鸡毒支原体醛缩酶的原核表达及膜定位鉴定

Prokaryotic expression and membrane localization of aldolase of Mycoplasma gallisepticum

为了探明醛缩酶在支原体中的定位,根据已发表的鸡毒支原体醛缩酶(fba)基因序列设计特异性的引物,以鸡毒支原体Rlow株基因组为模板,通过Overlap PCR点突变扩增鸡毒支原体醛缩酶fba基因,将fba克隆至pET-28a(+)载体后进行序列测定和分析。结果表明,fba基因全长873bp,编码290个氨基酸。将构建的重组表达质粒pET28a-fba转化至大肠杆菌BL21(DE3),在IPTG诱导下成功获得表达融合蛋白rMGfba,大小约为33ku。纯化蛋白并对蛋白进行酶活性检测,结果显示该酶在体外具有与阳性对照醛缩酶相同的催化功能。用该蛋白免疫小鼠制备多克隆抗体,提取鸡毒支原体膜蛋白,Western-blot分析结果显示FBA多抗可与疏水性膜蛋白特异性结合,说明FBA位于鸡毒支原体膜表面。

On the basis of fructose-bisphosphate aldolase（fba） gene sequences of Mycoplasma gallisepticum available in GenBank,specific primers were designed and used for overlap PCR amplification from fba gene from M.gallisepticum strain Rlow.The PCR product was cloned into the expression vector pET-28a（＋） and subsequently sequenced.The result showed that the open reading frame of fba gene was 873 bp in length,encoding 290 amino acids.The recombinant plasmid pET28a-fba was then transformed into Escherichia coli BL21（DE3） competent cells for expression via induction using IPTG.The rMGfba exhi-bited aldolase catalytic activity indicating that it was correctly expressed in vitro.Mouse antiserum was generated by immunization mouse with rMGfba.Western-blot assay showed that the antiserum could re-cognize a protein presented on the surface of M.gallisepticum.