蓝氏贾第鞭毛虫甲硫氨酸硫氧化物还原酶基因的克隆、表达与纯化

Cloning,expression and purification of the methionine-S-sulfoxide reductase gene of Giardia lamblia

目的蓝氏贾第鞭毛虫(简称为贾第虫)甲硫氨酸硫氧化物还原酶基因(GlMSR),进行原核表达获得其重组蛋白。方法依据GenBank中GlMSR序列设计引物,以贾第虫中国C2克隆株基因组DNA为模板,通过PCR扩增编码序列,将所得片段连接至质粒pET28a以构建原核表达载体pET28a-GlMSR,将pET28a-GlMSR导入大肠杆菌E.coli JM109并挑取阳性克隆进行基因测序和鉴定。用化学方法将经测序验证的重组质粒pET28a-GlMSR转化至E.coli BL21(DE3)。经异丙基硫代半乳糖苷(Isopropylβ-D-Thiogalactoside,IPTG)诱导表达后,聚丙烯酰胺凝胶电泳检测重组蛋白表达结果。收集沉淀中的重组表达产物,镍柱亲和层析纯化带组氨酸标签的重组蛋白,并通过免疫印迹鉴定纯化结果。结果电泳结果显示在约624 bp处出现目的DNA条带,表明成功得到GlMSR编码序列;PCR鉴定、酶切鉴定以及基因测序结果表明成功构建重组表达载体pET28a-GlMSR,经IPTG诱导后在沉淀中获得目的蛋白,目的蛋白分子量(Mr)约为25000,与预期分子量(Mr)22800基本相符。经Western blot分析显示该重组蛋白含有His6标签。结论本研究克隆、表达了蓝氏贾第虫GlMSR基因,并纯化获得重组蛋白,为进一步探索该酶的功能奠定了基础。

Objective To clone and express the methionine-S-sulfoxide reductase gene of the Giardia lamblia(GLMSR),and obtain the recombinant GlMSR protein.Methods According to the GLMSR gene homologous sequences in GenBank,the specific primers were designed.Using the genomic DNA of Giardia lamblia Chinese C2 as template,the MSR gene was amplified by PCR and was inserted into the expression vector pET28a to generate pET28a-Glmsr.The pET28a-Glmsr was transformed into E.coli JM109.The positive clone was selected and identified by sequencing.The expression vector pET28a-Glmsr was transformed into E.coli BL21(DE3)by chemical method.The expression of Glmsr was induced by isopropyl thiogalactoside(IPTG).The expression of the recombinant protein was analyzed by SDS-PAGE.Recombinant proteinin the sediment was collected and purified by Ni2+affinity chromatography and verified by Western blotting.Results The electrophoresis result showed that there was a DNA band of approximately 624 bp,meant that the GLMSR gene sequences were obtained.The recombinant expression vector was constructed and identified by PCR,enzyme digestion and sequencing.The above results were all consistent with expectation.The recombinant protein′s molecular mass(Mr)is approximately 25000,which was consistent with expectation(22800).The results of SDS-PAGE showed that the recombinant protein was expressed in the sediment.The result of Western blotting showed that the recombinant protein can be recognized by anti-His-tag antibody.Conclusion In this study,we successfully cloned the GLMSR gene coding sequence of Giardia lamblia and obtained the purified recombinant protein.