蓝氏贾第鞭毛虫沉默信息调节因子2(Sir2)基因的克隆、表达与纯化

Cloning,Expression and Purification of Silent Information Regulator 2 from Giardia lamblia

目的获取蓝氏贾第鞭毛虫(Giardia lamblia,Gl)沉默信息调节因子2(Sir2)基因序列及其重组蛋白。方法以蓝氏贾第鞭毛虫中国C2克隆株基因组DNA为模板,PCR扩增GlSir2基因编码序列,将所得片段连接至pMD-19T质粒。转化大肠埃希菌(E.coli)JM109,挑取阳性克隆进行测序。将GlSir2基因插入原核表达载体pET28b,构建表达载体pET28b-GlSir2,转化E.coli BL21(DE3),用异丙基硫代半乳糖苷(IPTG)诱导表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析重组蛋白的表达情况。收集重组表达产物,用8 mol/L尿素溶解包涵体并收集上清,镍离子亲和层析进行纯化。将纯化产物透析复性,蛋白质印迹(Western blotting)进行免疫学鉴定。结果获得了蓝氏贾第鞭毛虫中国C2克隆株GlSir2基因编码序列,该序列包含一个1 680 bp的开放阅读框架,可编码559个氨基酸,预测其蛋白的相对分子质量(Mr)约为62 800。构建表达载体pET28b-GlSir2,经IPTG诱导,重组蛋白以包涵体形式表达。溶解包涵体并经纯化后的目的蛋白纯度达80%以上,经透析复性后获得可溶蛋白。Western blotting分析显示,该重组蛋白可被His标签抗体识别。结论克隆了蓝氏贾第鞭毛虫GlSir2基因编码序列,并获得重组表达,重组蛋白可被His标签抗体识别。

Objective To clone and express silent information regulator 2（Sir2） gene from Giardia lamblia.Methods The GlSir2 gene was amplified by PCR from genomic DNA of Giardia lamblia（Chinese strain C2 clone）.PCR product was cloned into pMD-19T vector and transformed into E.coli JM109.The recombinant plasmid was sequenced and then cloned into the pET28b vector.The pET28b-GllSir2 recombinant plasmid was transformed into E.coli BL21（DE3）,followed by expression of the protein induced by IPTG.The recombinant protein was analyzed by SDS-PAGE.Inclusion bodies were dissolved with 8 mol/L urea,and the supernatant was collected and applied to Ni2＋ affinity chromatography.The purified recombinant protein was renatured by dialysis and verified by Western blotting using anti-His tag antibody.Results GlSir2 gene sequence was cloned.The GlSir2 open reading frame（1 680 bp） encoded a 559-amino acid protein with Mr 62 800.The recombinant plasmid pET28b-GlSir2 expressed an inclusion body protein of GlSir2 after being induced with IPTG.The protein purity reached above 80% after purification.The purified protein was renatured by dialysis.The recombinant GlSir2 was recognized by anti-His tag antibody.Conclusion The coding sequence of GlSir2 gene was cloned and expressed in vitro.The recombinant protein was identified by anti-His tag antibody.