摘要
[目的]对布鲁氏菌分泌蛋白BspJ进行亚细胞定位分析,为深入探索该分泌蛋白的功能奠定基础。[方法]使用生物信息学方法对分泌蛋白BspJ的核酸和氨基酸序列进行生物信息学分析;利用常规PCR方法扩增BspJ目的基因并克隆到pMD19-T载体;构建真核表达载体pDsRed2-C1-BspJ并转染HEK293T细胞,制作细胞爬片后进行激光共聚焦显微镜分析;利用RT-PCR方法分析HEK293T转染细胞中BspJ转录情况。[结果]生物信息学分析结果显示,BspJ基因ORF全长是531bp,共编码177个氨基酸,BspJ蛋白无跨膜结构,有7个抗原决定簇,具有核定位信号(NLS)和核输出信号(NES),据此推测BspJ蛋白亚细胞定位可能具有核—胞浆穿梭过程;PCR方法扩增出BspJ基因;成功构建pDsRed2-C1-BspJ重组质粒;激光共聚焦分析显示BspJ蛋白定位于宿主细胞核及核周围;RT-PCR分析显示BspJ基因于宿主细胞中进行转录。[结论]布鲁氏菌分泌蛋白BspJ分布于宿主细胞核及核周围,可能具有胞核—胞质穿梭过程。
[Objective]To analyze the subcellular localization of the secreted protein BspJ of Brucella,and lay a foundation for further exploration of the function of the secreted protein.[Methods]The bioinformatics analysis of the nucleic acid and amino acid sequences of the secreted protein BspJ was carried out by bioinformatics method.The BspJ gene was amplified by conventional PCR and cloned into pMD19-T vector.The eukaryotic expression vector pDsRed2-C1-BspJ was constructed and transfected into the HEK293T cells which were sliced and analyzed by laser confocal microscopy.The transcription of BspJ in HEK293T transfected cells was analyzed by RT-PCR.[Results] In bioinformatics analysis results,The BspJ gene ORF was 531 bp in length and encoded a total of 177 amino acids.The BspJ protein has no transmembrane structure and has seven antigenic determinants with nuclear localization signal(NLS)and nuclear output signal(NES).So,subcellular localization of the BspJ protein may have a nuclear-cytoplasmic shuttle process.The target gene was amplified by PCR and the recombinant plasmid pDsRed2-C1-BspJ was successfully constructed.The laser confocal analysis of BspJ protein was localized in the host cell nucleus and around the nucleus;RT-PCR analysis of BspJ gene was carried out in host cells for transcription.[Conclusion] The secreted protein BspJ of Brucella is distributed around the host cell nucleus and nucleus,and may have a nuclear-cytoplasmic shuttle process.
作者
马忠臣
胡尊楠
杨亚军
何金科
荆明龙
王勇
陈创夫
Zhongchen Ma;Zunnan Hu;Yajun Yang;Jinke He;Minglong Jing;Yong Wang;Chuangfu Chen(College of Animal Science and Technology Shihezi University,Shihezi 832000,China;College of Life Science,Shihezi University,Shihezi 832000,China)
出处
《生物技术》
CAS
2018年第6期572-578,共7页
Biotechnology
基金
国家重大研发计划项目子课题(“病原菌感染与入侵的分子机制”,No.2017YFD0500304)
