摘要
目的:利用自主知识产权抗体3E1,确定RTB与细胞特异性结合的活性肽部位,基因工程合成后,动物实验评价绿色荧光蛋白在基于RTB活性肽靶向下在体内的分布,为后续研究奠定基础。方法:用生物信息学技术,确定RTB与细胞特异性结合的活性肽部位;分子克隆技术构建含不同肽的绿色荧光蛋白原核表达载体,实现肽-GFP融合蛋白在大肠杆菌中的高效、可溶性表达;动物实验评价小鼠舌下口服肽-GFP融合蛋白后GFP在体内的分布。结果:建立了自主知识产权单抗3E1与RTB相互作用的空间构象,判定出四个活性区域,构建了4个含不同活性肽的GFP原核表达载体,获得了高纯度蛋白;经舌下口服免疫BALB/c小鼠后,免疫组化和流式细胞术证实,P1-GFP免疫组GFP和CD11c在不同组织中的分布与对照组有明显区别。P1-GFP初次免疫后,GFP主要分布于空肠,再次免疫后,GFP主要集中于回肠、肠相关淋巴组织和小肠PP结;初次免疫后CD11c+细胞主要分布于肠系膜淋巴结,胃和空肠,再次免疫后肠系膜淋巴结中的CD11c+细胞不再占优势,空肠中CD11c+细胞明显增加。结论:基于RTB的活性肽P1与GFP融合蛋白口服免疫小鼠后,P1-GFP经P1肽的导向,初次和再次免疫后GFP在体内的分布不同,再次免疫后范围更广。P1的导向使GFP直接进入小肠PP结和肠系膜淋巴结两个黏膜免疫应答的主要诱导及效应部位,利于免疫应答的发生。
Objective：Based on independent intellectual property rights monoclonal antibody 3E1,specific active sites of RTB binding with mononuclear macrophage are identified.After synthesis peptide GFP fusion protein by Gene Engineering Technique,immunopotency assessments were performed after oral vaccination peptide GFP fusion protein in BALB/c mice by hypoglossal.Methods：Specific active sites of RTB binding with mononuclear macrophage were identified by bioinformatics technique;prokaryotic expression vectors including 4 individual peptide fusion with GFP be constructed by molecular cloning.High purity and soluble peptide-GFP fusion protein be obtained by expression in E.Coli;distribution of GFP be detected by immunohistochemisty and flow cytometry.Results：A specific spatial conformation of independent intellectual property right monoclonal antibody 3E1 binding with galactose binding sites of RTB would be established and 4 active region be identified too.4 prokaryotic expression vectors including different active region and green fluorescent protein（GFP） genes be constructed and high purity target protein obtained.After oral vaccination BALB/c mice by hypoglossal,distribution of GFP and CD11c in different tissues had no difference assured by immunohistochemisty and flow cytometry.GFP distributed in jejunum after primary immunization and distributed in ileum,gut associated lymphoid tissue and small intestinal lymph node after secondary immunization.The distribution of CD11c＋cells major in mesenteric lymph node,stomach and jejunum after primary immunization.And the dominance in mesenteric lymph node to substitute with jejunum after secondary immunization.Conclusion：After oral vaccination BALB /c mice with peptide-GFP fusion protein which targeting with P1 peptide by hypoglossal,the distribution between primary and secondary immunization was different and had a more wide distribution after secondary immunization.Targeting of the P1 peptide made the GFP into the small intestinal lymph node and the mesenteric lymph node more easily which were the major induced and effective part of Mucosal Immunity response.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2013年第9期974-980,988,共8页
Chinese Journal of Immunology
基金
国家自然科学基金资助项目(No.30872394)
