昆虫细胞中的不同表达效率载体的元件优化及构建  

Element Optimization and Construction of Vectors with Different Expression Efficiency in Insect Cells

作　　者：王金武 徐一夫 赵亚琦 薛霆 宋柳 郭华荣[1,2] Wang Jinwu;Xu Yifu;Zhao Yaqi;Xue Ting;Song Liu;Guo Huarong(Key Laboratory of Marine Genetics and Breeding,Ministry of Education,College of Marine Life Sciences,Ocean University of China,Qingdao 266003,China;Institute of Evolution and Marine Biodiversity,Ocean University of China,Qingdao 266003,China)

机构地区：[1]中国海洋大学海洋生命学院海洋生物遗传学与育种教育部重点实验室,山东青岛266003 [2]中国海洋大学海洋生物多样性与进化研究所,山东青岛266003

出　　处：《中国海洋大学学报（自然科学版）》2024年第12期63-71,共9页Periodical of Ocean University of China

基　　金：国家自然科学基金项目(32273116);山东省自然科学基金项目(ZR2020MC189)资助。

摘　　要：为了构建昆虫细胞高效表达载体,本文深入探讨了昆虫Sf9细胞中,3种不同种属来源的病毒早期启动子(OpIE2、P2和CMV)、2种荧光报告基因(EGFP和TagBFP)和2种转录终止子(SV40-pA和OpIE2-pA)的12种不同组合方式对所构建的表达质粒表达效率的影响。研究结果发现,上述3种表达元件本身都可对表达质粒的表达效率产生严重影响,其中启动子的影响效果尤为突出,而且三者之间的搭配要慎重,否则可能导致载体表达效率的显著降低。结果表明:在Sf9细胞中,OpIE2-EGFP-OpIE2-pA组合展现出最高的表达效率;启动子活性方面,OpIE2启动子的活性最高,P2次之,CMV最差;EGFP的荧光信号明显高于TagBFP的荧光信号;OpIE2-pA的转录终止活性高于SV40-pA;双启动子(OpIE2-P2)驱动的表达质粒的转染结果表明,P2启动子的存在可抑制OpIE2启动子的活性。本结果可为昆虫细胞高效表达载体的构建提供重要参考。In order to understand the effect of different combinations among the three kinds of expression elements(promoter,reporter and transcription terminator)on the expression efficiencies of insect cell expression vectors,twelve kinds of single promoter-driven expression vectors were constructed from three kinds of viral early-promoters(OpIE2,P2 and CMV)with different species origins,two kinds of fluorescent reporter genes(EGFP and TagBFP)and two kinds of transcriptional terminator(SV40-pA and OpIE2-pA)by different arrangements and combinations among the above-mentioned three kinds of expression elements in this study.It was found that all the three kinds of expression elements themselves could impose a serious impact on the expression efficiencies,but the promoter produced the highest effect among them.Moreover,the combination among these three expression elements should be careful,otherwise,the expression efficiency of the vector will be greatly impaired by a wrong combination.In Sf9 cells,OpIE2-EGFP-OpIE2-pA was found to be the best combination which could produce the highest expression efficiency.In terms of promoter activity,the activity of OpIE2 promoter was the highest,followed by P2 and CMV was the worst.The reporter gene of EGFP could produce higher intensity of fluorescence signal than TagBFP did.The transcriptional termination activity of OpIE2-pA was higher than SV40-pA.In addition,the transfection results of double promoters(OpIE2-P2)-driven expression plasmid in Sf9 cells showed that the insertion of P2 promoter had inhibited the activity of OpIE2 promoter.The obtained results in this study will serve as an important reference for the construction of efficient expression vector for insect cells.

关 键 词：SF9细胞 表达质粒 表达效率 启动子 报告基因 转录终止子 

分 类 号：Q291[生物学—细胞生物学]