卡他莫拉菌UspA1蛋白多克隆抗体的制备及鉴定

Preparation and identification of polyclonal antibodies against Moraxella catarrhalis UspA1

摘要:

为制备抗卡他莫拉菌 (Moraxella catarrhalis，Mc)表面蛋白UspA1胞外结构域的多克隆抗体 (PcAb)，对UspA1蛋白进行生物信息学分析，获取胞外结构域中抗原表位最为丰富的肽段，找到其对应的基因序列并引入大肠杆菌偏好性密码子，对其优化后化学合成全基因序列。将该基因序列按常规方法克隆入表达载体pET-28a(+)后表达重组UspA1-His融合蛋白并纯化。以该纯化抗原免疫新西兰大白兔，经4次免疫后，用Protein A亲和层析柱从抗血清中纯化出抗UspA1-His融合蛋白PcAb IgG。经免疫荧光法、酶联免疫吸附法及Western blotting鉴定，抗UspA1-His融合蛋白PcAb能特异性识别UspA1蛋白的表面暴露区。该多抗的制备为下一步建立卡他莫拉菌快速检测技术奠定了基础。

Abstract:

To prepare polyclonal antibodies (PcAb) against UspA1 of Moraxella catarrhalis (Mc), we used bioinformatic analysis to determine the surface exposed region in this protein that holds the antigen epitopes. Then the corresponding coding sequences for this fragment was artificially synthesized according to the codon usage of Escherichia coli. The gene fragment was then subcloned into the prokaryotic expression vector pET-28a(+) and expressed in E. coli rosseta (DE3), and then the recombinant UspA1-His proteins were purified. Two New Zealand?white?rabbits were immunized with this protein to prepare antiserum. The resulting PcAb was then purified from the?antiserum with Protein A affinity?column. The results of fluorescence antibody assay, enzyme linked immunosorbent?assay and Western blotting analysis showed that the PcAb could specifically recognize the surface exposed region of UspA1 on Mc. The preparation of the PcAb laid a foundation of further development of rapid detection technique for M. catarrhalis.