应用噬菌体随机十二肽库筛选猪传染性胸膜肺炎菌体抗原模拟表位

Screening Mimotopes of Actinobacillus Pleuropneumoniae with Phage Display 12-mer Random Peptide Library

利用猪传染性胸膜肺炎灭活菌苗免疫日本大耳兔得到猪传染性胸膜肺炎抗血清,将血清用饱和硫酸铵粗提IgG后,经DEAE-sephadex-A50低压层析系统纯化,以此得到IgG作为配基包被酶标板,对噬菌体随机十二肽库进行3轮不同条件的富集筛选,通过改变筛选条件,噬菌体的回收率从5.76×10-5增加到了1.69×10-2,P/N值逐步提高;随机挑取10个噬菌斑进行扩增,用ELISA检测其免疫活性,结果有6个阳性克隆与纯化的IgG有较强的特异性结合能力;对筛选到的6个阳性克隆提取ssDNA进行测序,并分析其氨基酸序列,其中5个克隆所递呈的序列一致,用Blastp软件进行分析,2种类型的氨基酸序列之间无同源性,且未发现同源性50%以上的序列,提示该短肽可能模拟了猪传染性胸膜肺炎菌体的特异的抗原表位。

The specific sera were obtained by immunizing the rabbit with Actinobacillus pleuropneumoniae vaccine. IgG was purified through DEAE-sephadex-A50 low pressure chromatography system after rude purification. This IgG was used as target molecular to screen the phage display 12-mer random peptide library in different conditions for 3 rounds. By altering the condition of screening, the yield ratio increased from 5. 76×10^-5 to 1.69×10^-2, and the P/N increased gradually. Then 10 clones were picked out randomly and amplified to test their immunoactivity by ELISA. ssDNA of 6 positive clones which had specific combinations with the IgG were purified for DNA sequencing and the sequences of peptides displayed on the positive clones were analyzed by Blastp . The results showed that the sequences of peptides displayed on 5 positive clones were identical, and there were no homologies between o those two types of sequences, which meant that those peptides might be mimotopes of Actinobacillus pleuropneumoniae .