蓝氏贾第鞭毛虫中国株组蛋白H2A基因的克隆与重组表达

目的获取蓝氏贾第鞭毛虫中国株GlH2A基因及重组蛋白。方法 PCR扩增获取GlH2A基因,连接至pMD-19T并进行测序分析。连接至pET28a构建表达载体,并转化宿主菌E.coli BL21（DE3）,然后进行异丙基硫代半乳糖苷酶（IPTG）诱导表达。表达产物进行亲和层析纯化,再用Western blotting进行免疫学鉴定。结果序列测定获得蓝氏贾第鞭毛虫中国株GlH2A基因的编码序列,与美国WB株H2A（GL50803_14256,Accession：NW_001844132）序列完全一致。该基因编码124个氨基酸,预测分子量13900Mr,保留了与核小体形成相关的重要氨基酸位点,在大肠埃希菌中获得表达,纯化后纯度达90%以上。Western blotting检测表明重组蛋白能够被抗His6标签抗体识别。结论克隆得到GlH2A基因编码序列;得到了重组GlH2A蛋白,并进行了纯化,为进一步研究组蛋白及其修饰酶在基因转录调控中的作用奠定了基础。

Cloning and expression of H2A gene from Giardia lamblia

Objective To obtain GlH2A gene from Giardia lamblia and relevant recombinant proteins.Methods The GlH2A gene of Giardia lamblia was amplified from genomic DNA by PCR.A His6-tag coding sequence and a Nco I site were included in upstream primer and a stop codon and a BamH I site were included in downstream primer.The PCR products were subcloned into pMD-19T vector and the positive clones were selected by colony PCR.The recombinant plasmid was sequenced.The resulting sequence was queried in GenBank.The coding sequence of GlH2A was aligned with H2A sequences from the other organisms.The plasmid verified by sequencing was digested by Nco I and BamH I and then inserted into the expression vector pET28a,yielding pET28a-GlH2A.The E.coli BL21（DE3） was transformed and induced with IPTG.The cells were collected and dispersed with sonication.The recombinant protein was purified by nickel affinity chromatography.Recombinant GlH2A was detected using Western blotting method.Results The gene sequence obtained was identical to GlH2A GL50803_14256 （Accession：NW_001844132）,consisting of 375 bp and coding for 124 amino acids.The predicted molecular weight of GlH2A was 13 900 Mr,with PI value of 10.45.The amino acid sequence of H2A from different organisms was aligned and the result showed the divergent of GlH2A to the others H2A genes.The result also revealed conservation of a number of residues at some unique position such as H2A-H2A interface,H2A-H2B interface and DNA-binding site.The expression vector pET28a-GlH2A was constructed.rGlH2A was expressed in E.coli and accounted for about 20% of total protein.The recombinant protein was purified by nickel affinity chromatography and the purity reached 90%.The product was verified by Western blotting using anti-His6 tag antibody.Conclusions GlH2A gene of G.lamblia was cloned and sequenced.It showed conservation at some positions and may play similar role as that of in other organisms.Recombinant GlH2A was expressed in E.coli and purified,which set foundation for further studies on GlH2A and its modification enzyme.