嗜水气单胞菌溶血素基因的克隆表达及其类毒素的免疫原性分析

Cloning and expression of a hemolysin gene of Aeromonas hydrophila and the immunogenicity of the toxoid

根据嗜水气单胞菌溶血素保护性抗原基因序列(GenBank Accession No.AF539467)设计一对引物,以嗜水气单胞菌河北分离株基因组为模板,经PCR扩增得到hly基因。序列分析表明,该基因产物大小为1485 bp,经测序与GenBank报道的多个嗜水气单胞菌hly基因序列一致性高于99%。将得到的hly基因定向克隆到原核表达载体pET-28a中构建原核重组质粒pET-28a-hly,转化大肠杆菌BL21(DE3)中,得到重组菌株BL21(DE3)(pET-28a-hly),经IPTG诱导后,SDS-PAGE分析可见一条56 kD的特异条带。Western blotting分析结果显示表达的Hly蛋白能与抗体发生特异性结合,说明其具有较好的免疫原性。将表达的溶血素蛋白制成类毒素疫苗免疫小鼠后,具有较高的保护效力,表明该类毒素疫苗有望作为预防由嗜水气单胞菌引起疾病的基因工程类毒素疫苗。

According to the GenBank sequences （GenBank Accession No. AF539467）, one pair of primers was designed to amplify hly gene of Aeromonas hydrophila by PCR. After sequencing, homology analysis indicated that a DNA fragment of 1485 bp was amplified from isolated DNA from Aeromonas hydrophila, and it shared more than 99% homology in nucleotide sequence compared with other reference strains in GenBank. The gene was cloned in pET-28a vector to construct a recombinant plasmid pET-28a-hly, which was transformed into Escherichia coli BL21 （DE3）, and the recombinant strain BL21（DE3）（pET-28a-hly） was obtained. The hemolysin was highly expressed when the recombinant strain BL21 （DE3） （pET-28a-hly） was induced by IPTG. The expressed protein was 56 kD as estimated by 15% SDS-polyacrylamide gel electrophoresis （SDS-PAGE）. The immunogenicity of the expressed Hly protein was confirmed by Western blotting. Mice were immunized with inactivated whole bacteria vaccine and the genetic engineering vaccines showing promise that all these vaccines have a high protective ability. The results showed that the recombinant strain BL21 （DE3）（pET-28a-hly） could be candidate of hemolysin toxoid vaccine to provide protective immunity against diseases caused by Aeromonas hydrophila.