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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, the green fluorescent protein (EGFP) gene in pEGFP-N1 vector was used as the reporter gene to optimize the conditions of voltage, pulse time, the amount of plasmid and the number of electroporation. Experiments showed that when the cell density was 1.5 × 107 / mL, 200μl was electroporated in a 0.2cm electric shock cup and the voltage was 200V. The pulse time was 45ms. When the plasmid was added 30μg and the electric shock was applied for 1 time, the transfection efficiency was the best. The total RNA of grass carp reovirus was extracted and its reverse transcribed cDNA was used as the amplification template. The specific primers were designed to amplify the gene fragment of GCRV non-structural protein NS26 and recombined into pEGFP-N1 vector to obtain recombinant plasmid pEGFP-NS26. Introduced into CIK cells electroporated EGFPNS26 fusion protein was highly expressed. This study laid the foundation for further study on the function of NS26.