新疆家蚕抗菌肽的抗体制备及鉴定

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.

Preparation and identification of antibody against Cecropin-XJ

AIM: To prepare the mouse antibody against Cecropin-XJ, identify its specificity and use it in cellular localization of Cecropin-XJ in vitro. METHODS: The cDNA sequence of Cecropin-XJ with signal peptide sequence was subcloned into the eukaryotic expression vector pcDNA3. The recombinant plasmid was named pcDNA3-Cecropin-XJ, which was used as an antigen to immune the Kunming white mouse. Meanwhile, the cDNA sequence was subcloned into the fusion expression vector and the fusion protein was expressed as a test antigen. RESULTS: Indirect ELISA showed that the fifth immunity's titer was highest. The immune gold-conjugated experiment showed that the prepared antibody was clearly and efficiently localized in prokaryotic cells where the Cecropin-XJ worked. CONCLUSION: The antibody against Cecropin-XJ has high immune reactivity and specificity, which is beneficial to further study on Cecropin-XJ function and antibody preparation of small peptides.