重组Thermus thermophilus DNA聚合酶表达纯化及其在RT-PCR中的应用

目的研究重组ThermusthermophilusDNA聚合酶的表达、纯化和应用。方法提取Thermusthermophilushb8基因组DNA作为模板,PCR扩增TthDNA聚合酶基因后,克隆至pET28a表达载体上,转化大肠杆菌BL21(DE3),经IPTG诱导表达,用His-BindQuickCartridge300纯化重组TthDNA聚合酶His-tag融合蛋白。提取Thermomonosporafusca总RNA,用一管法RT-PCR扩增E2cd基因,检测重组TthDNA聚合酶RT-PCR活性。结果大肠杆菌表达TthDNA聚合酶,分离出的电泳纯重组TthDNA聚合酶His-tag融合蛋白相对分子质量为94000,表达产量为200000U/L。一管法RT-PCR可扩增出850bp长度的E2cd基因。结论重组TthDNA聚合酶已成功地表达和纯化,并用于RT-PCR。

Expression and Purification of Recombinant Thermus thermophilus DNA Polymerase and Its Application in RT-PCR

Objective To study the expression and purification of recombinant Thermos theromphilus DNA polymerase and its application in RT-PCR.Methods Amplify the gene encoding Thermos thermophilus(Tth) DNA polymerase by PCR using the extracted Tth hb8 genomic DNA as template and insert into expression vector pET28a,then transform to E.coli BL21(DE3) for expression under induction of IPTG.Purify the expressed product by His-Bind Quick Cartidge 300.Determine the activity of Tth DNA polymerase by amplification of E2 cd gene by RT-PCR using the total RNA extracted from Thrmomonospora fusca as template.Results Electrophoretically pure Tth DNA polymerase with a relative molecular weight of 94 000 was expressed in E.coli.The yield of expressed product was 200 000 U/L .The E2 cd gene at a length of 850 bp was amplified by RT-PCR.Conclusion Recombinant Tth DNA polymerase was successfully expressed,purified and applied in RT-PCR.