鳗源嗜水气单胞菌主要粘附素基因克隆表达及产物粘附功能分析

摘要

目的 对致病性嗜水气单胞菌(Aeromonas hydrophila)ZN1的主要粘附素基因(major adhesin gene,Mah)进行克隆表达及产物粘附功能分析.方法 在OenBank上对PCR产物进行序列分析和同源性序列比对.将Mah重组到pET-32a表达质粒并转化E.coliDE3(BL21),采用ELISA和Western-blot对表达产物进行分析.结果 DNA序列分析表明PCR产物包含一个1 074bp的完整阅读框架,编码357个氨基酸,N-端22个氨基酸残基构成疏水信号肽.与已知的嗜水气单胞菌主要粘附素基因同源率为70%.ELISA和Western-blot分析表明表达产物能被鼠抗ZN1主要外膜蛋白血清特异性识别.同时,表达产物及其免疫血清能够竞争性抑制和阻断ZN1菌对EPC细胞的粘附作用.结论 Mah是嗜水气单胞菌的主要粘附因子;基因表达的融合蛋白Mah-TrxA具有天然粘附素的粘附活性.%Aeromonas hydrophila strain ZN1 was isolated from European eel (Anguilla anguilla).Its major adhesin gene (Mah) was amplified by PCR and the DNA sequence analysis showed that Mah had a full-lenghth open reading frame (ORF) of 1074 nt, encoding a 357-aa peptide, and which N-terminal 22 amino-acid was highly hydrophobic signal polypeptide.The homologous comparison proved that Mah had about 70％ homology with the other Mah sequences from A.hydrophila.The amplified Mah was inserted into vector pET-32a and transformed into E.coliDE3(BL21) for expression.The Mah-TrxA fusion protein was efficiently expressed in E.coliDE3 (BL21) which were recognized by mouse anti-ZN1-MOMP serum by ELISA and also Western-blotting to get the expected bands comparing with ZN1-MOMP, which was containing major adhesin.Adhesive assay indicated that ZN1-MOMP and Mah-TrxA fusion protein were able to inhibit the adhesive fuction of the bacteria and the rabbit anti-Mah-TrxA serum could significantly block the ZN1 to adhere to EPC monolayers.So that, we could have the conclusion that the cloned gene represented the major adhesin gene of strain ZN1, and the gene products shared similar antigenicity with the natural major adhesion.