FMDV抗原表位多肽及分子佐劑對小鼠上呼吸道免疫的影響
發(fā)布時間：2018-08-27 18:55
【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防醫(yī)學研究工作者所重視。很大一部分流行病都是由呼吸道感染機體,因此是否能夠建立起上呼吸道黏膜免疫保護是機體能否抵御病毒入侵的關鍵,上呼吸道粘膜免疫保護機制也為預防醫(yī)學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重要組成部分能夠顯著增強粘膜免疫保護效果。很多免疫增強劑都被應用到黏膜免疫的研究中來,但絕大多數佐劑都是經驗總結的產物,作用機理難以透徹研究。FMDV經濟上具有重大的影響,其每次大規(guī)模爆發(fā)流行都會給人類帶來了數以億計的經濟損失,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、核酸佐劑和細胞因子佐劑等研究內容在小鼠上進行了對比試驗。利用分子生物學技術、基因工程技術、生物信息學技術和DNA逆向工程技術構建Asia 1 FMDV抗原表位多肽基因經Linker蛋白基因與CTB多肽基因串聯構建了抗原表位多肽基因31c。反轉錄法克隆了成年牛干擾素cDNA即BoIFN-γ基因,將表達純化得到的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,應用微量血清中和試驗檢測體液免疫水平,淋巴細胞增殖實驗檢測細胞免疫水平以及應用反轉錄熒光定量PCR檢測IL-2、IL-4、IL-10和IFN-γ在頸部淋巴、肺臟及脾臟內的變化。通過以上數據統計分析得出黏膜免疫保護水平、全身體液免疫水平、全身細胞免疫水平和黏膜免疫誘導應答調節(jié)各個器官的細胞因子調節(jié)變化規(guī)律。 實驗數據表明人工合成的抗原表位級聯多肽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可以誘導小鼠產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口腔及鼻腔內產生分泌型抗體,其還可以誘導遠端生殖道內產生分泌型抗體。鼻腔免疫具有較皮下免疫更早誘導免疫記憶性細胞的能力。驗證了CTB佐劑可以輔助融合表達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作為效應細胞因子可以顯著增強注入部位的IFN-γ表達水平和sIgA表達水平,并能增強全身的細胞免疫水平。pBoIFN-γ基因在作為粘膜佐劑注入機體后能持續(xù)發(fā)揮免疫增強效應,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胞對IFN-γ反應產生耐受并顯著降低免疫位點的局部免疫水平。
[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.
【學位授予單位】：東北農業(yè)大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R392
本文編號：2208139
[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.
【學位授予單位】：東北農業(yè)大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R392
【參考文獻】
相關期刊論文 前9條
1 李伍舉,吳加金;pBV220載體中外源基因表達水平定量分析[J];病毒學報;1997年02期
2 易建中,黃海斌,楊志軍,黃妤,鄧余,嚴維耀,丁恩雨,龍永進,徐泉興,鄭兆鑫;A型口蹄疫病毒VP1免疫活性肽基因串聯片段的化學合成及克隆與表達[J];復旦學報(自然科學版);1997年05期
3 楊志軍,林焱,李光金,嚴維耀,徐泉興,尤永進,鄭兆鑫;含T細胞表位和B細胞表位的抗O型FMDV基因工程疫苗誘導豚鼠產生免疫反應[J];復旦學報(自然科學版);2000年05期
4 李光金,吳永慶,嚴維耀,徐泉興,盛祖恬,尤永進,葛燕,鄭兆鑫;聯用抗口蹄疫病毒重組多肽及DNA疫苗誘發(fā)豚鼠產生特異免疫應答[J];高技術通訊;2001年06期
5 趙凱,陳光輝,張震宇,嚴維耀,鄭兆鑫,孫理云,盛祖恬,尤永進,徐泉興,朱彩珠,馮霞,張強,盧永干;以免疫球蛋白為載體的抗O型口蹄疫病毒基因工程疫苗的構建[J];生物工程學報;2000年06期
6 李光金,嚴維耀,徐泉興,盛祖恬,鄭兆鑫;以豬IgG重鏈恒定區(qū)為抗原載體的抗口蹄疫病毒DNA疫苗的研制[J];生物工程學報;2001年03期
7 李伍舉,吳加金;pBV220載體中外源基因二級結構與表達水平關系[J];生物技術通訊;1996年04期
8 呂宏亮;劉松友;董小曼;沈心亮;畢勝利;馮玉萍;馬忠仁;;干擾素對M2e-HBc疫苗鼻內免疫的佐劑作用[J];醫(yī)學研究雜志;2008年11期
9 盧曾軍,曹軼梅,包惠芳,劉在新,趙啟祖,陳應理,�；菔|,謝慶閣;同源重組法制備口蹄疫病毒多基因重組腺病毒[J];中國病毒學;2004年02期
，本文編號：2208139
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2208139.html
最近更新
教材專著
