肉鸡谷胱甘肽S-转移酶A3基因的克隆及蛋白的表达和纯化

Cloning, expression and purification of glutathione-s-transferase A3 genes of broilers

[目的]谷胱甘肽S-转移酶A3(GSTA3)是GSTs超家族中α家族的一员,具有解毒功能,同时还参与抗氧化应激引起的信号通路调节,另外在类固醇和前列腺素的合成中也必不可少。[方法]本试验采用RT-PCR方法扩增肉鸡GSTA3基因,连接pZeroBack/blunk克隆质粒,再与表达载体pET-28a连接,并转入E.coli BL21(DE3)中获得pET-28a-GSTA3重组表达质粒,使用IPTG诱导其蛋白的表达,应用镍柱亲和层析法纯化蛋白,并对其进行SDS-PAGE鉴定,使用谷胱甘肽-S转移酶活性测定试剂盒对重组蛋白进行活性检测。[结果]试验成功构建重组表达质粒pET-28a-GSTA3,并在大肠杆菌中成功表达,其大小为29.6 kDa,与预期一致,并且表达的重组GSTA3蛋白具有生物活性。[结论]本试验成功获得高纯度的可溶性GSTA3融合蛋白,为其在家禽领域的进一步研究奠定了基础。

[Objective]Glutathione S-transferase A3(GSTA3)is a major part of GST alpha subfamily,which possesses alexipharmic property.It is also involved in the regulation of signal pathways induced by oxidative stress and in the synthesis of steroids and prostaglandin.The proposed study was aimed to…..[Methods]The GSTA3 gene was amplified by RT-PCR method and the amplified ingredients were inserted into the pZeroBack/blunk cloning vector,and the product was connected to the pET-28a.The construct was then transformed into E.coli BL21 to obtain recombinant expression plasmid pET-28a-GSTA3.Protein expression was induced by IPTG.The GSTA3 fusion protein was purified by nickel column affinity chromatography,and the purified protein was identified by Western Blot and SDS-PAGE.The bioactivity of GSTs was examined using assay kit.[Results]The test successfully exhibited the recombinant expression vector pET-28a-GSTA3.GSTA3 gene was successfully expressed in E.coli and the recombinant protein was around 29.6 kDa,and the expressed recombinant GSTA3 protein was biologically active.[Conclusion]This experiment successfully obtained high-purity soluble GSTA3 fusion protein,which established solid basis for further research in the field of poultry.