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目的:制备鼠抗新疆家蚕抗菌肽(Cecropin-XJ)的抗体,用于抗菌肽的体外细胞定位分析。方法:将编码抗菌肽的cDNA序列亚克隆到真核表达载体pcDNA3上,构建重组真核表达载体pcDNA3/Cecropin-XJ,进行核酸免疫昆明白小鼠;同时构建原核表达载体,对抗菌肽进行融合表达,以表达的融合蛋白作为检测抗原。采用免疫电镜技术分析Cecropin-XJ的作用部位。结果:间接ELISA表明,重组质粒第5次免疫的效价最高;免疫胶体金实验显示,所制备的抗血清能够对抗菌肽作用部位进行准确清晰的定位。结论:所制备的鼠抗Cecropin-XJ的抗血清具有较高的免疫反应性和特异性,为Cecropin-XJ的抑菌机制研究提供了有力的工具,同时也为小分子肽抗体的制备提供了参考。
OBJECTIVE: To prepare antibodies against Cecropin-XJ in Xinjiang Silkworm (Bombyx mori) for the in vitro cell localization of antimicrobial peptides. Methods: The cDNA encoding antimicrobial peptide was subcloned into the eukaryotic expression vector pcDNA3 to construct the recombinant eukaryotic expression vector pcDNA3 / Cecropin-XJ and the nucleic acid was used to immunize Kunming white mice. At the same time, the prokaryotic expression vector was constructed and the antimicrobial peptides were fused The expressed fusion protein was used as detection antigen. Immuno-electron microscopy was used to analyze the site of Cecropin-XJ. Results: Indirect ELISA showed that the titer of recombinant plasmid was the highest at the fifth immunization. Immunogold gold assay showed that the prepared antiserum could locate the active site of antibacterial peptide accurately and clearly. CONCLUSION: The prepared antisera of mouse anti-Cecropin-XJ has high immunoreactivity and specificity, which provides a powerful tool for studying the antibacterial mechanism of Cecropin-XJ and also provides the preparation of small molecule peptide antibody reference.