慢病毒包装体系的建立与体外表达  

The Construction of Packaging System and Expression in Vitro of Lentivirus

作　　者：王雪晗[1] 贾俊龙[1] 王维[1] 朱玉博[1] 许诺[1] 白文林[1] 罗光彬[1] 

机构地区：[1]沈阳农业大学畜牧兽医学院,沈阳110161

出　　处：《沈阳农业大学学报》2016年第4期432-437,共6页Journal of Shenyang Agricultural University

基　　金：农业部转基因生物新品种培育重大专项基金项目(H022020060420)

摘　　要：为筛选出最佳的慢病毒转染处理时间,研究三质粒对慢病毒包装的最佳浓度配比,以及对293T细胞的细胞转染率和滴度,通过脂质体与Fugw质粒混合后转染293T细胞,观察293T细胞的形态和荧光表达量。结果表明:转染6h组的细胞形态略好于8h;转染8h组和6h组的转染率均极显著高于10h组(p<0.01),但彼此间差异不显著(p>0.05);质粒配比为4:3:2时,慢病毒滴度最高,达3.795×107TU·m L-1,经离心后滴度与离心之前相比能提高100倍。不同浓度的质粒配比对293T细胞转染率和滴度方面,B组(4∶3∶2)的转染率最高、C组(3∶2∶1)最低、A组(5∶4∶3)介于二者之间,3组间彼此差异极显著(p<0.01);转染后B组的滴度极显著高于A组和C组(p<0.01),而A组和C组间差异不显著(p>0.05)。感染293T靶细胞存在EGFP基因表达。利用10μL脂质体2000介导4μg的Fugw质粒转染293T细胞的最佳时间为6h;当Fugw∶△8.2∶Vsvg为4∶3∶2时,慢病毒转染293T细胞的效果最好。In order to select the best processing time of lentivirus transfection, to study the optimum concentration of threeplasmids about lentiviral packaging, as well as transfected rate and titer of the 293T cells, this experiment was conducted.Liposome mixes with Fugw plasmid were transfected into 293T cell line, morphology and fluorescence expression of 293T cellwere observed. The results showed that cell morphology after 6h transfection was slightly better than that 8h transfection. Therates of 8h transfection and 6h transfection were significantly higher than that of 10h transfection （p〈0.01）, but there was nosignificant difference between 8h and 6h after transfection （p〉0.05）. The ratio of plasmid of 4：32 showed that lentivirus obtainedthe highest titer, up to 3.795×10^7 TU.mL-1, titer could be improve 100 times after centrifugal. The ratio of different concentrationsof plasmid transfection of 293T cell line and titer, group B （4：3：2） has the highest rate of transfection, group A （5：4：3） was thesecond, the lowest （3：2：1） was group C. There were significant differences among the three groups （p〈0.01）. Group B aftertransfection was significantly higher than that of group A and group C （p〈0.01）, while there was no significant difference betweengroup A and group C （p〉0.05）. EGFP gene could be expressed in transfected 293T cells line. The best time that 293T cellswere transfected by 10μL Liposome 2000 mediating 4μg Fugw plasmid was 6h. When Fugw：△8.2：Vsvg was 4：3：2,293T cellscould be transfected best by lentivitus.

关 键 词：慢病毒 转染 293T细胞 滴度 

分 类 号：Q786[生物学—分子生物学]