Zhu Fu-Xiang, Liu Ze-Long, Miao Jing, Qu Hui-Ge, Chi Xiao-Yan
Life Science College of Ludong University, Yantai 264025, China.
Yao Xue Xue Bao. 2012 Jun;47(6):734-8.
To investigate the improving effect of inter-chain disulfide formation on protein trans-splicing, we introduce a Cys point mutation at Tyr(664) in heavy chain and at Thr(1826) in light chain of B-domain-deleted FVIII (BDD-FVIII). By co-transfection of COS-7 cell with the two Cys mutated chain genes, the intracellular protein splicing, inter-chain disulfide formation, secreted BDD-FVIII and bioactivity in culture supernatant were observed. The data showed that a strengthened spliced BDD-FVIII with an inter-chain disulfide detected by Western blotting and an elevated secretion of spliced BDD-FVIII (128 +/- 24 ng mL(-1)) compared to control (89 +/- 15 ng mL(-1)), assayed by a sandwich ELISA. A Coatest was performed to assay the secretion of bioactivity in culture supernatant and shown a much higher value (0.94 +/- 0.08 u mL(-1)) compared to that of control (0.62 +/- 0.15 u mL(-1)). It suggests that inter-chain disulfide formation could improve protein trans-splicing based dual-vector delivery of BDD-FVIII gene providing experimental evidence for ongoing in vivo study.
为了研究链间二硫键形成对蛋白质反式剪接的改善作用，我们在缺失B结构域的FVIII（BDD-FVIII）重链的Tyr(664)和轻链的Thr(1826)处引入了半胱氨酸点突变。通过将两个半胱氨酸突变链基因共转染COS-7细胞，观察细胞内蛋白质剪接、链间二硫键形成、分泌的BDD-FVIII以及培养上清液中的生物活性。数据显示，通过蛋白质免疫印迹法检测到链间二硫键增强的剪接BDD-FVIII，并且通过夹心酶联免疫吸附测定法测定，与对照（89±15 ng mL(-1)）相比，剪接BDD-FVIII的分泌量升高（128±24 ng mL(-1)）。进行Coatest检测培养上清液中的生物活性分泌，结果显示与对照（0.62±0.15 u mL(-1)）相比，其值要高得多（0.94±0.08 u mL(-1)）。这表明链间二硫键的形成可以改善基于BDD-FVIII基因双载体递送的蛋白质反式剪接，为正在进行的体内研究提供实验证据。
