A型流感病毒PB2蛋白帽子结合结构域的原核表达与鉴定  

Prokaryotic expression and characterization of PB2 cap-binding domain of influenza A virus

作　　者：林维鹏 崔鹏飞[1] 王思文 邓国华[1] 陈化兰[1] LIN Wei-peng;CUI Peng-fei;WANG Si-wen;DENG Guo-hua;CHEN Hua-lan(Influenza Laboratory of the Ministry of Agricultural and Rural Affairs,State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室/农业农村部动物流感重点开放实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2024年第2期107-112,139,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：十四五国家重点研发计划(2021YFD1800200)。

摘　　要：A型流感病毒是一种重要的人畜共患病病原,严重威胁全球公共卫生安全。PB2蛋白的帽子结合结构域(CBD)在流感病毒的转录过程中发挥重要功能,是抗流感病毒药物的重要靶点之一。为获得高纯度原核表达的A型流感病毒的CBD蛋白,本研究利用同源重组策略将8×His标签与一株H5N1亚型高致病性禽流感病毒的CBD区基因序列克隆至原核表达载体pET28a中,构建重组质粒pET28a-8×His-CBD,经PCR及测序鉴定正确后转化大肠杆菌BL21(DE3)感受态细胞,经不同浓度IPTG诱导不同时间后,经SDS-PAGE检测蛋白表达后使用镍柱纯化。结果显示,菌体裂解上清中在21 ku处可见目的条带,与8×His-CBD蛋白预期大小符合,且以0.5 mmol/L IPTG于37℃诱导6 h时蛋白表达量最高,镍柱纯化后可获得纯度大于95%的8×His-CBD蛋白。为确定该蛋白是否具有生物学活性,本研究以帽子结构类似物m7GTP对其进行等温滴定量热试验,结果显示8×His-CBD蛋白和m~7GTP结合时存在明显热峰,表明8×His-CBD蛋白具有生物学活性;经计算,其Kd值为1.71×10^(-5)(±2.92×10^(-6))mol/L。本研究结果对流感病毒的基础研究和抗流感病毒药物的研发具有借鉴意义。As a major zoonotic pathogen,influenza A virus(IAV) poses serious threats to global public health security.The cap-binding domain(CBD) of PB2 protein plays an important role in IAV transcription process,and is therefore one of the important targets for anti-influenza drugs.To obtain high-purity CBD protein by prokaryotic expression system,the CBD gene sequence of a highly pathogenic IAV H5N1 and an 8×His tag were constructed into the prokaryotic expression vector pET28a by homologous recombination strategy.The constructed recombinant plasmid pET28a-8×His-CBD was identified by PCR and sequencing,and then transformed into Escherichia coli receptor cells BL21(DE3).The expression of CBD protein was detected by SDS-PAGE after induction with different concentrations of IPTG for different hours,and then purified by Ni-NTA.The results showed that a distinct band at 21ku was visible in the cell lysate supernatant of the bacterium,which was consistent with the expected size of 8×His-CBD protein.The highest expression of the protein was obtained under the condition of 0.5mmol/L IPTG,cultured for 6 hours at 37℃,and the purity of the 8 ×His-CBD protein was more than 95% after Ni-NTA purification.To determine whether the 8×His-CBD protein in this study was biologically active,it was subjected to an isothermal titration calorimetry(ITC) assay with m~7GTP,the cap structure analogue.Apparent thermal peaks were observed when the two were combined,indicating that the 8×His-CBD protein was biologically active,and the Kd value was calculated to be 1.71 ×10^(-5)(±2.92×10^(-6)) mol/L.In this study,the recombinant plasmid p ET28a-8 ×His-CBD was correctly constructed,and the optimal induction conditions were determined.The high-purity 8 ×His-CBD protein was obtained with biological activity.This study could have reference significance for the basic research of influenza virus and for the development of anti-influenza virus drugs.

关 键 词：A型流感病毒 PB2蛋白 帽子结合结构域 原核表达 纯化 

分 类 号：S852.65[农业科学—基础兽医学]