弓形虫沉默信号调节子2(TgSir2)的克隆表达与多克隆抗体制备

Cloning and expression of Toxoplasma gondii silent information regulator 2(TgSir2) and preparation of its polyclonal antibody

目的制备特异识别弓形虫沉默信号调节子2(TgSir2)多克隆抗体。方法以酵母Sir2氨基酸序列为模板,寻找弓形虫基因组中同源序列,并进行信号肽预测。以弓形虫RH株cDNA为模板,构建pET28b-TgSir2重组质粒,转化入大肠埃希菌(E.coli)BL21,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达后,用镍柱亲和层析法纯化诱导表达产物,并以小鼠抗His标签单克隆抗体作为一抗,蛋白质印迹(Western blotting)进行鉴定。以纯化的TgSir2蛋白作为抗原免疫新西兰大白兔,获得特异性抗TgSir2多克隆抗体。最后,以此多克隆抗体作为一抗,Western blotting检测与虫体蛋白的反应情况。结果同源比对找到弓形虫基因组中TgSir2,信号肽分析提示1-15位氨基酸为其信号肽序列。PCR、双酶切及测序结果证实原核表达质粒构建成功。SDS-PAGE结果表明,IPTG可诱导TgSir2融合蛋白的大量表达。Western blotting结果显示,制备的多克隆抗体既能特异性识别原核表达的TgSir2蛋白,也能识别弓形虫体内的内源性TgSir2蛋白。结论制备的抗重组TgSir2蛋白多克隆抗体能特异性识别弓形虫RH株的TgSir2蛋白。

To obtain the specific polyclonal antibody against Toxoplasma gondii silent information regulator 2(TgSir2).Searching the homologous sequence in Toxoplasma genome used the amino acid sequence of Saccharomyces cerevisiae silent information regulator 2(ScSir2)as template and predicting the signal peptide.TgSir2 cDNA was inserted into prokaryotic expression vector pET28b.After identification,the constructed plasmid pET28b-TgSir2 was transformed into E.coli BL21 cells and induced by isopropyl-β-D-thinoglucoside(IPTG)for expression of the protein.The recombinant protein was purified via Ni-NTA affinity chromatography.Western blotting assay was conducted with anti-His tag monoclonal antibody as the primary antibody.Rabbits were immunized with purified recombinant protein.The specific anti-TgSir2 polyclonal antibody was obtained and Western blotting was performed to detect the reaction by the antibody.The TgSir2 in Toxoplasma genome was found through homologous comparison,signal peptide analysis indicated that 1-15 amino acids were the sequence of its signal peptide.pET28b-TgSir2 plasmid was identified by PCR amplification,restriction enzyme digestion and sequencing.SDS-PAGE analysis showed that the recombinant TgSir2 protein was expressed highly induced by IPTG.Western blotting showed that both TgSir2 protein from prokaryotic expression and tachyzoite lysates can be recognized by the specific anti-TgSir2 polyclonal antibody.The obtained polyclonal antibody against TgSir2 can specifically react with the endogenous TgSir2 protein.