目的:利用噬菌体随机肽库筛选与人肺泡Ⅱ型上皮A549细胞存在特异性结合的多肽,为实现对肺泡Ⅱ型上皮细胞的靶向干预和开发针对急性肺损伤等呼吸系统疾病治疗的导向药物载体提供实验依据。方法:采用全细胞筛选的方式,通过对人肺泡Ⅱ型上...目的:利用噬菌体随机肽库筛选与人肺泡Ⅱ型上皮A549细胞存在特异性结合的多肽,为实现对肺泡Ⅱ型上皮细胞的靶向干预和开发针对急性肺损伤等呼吸系统疾病治疗的导向药物载体提供实验依据。方法:采用全细胞筛选的方式,通过对人肺泡Ⅱ型上皮A549细胞的4轮筛选和与人前列腺癌上皮PC-3细胞的差减筛选,从噬菌体随机肽库中筛选出与人肺泡Ⅱ型上皮A549细胞存在特异性结合的多肽,选择丰度较高的一条命名为NTG,用其构建含有增强绿色荧光蛋白(EGFP)的原核表达载体,转化感受态细菌,层析法获得重组蛋白His-NTG-EGFP,测定其分子质量。向A549细胞和PC-3细胞分别加入含有重组蛋白His-NTG-EGFP或His-EGFP的培养基,荧光显微镜下观察。结果:从第1轮至第4轮,淘选所得的噬菌体回收率逐步提高,经过PC-3细胞差减筛选,少量的与A549细胞存在特异性结合的噬菌体得到了回收。重组表达并纯化得到的His-NTG-EGFP,测定其分子质量为33 k D,与预期相符。荧光显微镜下观察发现,经融合蛋白His-NTG-EGFP处理后的A549细胞表面有绿色荧光分布,经融合蛋白His-NTG-EGFP处理后的PC-3细胞以及经对照蛋白处理的A549细胞和PC-3细胞表面均未出现绿色荧光,说明这一融合蛋白可以特异性结合A549细胞。结论:人肺泡Ⅱ型上皮A549细胞特异性靶向肽被成功获得。展开更多
Allergen specific IgE response is the major cause of immediate hypersensitivity. However the number of IgEproducing B cells and the amount of IgE, especially the specific IgE, are so low, it greatly impedes the study ...Allergen specific IgE response is the major cause of immediate hypersensitivity. However the number of IgEproducing B cells and the amount of IgE, especially the specific IgE, are so low, it greatly impedes the study of the allergic-specifc antibody responses. Here we report the construction of a normal human IgE combinatorial library The repertoire of IgE VH genes and of K genes were separately amplified from normal human peripheral blood lymphocytes through RT-PCR, and were then constructed to form the phage surface display human Fab(IgEVH) library. A plant protein allergen, trichosanthin(TCS), was used to affinity-enrich and to screen the anti-TCS phage HuFab clones from the library. Human IgE(Fab) to TCS were detected.展开更多
To select the immunogenic short peptide mimics of male worm origin of Schistosoma japonicum (Sj) and to explore their protection effect against schistosomiasis in mice, the random phage display peptide library of 12-m...To select the immunogenic short peptide mimics of male worm origin of Schistosoma japonicum (Sj) and to explore their protection effect against schistosomiasis in mice, the random phage display peptide library of 12-mer was screened with IgG to soluble male worm antigen of Sj, and the specific positive clones selected through three rounds of screenings were detected by Dot-ELISA, and then injected subcutaneously into mice for vaccination and protection assessment against Sj. It was found that 18 randomly picked phage displayed clones all showed definite antigenicity with various intensities. The pooled phages displayed clones could induce production of specific antibodies and cause 31.72% of worm reduction rate and 51.54% of egg reduction rate in mice, revealing a significant difference (P<0.001) in comparison with those of the controls. It concludes that the short peptide mimics of male worm origin of Sj obtained by affinity screening phage display peptide library can elicit partial protection against this pathogen.展开更多
文摘目的:利用噬菌体随机肽库筛选与人肺泡Ⅱ型上皮A549细胞存在特异性结合的多肽,为实现对肺泡Ⅱ型上皮细胞的靶向干预和开发针对急性肺损伤等呼吸系统疾病治疗的导向药物载体提供实验依据。方法:采用全细胞筛选的方式,通过对人肺泡Ⅱ型上皮A549细胞的4轮筛选和与人前列腺癌上皮PC-3细胞的差减筛选,从噬菌体随机肽库中筛选出与人肺泡Ⅱ型上皮A549细胞存在特异性结合的多肽,选择丰度较高的一条命名为NTG,用其构建含有增强绿色荧光蛋白(EGFP)的原核表达载体,转化感受态细菌,层析法获得重组蛋白His-NTG-EGFP,测定其分子质量。向A549细胞和PC-3细胞分别加入含有重组蛋白His-NTG-EGFP或His-EGFP的培养基,荧光显微镜下观察。结果:从第1轮至第4轮,淘选所得的噬菌体回收率逐步提高,经过PC-3细胞差减筛选,少量的与A549细胞存在特异性结合的噬菌体得到了回收。重组表达并纯化得到的His-NTG-EGFP,测定其分子质量为33 k D,与预期相符。荧光显微镜下观察发现,经融合蛋白His-NTG-EGFP处理后的A549细胞表面有绿色荧光分布,经融合蛋白His-NTG-EGFP处理后的PC-3细胞以及经对照蛋白处理的A549细胞和PC-3细胞表面均未出现绿色荧光,说明这一融合蛋白可以特异性结合A549细胞。结论:人肺泡Ⅱ型上皮A549细胞特异性靶向肽被成功获得。
文摘Allergen specific IgE response is the major cause of immediate hypersensitivity. However the number of IgEproducing B cells and the amount of IgE, especially the specific IgE, are so low, it greatly impedes the study of the allergic-specifc antibody responses. Here we report the construction of a normal human IgE combinatorial library The repertoire of IgE VH genes and of K genes were separately amplified from normal human peripheral blood lymphocytes through RT-PCR, and were then constructed to form the phage surface display human Fab(IgEVH) library. A plant protein allergen, trichosanthin(TCS), was used to affinity-enrich and to screen the anti-TCS phage HuFab clones from the library. Human IgE(Fab) to TCS were detected.
文摘To select the immunogenic short peptide mimics of male worm origin of Schistosoma japonicum (Sj) and to explore their protection effect against schistosomiasis in mice, the random phage display peptide library of 12-mer was screened with IgG to soluble male worm antigen of Sj, and the specific positive clones selected through three rounds of screenings were detected by Dot-ELISA, and then injected subcutaneously into mice for vaccination and protection assessment against Sj. It was found that 18 randomly picked phage displayed clones all showed definite antigenicity with various intensities. The pooled phages displayed clones could induce production of specific antibodies and cause 31.72% of worm reduction rate and 51.54% of egg reduction rate in mice, revealing a significant difference (P<0.001) in comparison with those of the controls. It concludes that the short peptide mimics of male worm origin of Sj obtained by affinity screening phage display peptide library can elicit partial protection against this pathogen.
