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目的 :将肽库筛选到的正调控金黄色葡萄球菌 (金葡菌 )外毒素分泌的关键分子RAP(RNAⅢactivatingpro tein)的结合肽与人IgG1抗体Fc片段 (hFcγ1)融合表达 ,检测其干扰金葡菌外分泌毒素产生的活性。方法 :根据噬菌体肽库筛选到的RAP结合肽序列 ,选用大肠杆菌偏爱的密码子合成基因片段 ,将其融合在人IgG1抗体Fc片段基因序列的 5′端 ,构建pET rapbp fc融合表达载体 ,转化大肠杆菌并诱导表达。以包涵体形式存在的目的蛋白经变性、复性后 ,ELISA实验分别证实其与抗人IgG抗体及纯化RAP的结合活性。以MDBK细胞株为模型 ,MTT实验观察融合蛋白对金葡菌 196菌株外分泌毒素水平的影响。结果与结论 :融合基因在大肠杆菌中获得表达。复性后 ,融合蛋白的Fc片段及结合肽部分分别能够与抗人IgG抗体及RAP结合 ,且能在一定程度上降低金葡菌 196株上清中的毒素水平。
OBJECTIVE: To fuse and express the binding peptide of RAP (RNAⅢactivatingpro tein) secreted by Staphylococcus aureus exotoxin secreted by peptide library with the Fc fragment of human IgG1 antibody (hFcγ1) Bacterial exocrine toxin production activity. Methods: According to the RAP binding peptide sequences screened by phage peptide library, the preferred codon usage gene of E. coli was selected and fused to the 5 ’end of the Fc region of human IgG1 antibody to construct the fusion expression vector pET rapbp fc. E. coli and induce expression. The target protein in the form of inclusion bodies was denatured and refolded, and its binding activity to anti-human IgG antibody and purified RAP was confirmed by ELISA. Using MDBK cell line as a model, MTT assay was used to observe the effect of the fusion protein on the exocrine toxin level of Staphylococcus aureus 196 strain. RESULTS AND CONCLUSION: The fusion gene was expressed in E. coli. After refolding, the Fc fragment and the binding peptide of the fusion protein could bind to anti-human IgG antibody and RAP, respectively, and could reduce the level of toxin in the supernatant of S. aureus 196 to a certain extent.