草鱼肾细胞电转条件的优化及草鱼呼肠孤病毒NS26蛋白的瞬时表达  

Optimization of Electroporation Parameters for Ctenopharyngodon Idellus Kidney Cells and Transient Expression of Grass Carp Reovirus NS26 Protein

作　　者：李炎[1] 张娅楠[1] 吕利群[1] 

机构地区：[1]上海海洋大学农业部淡水种质资源重点实验室,上海201306

出　　处：《病毒学报》2014年第3期278-284,共7页Chinese Journal of Virology

基　　金：国家自然科学基金(No.31372561);现代农业产业技术体系建设专项资金(CARS-46-12);上海海洋大学水产学一流学科

摘　　要：本研究先以pEGFP-N1载体质粒中的绿色荧光蛋白基因为报道基因进行电转实验,优化电压、脉冲时间、质粒添加量和电击次数等电转条件,确立最佳条件。实验表明,细胞密度为1.5×107/mL时,取200μl在0.2cm电击杯中进行电击转染,电压为200V,脉冲时间为45ms,添加质粒30μg,电击1次时,转染效果最佳。提取草鱼呼肠孤病毒基因组总RNA,以其逆转录的cDNA作为扩增模板,设计特异引物扩增出GCRV非结构蛋白NS26的基因片段,重组到pEGFP-N1载体上得到重组质粒pEGFP-NS26,将其导入CIK细胞中用电转法高效表达了EGFPNS26融合蛋白。本研究为进一步研究NS26的功能奠定了基础。In this study, pEGFP-N1 was chosen as the reporter plasmid and transferred into Ctenopharyngodon idellus kidney （CIK） ceils by electroporation, and the optimal eleetroporation conditions were determined by testing the transfection efficiency with different voltages, pulse times, plasmid amounts, and numbers of shocks. The results showed that the maximum electroporation efficiency was achieved under the following conditions in a 0.2 cm electroporation cuvette containing CIK cells （1.5×10^7/mL, 200 μl）： electric voltage 200 V, pulse time 45 ms, plasmid 30 μg, and one electric shock. The total genomic RNA of grass carp reovirus （GCRV） was extracted in this experiment and reversely transcribed into cDNA, which was used to amplify the gene segment of GCRV nonstructural protein NS26 using designed specific primers. The PCR product was recombined into pEGFP-N1 vector. The fusion protein EGFP-NS26 was successfully and efficiently expressed in the CIK cells by electroporation, which was confirmed by both fluorescent imaging and Western blot analysis. This experiment laid a foundation for further functional studies of the non-structural protein NS26 of GCRV.

关 键 词：电转染 草鱼呼肠孤病毒 草鱼肾细胞 重组质粒 

分 类 号：S943[农业科学—水产养殖]