摘要
目的利用自主知识产权抗体3E1,确定蓖麻毒素B链与细胞特异性结合的活性肽部位,通过构建含肽的绿色荧光蛋白原核表达载体,实现含肽的绿色荧光蛋白在大肠杆菌中的高效、可溶性表达,评价小鼠舌下口服含蓖麻毒素B链肽的绿色荧光蛋白后黏膜免疫应答的能力。方法用生物信息学技术,确定蓖麻毒素B链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;镍金属离子鏊合柱纯化目的蛋白;间接ELISA测定小鼠舌下口服不同肽-GFP融合蛋白后各种抗体水平的变化。结果实现了含不同肽的GFP在大肠杆菌中的高效、可溶性表达,获得了纯度大于90%的肽-GFP融合蛋白。免疫小鼠后,小鼠血清中均检测到了较高水平的IgG抗体,其中的P1肽-GFP融合蛋白免疫小鼠血清中检测到了较高水平的IgG2a和IgM抗体。免疫组血清中IgG2b、IgG3、IgA水平与对照组无明显差异。结论分别用4个小分子肽-GFP融合蛋白免疫小鼠后,小鼠血清中均检测到了较高水平的IgG抗体,其中的P1肽-GFP融合蛋白免疫小鼠的血清中检测到了较高水平的IgG2a和IgM抗体,激发了Th1型免疫应答。
This paper is aimed to develop a new immunopotentiator peptide binding with mononuclear macrophage and determine the immunological effects of peptide-green fluorescent protein （GFP） fusion protein. Specific active sites of ricin toxin B binding with mononuclear macrophage were identified by bioinformatics technique, named P. Prokaryotic expression vectors were constructed and series high purity P-GFP fusion proteins were purified by Ni metal chelate chromatography. High levels IgG antibodies were detected by indirect ELISA after hypoglossal vaccination of Balb/c mice and high levels of IgG2a and IgM antibodies were detected in P1-GFP fusion protein inoculated mice too. Furthermore, antibody levels of IgG2b, IgG3 and IgA in hypoglossal vaccination Balb/c mice have no difference between experiment groups and control groups. Thus, we concluded that P1-GFP fusion protein can arouse a Thl type immune response.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2013年第11期966-969,共4页
Immunological Journal
基金
国家自然科学基金(30872394)
