摘要
目的:研究耐热DNA聚合酶GBDpol在大肠杆菌中的异源表达情况,优化其表达条件,并验证其DNA聚合酶活性。方法:将GBDpol在大肠杆菌中进行重组表达,测试不同异丙基-β-D硫代半乳糖苷(IPTG)浓度、培养温度和培养时间对GBDpol表达量的影响,并利用His标签亲和层析纯化GBDpol,透析后通过SDS-PAGE分析蛋白纯度,Bradford法测定蛋白浓度;以纯化的GBDpol进行PCR扩增检测其活性。结果:当IPTG浓度为0.2 mmol/L,并在35℃培养15 h时,GBDpol的可溶性表达量最高;纯化后的GBDpol浓度为0.429 mg/mL,SDS-PAGE显示目标蛋白条带单一;聚合酶链式反应(PCR)实验证实重组GBDpol能成功扩增目的基因。结论:本研究成功在大肠杆菌中实现GBDpol的高效可溶性表达,纯化的GBDpol表现出DNA聚合酶活性,可用于基因工程中的PCR扩增。这为其进一步应用提供了数据基础。
Objective:To study the heterologous expression of heat-resistant DNA polymerase GBDpol in Escherichia coli,optimize its expression conditions,and verify its DNA polymerase activity.Methods:GBDpol is recombinantly expressed in Escherichia coli,and the effects of different isopropyl-β-D thiogalactoside(IPTG)concentrations,incubation temperature and incubation time on the expression of GBDpol are tested,and GBDpol is purified by His-tagged affinity chromatography,and the protein purity is analyzed by SDS-PAGE after dialysis,and the protein concentration is determined by Bradford method.Purified GBDpol is used for polymerase chain reaction(PCR)amplification to detect its activity.Results:When the concentration of IPTG is 0.2 mmol/L and incubated at 35℃for 15 h,the soluble expression of GBDpol is the highest.The purified GBDpol concentration is 0.429 mg/mL,and SDS-PAGE shows that the target protein band is single.PCR experiments confirm that the recombinant GBDpol can successfully amplify the target gene.Conclusion:In this study,the efficient soluble expression of GBDpol is successfully achieved in Escherichia coli,and the purified GBDpol exhibits DNA polymerase activity,which can be used for PCR amplification in genetic engineering.This provides a data basis for its further application.
作者
严鹤松
鲁奕航
李婵娟
YAN Hesong;LU Yihang;LI Chanjuan(Hubei Light Industry Technology Institute,Wuhan 430070,China;Wuhan Institute of Design and Sciences,Wuhan 430205,China)
出处
《生物化工》
2025年第3期12-16,21,共6页
Biological Chemical Engineering
基金
湖北省自然科学基金项目(2022CFB567)。
关键词
耐热DNA聚合酶
PCR反应
诱导表达
蛋白纯化
thermostable DNA polymerase
PCR reaction
induced expression
protein purification
