嗜水气单胞菌溶血素A表达、免疫原性及活性检测

EXPRESSON,IMMUNOGENICITY AND HEMOLYTIC ACTIVITY OF AEROMONAS HYDROPHILA HEMLYSIN A

根据已发表的嗜水气单胞菌(Aeromonas hydrophila,Ah)溶血素A基因序列设计一对特异性引物,以TPS30菌株基因组为模板,通过PCR扩增溶血素A基因,经T-A克隆、序列测定和分析,结果表明,该基因包含1482 bp碱基,编码494个氨基酸。将溶血素A定向克隆至表达载体pET32a(+)中,构建重组表达质粒pET-THA,在IPTG诱导下成功获得重组表达蛋白His-GroEL,大小为68 kDa。对该质粒在大肠杆菌BL21(DE3)中的表达特性分析表明,最佳的诱导表达条件为:在0.1 mmol/L的IPTG浓度下,21℃诱导表达3 h。表达的溶血素A蛋白纯化后免疫小鼠,经Western blot检测,表明该蛋白具有免疫原性。对纯化的溶血素A蛋白复性后,进行溶血实验,结果表明该蛋白具有溶血活性。通过本实验的研究,为进一步研究嗜水气单胞菌溶血素的生物学功能奠定了基础。

A pair of specifical primers was designed according to gene sequence of Aermonas hydrophila hemolysin A which has been reported.Using Aermonas hydrophila strain TPS30 genomic DNA as template,Aermonas hydrophila hemolysin A was amplied by PCR and cloned into pGEM-T vector.The result of sequence analysis shown that the length of hemolysin A is 1482 bp,encoding a 494-amino-acid protein.This gene was cloned into pET-32a（＋） vector and generated recombinant express plasmid pET-THA,a recombinant protein His-GroEL with molecular weight of 68 kDa was obtained after induction of IPTG.Mice were immunized with the purified hemolysin A protein,Western bolt analysis with mouse sera indicated that this recombinant protein has immunogenic activity.The hemolytic test indicated that renatured hemolysin A protein possessed hemolytic activity.