant, nucleic acid adjuvant and cytokine adjuvant. An antigenic epitope polypeptide gene 31C was constructed by connecting the R protein gene with the CTB polypeptide gene. BoIFN-gamma gene was cloned by reverse transcription. The BoIFN-gamma mature polypeptide protein was used as an adjuvant to assist 31C polypeptide. In the polypeptide and whole virus control group, cytokine nucleic acid adjuvant and active polypeptide were established. In a series of studies, such as adjuvant control group, upper respiratory tract immunization and subcutaneous immunization control group and CTB adjuvant research group, secretory IgA antibody level was detected by indirect ELISA, humoral immunity level was detected by microserum neutralization test, cellular immunity level was detected by lymphocyte proliferation test, and reverse transcription fluorescence quantitative PCR was used. The changes of IL-2, IL-4, IL-10 and IFN-gamma in cervical lymph nodes, lungs and spleens were measured.
The experimental data showed that the immune activity of synthesized antigen epitope cascade polypeptide 31C was significantly higher than that of commercial polypeptide, which proved that antigen epitope polypeptide 31C could induce neutral antibody and secretory antibody in mice. Nasal immunization has the ability to induce immune memory cells earlier than subcutaneous immunization. It is verified that CTB adjuvant can significantly enhance the immunogenicity of fusion-expressed peptides. It is also proved that CTB can increase the level of sIgA antibody in oral and nasal secretions, and immunize mice through nasal cavity. CT The role of mBoIFN-gamma polypeptide pBoIFN-gamma gene in mucosal adjuvant was studied. As an effector cytokine, mBoIFN-gamma could significantly enhance the expression of IFN-gamma and sIgA at the site of injection, and enhance the cellular immunity of the whole body. Genes can continue to exert immunopotentiatory effects after being injected as mucosal adjuvants. However, for immune sites, their continuous immunity makes local immune cells tolerate IFN-gamma response and significantly reduces local immune levels at immune sites.
【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392

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