【摘要】 目的构建SSB蛋白原核表达质粒,在大肠埃希菌中诱导表达后进行纯化。方法以Hela细胞提取RNA后逆转录的cDNA为模板,利用聚合酶链反应(PCR)扩增出带酶切位点BamHⅠ和XhoⅠ的人SSB蛋白基因序列,经酶切后插入PET41a质粒中,构建成GST-SSB-6*His-pet41a重组表达质粒,转化入感受态大肠埃希菌OverExpress C41(DE3)中,经异丙基β-D-半乳糖苷(IPTG)诱导表达,利用C端的His标签(Histag)进行Ni-树脂柱亲和层析纯化,SDS-PAGE鉴定重组蛋白表达形式,蛋白印迹法(WB)鉴定重组蛋白的免疫原性,酶联免疫吸附试验(ELISA)鉴定其免疫反应性和特异性。结果 PCR扩增获得目的基因,菌落PCR示重组表达质粒构建成功,转化感受态OverExpress C41(DE3)后经诱导有高效表达,SDS-PAGE显示GST-SSB-6*His重组蛋白为上清可溶性表达,WB示重组蛋白有足够的免疫原性,能与外周血中的抗SSB抗体发生特异性反应,特异性达100%。结论成功构建了GST-SSB-6*His-PET41a重组表达质粒,经诱导表达后可纯化出目的蛋白,为进一步建立应用于国产仪器的全自动定量检测抗SSB自身抗体的磁微粒化学发光试剂,奠定了基础。更多还原

【Abstract】 Objective Construct the prokaryotic expression plasmid of SSB protein,and purify after induction of expression in Escherichia coli.Methods Using reverse-transcribed cDNA from Hela cells as a template,polymerase chain reaction(PCR)was used to amplify human SSB protein with restriction sites BamH Ⅰand XhoⅠ.The gene sequence was digested and inserted into the PET41 aplasmid to construct GST-SSB-6*His-pet41 arecombinant expression plasmid,which was transformed into competent Escherichia coli OverExpress C41(DE3),after isopropylβ-D-galactosin(IPTG)induced,Ni-resin column affinity chromatography purified by His-tag at the C-terminal.The recombinant protein expression was identified by SDS-PAGE,the immunogen of recombinant protein was identified by Western blot(WB),and the enzyme reactivity and specificity were identified by enzyme-linked immunosorbent assays(ELISA).Results The target gene was obtained by PCR amplification.The colony PCR showed that the recombinant expression plasmid was successfully constructed.After transforming competent overExpress C41(DE3),it was induced to express efficiently.SDSPAGE showed that GST-SSB-6*His recombinant protein was soluble in the supernatant.WB shows that the recombinant protein was sufficiently immunogenic to specifically react with anti-SSB antibodies in peripheral blood with a specificity of 100%.Conclusion The recombinant expression plasmid of GST-SSB-6*His-PET41 ais successfully constructed,and the target protein is purified after induction,which promotes the magnetic particle chemiluminescence reagent for the automatic detection of anti-SSB autoantibodies in domestic instruments.更多还原