日本鳗鲡Ⅱ型干扰素基因(IFN-γ和IFN-γrel)的原核表达与纯化研究  

Prokaryotic expression and purification of type Ⅱ interferon genes (IFN-γ and IFN-γrel) from Anguilla japonica

作　　者：李想 黄文树[1,3] 黄贝[1,3] 徐继松[1,3] 翟少伟[1,3] 梁英[1,2,3] 张婉婷[1] 刘海姿 LI Xiang;HUANG Wen-shu;HUANG Bei;XU Ji-song;ZHAI Shao-wei;LIANG Ying;ZHANG Wan-ting;LIU Hai-zi(College of Fisheries,Jimei University,Xiamen Fujian 361021,China;Fujian Province Key Laboratory of Special Aquatic Formula Feed,Fuqing Fujian 350308,China;Engineering Research Center of the Modern Technology for Eel Industry,Ministry of Education,Xiamen Fujian 361021,China)

机构地区：[1]集美大学水产学院,福建厦门361021 [2]福建省特种水产配合饲料重点实验室,福建福清350308 [3]鳗鲡现代产业技术教育部工程研究中心,福建厦门361021

出　　处：《海洋渔业》2019年第5期567-577,共11页Marine Fisheries

基　　金：福建省自然科学基金(2017J05054);现代农业产业技术体系专项资金(CARS-46);福建省特种水产配合饲料重点实验室开放课题(TMKJZ1703);技术服务项目(S18200);福建省科技厅资助省属高校专项(JK2014026);大学生创新训练计划项目(2018xj277);大学生创新训练计划项目(201710390256)

摘　　要：分别以日本鳗鲡(Anguilla japonica)pMD19-T-IFNγ和pMD19-T-IFN-γrel重组克隆质粒为模板,采用PCR方法扩增日本鳗鲡IFN-γ和IFN-γrel基因,而后分别将IFN-γ和IFN-γrel成熟肽的编码序列克隆至原核表达载体pQE30和pET32a上,成功构建了重组表达载体pQE30-IFN-γ和pET32a-IFN-γrel。将重组表达载体pQE30-IFN-γ和pET32a-IFN-γrel分别转化入Escherichia coli M15和Escherichia coli BL21感受态细胞进行表达,并优化IPTG诱导表达体系后用SDS-PAGE电泳鉴定IFN-γ与IFN-γrel重组蛋白表达形式。IFN-γ与IFN-γrel重组蛋白经镍柱纯化后进行SDS-PAGE电泳检测和Western blot鉴定。结果显示,重组表达载体pQE30-IFN-γ与pET32a-IFN-γrel分别在E.coli M15和E.coli BL21中得以正确表达。重组IFN-γ蛋白分子量为21 kD左右,表达的融合蛋白以可溶性形式存在;重组IFN-γrel蛋白分子量为31 kD左右,以包涵体形式存在。且两者均在1.0 mmol·L^-1 IPTG诱导6 h时表达量最高。经镍柱纯化和Western blot检测,成功获得高纯度的IFN-γ和IFN-γrel重组蛋白。建立了日本鳗鲡IFN-γ和IFN-γrel基因的原核表达系统,为进一步研究日本鳗鲡干扰素系统的功能及调控机制奠定了基础。Using recombination cloning plasmids of pMD19-T-IFNγand pMD19-T-IFN-γrel as template,IFN-γand IFN-γrel of Japanese eel Anguilla japonica were cloned by PCR.The sequences coding mature peptides of IFN-γand IFN-γrel were cloned into pQE30 and pET32a respectively,to construct the recombinant expression vectors of pQE30-IFN-γand pET32a-IFN-γrel.The recombinant expression vectors of pQE30-IFN-γand pET32a-IFN-γrel were transformed into Escherichia coli M15 and Escherichia coli BL21 respectively for protein expression,and IPTG induction system was optimized,and then SDS-PAGE was used to identify the expression patterns of IFN-γand IFN-γrel recombinant proteins.Recombinant IFN-γand IFN-γrel proteins were purified by Ni 2+-NTA spin column,and analyzed by SDS-PAGE and Western blot.The results indicated that pQE30-IFN-γand pET32a-IFN-γrel were correctly expressed in E.coli M15 and E.coli BL21,respectively.The molecular weight of pQE30-IFN-γrecombinant protein was about 21 kD,and the IFN-γwas expressed in soluble form.The molecular weight of pET32a-IFN-γrel recombinant protein was about 31 kD,and the IFN-γrel protein was expressed in inclusion bodies.The highest expression of IFN-γand IFN-γrel recombinant proteins were obtained when E.coli M15 transferred by pQE30-IFN-γand E.coli BL21 transferred by pET32a-IFN-γrel were induced with 1.0 mmol·L^-1 IPTG for 6 h.Then,the protein was purified by Ni column and tested by Western blot.The high purity proteins of IFN-γand IFN-γrel were finally obtained.In this study,recombinant expression systems of IFN-γand IFN-γrel of Anguilla japonica were successfully constructed to build foundation for further studies on the functions and regulation mechanism of Japanese eel interferon system.

关 键 词：日本鳗鲡 Ⅱ型干扰素 原核表达 

分 类 号：S917.4[农业科学—水产科学]