禽致病性大肠杆菌IMT5155株毒力相关基因片段E9的表达及生物学特性分析

Expression and biological characterizations of virulence-related gene fragment E9 of avian pathogenic Escherichia coli IMT5155

根据禽致病性大肠杆菌IMT5155毒力相关基因E9的序列,设计并合成了1对特异性引物,以IMT5155菌株的基因组DNA为模板扩增了E9基因所在ORF的部分序列。将PCR产物进行了T/A克隆,转化大肠杆菌,鉴定成功获得目的片段后,将其定向亚克隆到pET-28a(+)载体中,构建了原核表达质粒pET-28a-E9,并将其转化至大肠杆菌BL21(DE3)感受态细胞中,经1 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导和SDS-PAGE分析,出现了与预期目的蛋白一致的外源蛋白带(32.2 ku)。Western-blot分析表明,该融合蛋白具有免疫反应性。对重组蛋白进行纯化后免疫动物进行抗体效价测定,进一步分析重组蛋白的相关生物学功能,分别进行了细胞毒性试验、动物试验、细胞黏附与黏附影响试验。结果表明,该蛋白没有细胞毒性,对小鼠亦无毒性,有一定的黏附作用。

Avian colibacillosis caused by avian pathogenic Escherichia coli （APEC） is an acute or recurrent infectious disease. Based on the E9 gene sequence of E. coil published on the GenBank, a pair of primers was designed. The gene fragment of E9 was amplified from genomic DNA of APEC IMT5155 strain by polymerase chain reaction （PCR）. Subsequently, the PCR product was inserted into pMD18-T Simple vector routinely, and then subcloned into prokaryotic expression vector pET-28a （＋） , generating a recombinant expression plasmid pET- 28a-E9. The resulting plasmid was confirmed by direct DNA sequencing and transformed into E. coli BL21 （ DE3 ） competent cells. The SDS-PAGE analysis showed that a 32. 2 ku protein band could be observed under the induction of IPTG, and the Western-blotting analysis demonstrated clearly the recombinant protein had a strong specific antigenicity. Moreover, the protein with a single band of 32. 2 ku was obtained by HisTrapTM HP purification Kit. New Zealand rabbits were immunized with the purified protein. The antiserum titer to the recombinant protein was 1 ： 6400 by indirect enzyme-linked immunosorbent assay （ELISA）, HEp-2 cells, a kind of typical cell model, were used in the adhesion and adhesion inhibition experiments to evaluate the adhesion of the fusion protein from IMT5155. Meanwhile the fusion protein was co-cultured with HEp-2 cells to detect cytopathic effect （CPE）. The results demonstrated that the purified protein did not cause CPE and death of mice by intraperitoneal injection, but could inhibit the adhesion of IMT5155 strain to HEp-2 cells to some extent.