【摘要】： 上呼吸道接種免疫作為新型的FMDV接種途徑,正在被越來越多的預(yù)防醫(yī)學(xué)研究工作者所重視。很大一部分流行病都是由呼吸道感染機(jī)體,因此是否能夠建立起上呼吸道黏膜免疫保護(hù)是機(jī)體能否抵御病毒入侵的關(guān)鍵,上呼吸道粘膜免疫保護(hù)機(jī)制也為預(yù)防醫(yī)學(xué)提供了研究防控疾病的有效途徑。粘膜佐劑作為黏膜免疫研究的一個(gè)重要組成部分能夠顯著增強(qiáng)粘膜免疫保護(hù)效果。很多免疫增強(qiáng)劑都被應(yīng)用到黏膜免疫的研究中來,但絕大多數(shù)佐劑都是經(jīng)驗(yàn)總結(jié)的產(chǎn)物,作用機(jī)理難以透徹研究。FMDV經(jīng)濟(jì)上具有重大的影響,其每次大規(guī)模爆發(fā)流行都會給人類帶來了數(shù)以億計(jì)的經(jīng)濟(jì)損失,研究防控各亞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對防控該病流行是行之有效的手段之一。 本研究結(jié)合了上呼吸道黏膜免疫、FMDV抗原表位多肽、CTB佐劑、核酸佐劑和細(xì)胞因子佐劑等研究內(nèi)容在小鼠上進(jìn)行了對比試驗(yàn)。利用分子生物學(xué)技術(shù)、基因工程技術(shù)、生物信息學(xué)技術(shù)和DNA逆向工程技術(shù)構(gòu)建Asia 1 FMDV抗原表位多肽基因經(jīng)Linker蛋白基因與CTB多肽基因串聯(lián)構(gòu)建了抗原表位多肽基因31c。反轉(zhuǎn)錄法克隆了成年牛干擾素cDNA即BoIFN-γ基因,將表達(dá)純化得到的BoIFN-γ成熟多肽蛋白作為了輔助31C多肽的佐劑。在建立的多肽與全病毒對照組、細(xì)胞因子核酸佐劑與活性多肽佐劑對照組、上呼吸道免疫與皮下接種免疫對照組和CTB佐劑研究組等一系列研究中,通過間接ELISA檢測分泌型IgA抗體水平,應(yīng)用微量血清中和試驗(yàn)檢測體液免疫水平,淋巴細(xì)胞增殖實(shí)驗(yàn)檢測細(xì)胞免疫水平以及應(yīng)用反轉(zhuǎn)錄熒光定量PCR檢測IL-2、IL-4、IL-10和IFN-γ在頸部淋巴、肺臟及脾臟內(nèi)的變化。通過以上數(shù)據(jù)統(tǒng)計(jì)分析得出黏膜免疫保護(hù)水平、全身體液免疫水平、全身細(xì)胞免疫水平和黏膜免疫誘導(dǎo)應(yīng)答調(diào)節(jié)各個(gè)器官的細(xì)胞因子調(diào)節(jié)變化規(guī)律。 實(shí)驗(yàn)數(shù)據(jù)表明人工合成的抗原表位級聯(lián)多肽31C的免疫活性顯著高于商品化多肽,證明了抗原表位多肽31C可以誘導(dǎo)小鼠產(chǎn)生中和性抗體和分泌型抗體。證明了鼻腔免疫多肽抗原不僅可以誘導(dǎo)口腔及鼻腔內(nèi)產(chǎn)生分泌型抗體,其還可以誘導(dǎo)遠(yuǎn)端生殖道內(nèi)產(chǎn)生分泌型抗體。鼻腔免疫具有較皮下免疫更早誘導(dǎo)免疫記憶性細(xì)胞的能力。驗(yàn)證了CTB佐劑可以輔助融合表達(dá)多肽顯著提高后者的免疫原性。且證明了CTB具有提高口鼻分泌液中sIgA抗體水平的功效,通過鼻腔免疫小鼠,CTB可以顯著增加結(jié)合在其表面抗原表位的體液免疫抗體水平。研究了mBoIFN-γ多肽pBoIFN-γ基因在粘膜佐劑中的作用,mBoIFN-γ作為效應(yīng)細(xì)胞因子可以顯著增強(qiáng)注入部位的IFN-γ表達(dá)水平和sIgA表達(dá)水平,并能增強(qiáng)全身的細(xì)胞免疫水平。pBoIFN-γ基因在作為粘膜佐劑注入機(jī)體后能持續(xù)發(fā)揮免疫增強(qiáng)效應(yīng),但是對于免疫部位而言,由于其連續(xù)持續(xù)的免疫使得局部免疫細(xì)胞對IFN-γ反應(yīng)產(chǎn)生耐受并顯著降低免疫位點(diǎn)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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R392

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