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利用基因重组、PCR扩增、定点突变及转译优化等程序,构建了人白细胞介素6重组表达载体CSV-IL-6(1)和CSV-IL-6(2);并利用DEAE-Dextran法转染猴细胞系COS7获得了有生物活性IL-6的暂时性高表达。IL-6ELISA定量测定表达水平:转染后48小时收集的上清CSV-IL-6(1)2573pg/ml,72小时1476pg/ml;CSV-IL-6(2)分别为8261Pg/ml与7101pg/ml。利用IL-6依赖的杂交瘤细胞株7TD1检测转染后48小时收集的培养上清的IL-6生物活性结果为:CSV-IL-6(1)10~4U/ml、3.9×10~9U/mg;CSV-IL-6(2)6.3×10~4U/ml,7.6×10~9U/mg。
The recombinant human interleukin-6 (CSV-IL-6) and CSV-IL-6 (2) were constructed by gene recombination, PCR amplification, site-directed mutagenesis and translation optimization. The DEAE-Dextran Transfection of the monkey cell line COS7 resulted in a transiently high expression of bioactive IL-6. The levels of CSV-IL-6 (1) 2573pg / ml and 72hours 1476pg / ml in the supernatants collected at 48h after transfection were 8261pg / ml and 7101pg / ml. The IL-6 bioactivity of the culture supernatants collected 48 hours after transfection was detected using IL-6-dependent hybridoma cell line 7TD1 as 10-4U / ml as CSV-IL-6 (1), 3.9x10-9U /mg;CSV-IL-6(2)6.3 × 10 ~ 4U/ml, 7.6 × 10 ~ 9U / mg.