摘要
探讨携带SIRPα-Fc(SF)融合基因蛋白基因的重组腺病毒Ad35-SF感染T细胞后对T细胞功能的影响。腺病毒Ad35-SF感染Jurkat T细胞后,通过Western blotting及ELISA方法检测细胞上清中SF融合蛋白的大小与表达量;通过流式分析技术检测细胞表面CD47的封闭情况;通过CCK8方法检测细胞的增殖与对肿瘤细胞的杀伤作用。结果表明,Ad35-SF构建成功,其感染Jurkat T细胞后,能在其内表达并分泌产生大小约为48kD的SF蛋白,细胞上清中SF的表达量可到达19.1μg/mL。Ad35-SF(MOI=1)感染Jurkat细胞18h后,细胞CD47阳性比例从97.06%下降到15.32%;当Ad35-SF感染复数增加至5,10时,CD47阳性比率进一步降低至2.94%和1.73%。相对于空病毒对照组,Ad35-SF感染Jurkat细胞48h后其增殖效果提高了29%,对肝癌细胞株Hep 3B的杀伤作用提升25.6%(p<0.01)。结果表明构建的重组腺病毒Ad35-SF能有效感染Jurkat细胞并在其内表达分泌SF蛋白,封闭细胞表面的CD47,同时显著提高Jurkat细胞的增殖能力和对肝癌细胞的杀伤作用。
This paper investigates the effect of T cell functions after recombinant adenovirus Ad35-SF with SIRPα-Fc（SF）fusion gene and protein gene infects T cells.After adenovirus Ad35-SF infected Jurkat T cells,the magnitude and expression quantity of SF fusion protein in suspension of Jurkat T cells were detected through Western blotting and ELISA.The closure condition of CD47 was detected through flow cytometry.Cell proliferation and killing capacity to cancer cells were detected through CCK8.The results show that Ad35-SF was successfully constructed;after it infected Jurkat T cells,48 kD SF proteins could be expressed and secreted;SF expression quantity could reach 19.1μg/mL in the suspension of Jurkat T cells.After Ad35-SF（MOI=1）infected Jurkat cells for 18 h,the proportion of CD47-positive cells dropped to 15.32%from 97.06%.When infection multiplicity of Ad35-SF increased to 5hand 10 h,the proportion of CD47-positive cells further declined to 2.94% and 1.73%,respectively.Relative to the control group,the proliferation increased by 29%after Ad35-SF infected Jurkat cells for 48 h,and skilling effect on hepatocellular carcinoma cell line Hep3 Bincreased by 25.6%（p〈0.01）.The above results show that recombinant adenovirus Ad35-SF can effectively infect Jurkat cells,express and secrete SF protein as well as close CD 47 on cell surface.Meanwhile,it can significantly boost proliferation capacity of Jurkat cells and skilling effect on hepatoma carcinoma cells.
出处
《浙江理工大学学报（自然科学版）》
2015年第5期712-717,共6页
Journal of Zhejiang Sci-Tech University(Natural Sciences)
基金
国家自然科学基金项目(81071850)
