【摘要】 目的获得重组TsARG2基因蛋白,为进一步研究其功能和制备特异抗体奠定基础.方法根据已报道的TsARG2基因序列,采用RT-PCR的方法获得TsARG2基因.将所得的PCR产物插入原核表达载体pQE30中,得重组质粒(pQE/TSARG2)并转化大肠杆菌(E.coli)M15,通过IPTG诱导表达出带有六个组氨酸的融合蛋白,采用镍固定金属亲和层析法纯化.结果序列分析表明TSARG2基因成熟肽编码区含有918bp,编码305个氨基酸;与GenBank(AY040204)中已报道的TSARG2分离物的核苷酸序列有100%同源性.经IPTG诱导表达,SDS-PAGE电泳分析显示,表达的融合蛋白占菌体蛋白总量的66%,分子质量约为35kDa.经Ni2+-NTAagarose纯化获得SDS-PAGE电泳下单一条带.结论在大肠杆菌中获得了TsARG2基因蛋白的高效表达,为研究其生物学功能和制备单克隆抗体奠定了基础.更多还原

【Abstract】 Objective To obtain recombinant TSARG2 protein by prokaryotic expression for its functioned study.Methods The encoding sequence for mature peptide of human TSARG2 was amplified with RT-PCR and inserted into pQE30 vector to establish the prokaryotic expressing system coupled with 6×His.The competent cells of host strain of M15 were transformed by the recombinant plasmid(pQE30/TSARG2).Expression of the target protein was induced with IPTG and assayed by SDS-PAGE after sequencing.The recombinant products were purified by Ni 2+ -NTA agarose column.Results:The cloned fragment of human TSARG2 was 100% consistent with that in Genbank(AY040204),which was deduced to express 305 Aa mature peptide of human TSARG2 correctly.The expressed fusion protein was 35 kD in SDS-PAGE as expected and 66% in the total quatiy of germ proteins.Conclusion:The homologous recombinant human TSARG2 was obtained after Ni-affinity chromatograph,which could be used for its functioned study and preparation of specific antibodies.更多还原