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.
作者
李想
黄文树
黄贝
徐继松
翟少伟
梁英
张婉婷
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)
出处
《海洋渔业》
CSCD
北大核心
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)
关键词
日本鳗鲡
Ⅱ型干扰素
原核表达
Anguilla japonica
typeⅡinterferon
prokaryotic expression
