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作者最近证明intein的蛋白质剪接技术可用于双载体转B区缺失型FVIII(BDD-FVIII)基因,在此基础上,本研究将具有促FVIII分泌作用的L303E/F309S双突变和B结构域糖基化位点引入BDD-FVIII重链,观察重链变体对intein剪接的BDD-FVIII蛋白分泌和活性的影响。用分别融合Ssp DnaB intein的重链变体(DMN6HCIntN)和轻链(IntCLC)基因共转染培养的293细胞,用ELISA分析分泌至培养上清液中的剪接BDD-FVIII蛋白量,并用发色法检测培养上清液的凝血生物活性。结果显示,DMN6HCIntN和IntCLC共转基因细胞上清液中剪接BDD-FVIII的浓度为(149±23)ng.mL-1,活性为(1.12±0.14)u.mL-1,明显高于intein融合的野生型重链(HCIntN)与轻链(IntCLC)共转基因细胞[(99±14)ng.mL?1和(0.77±0.13)u.mL?1],提示重链变体能明显促进剪接BDD-FVIII蛋白的分泌和活性;另外,还检测到不依赖细胞机制的BDD-FVIII剪接。本研究为进一步动物体内双AAV载体转BDD-FVIII基因研究提供了依据。
The authors recently demonstrated that intein’s protein-splicing technique can be used to double-vector to B region deletion type FVIII (BDD-FVIII) gene. Based on this, L303E / F309S double mutation and B-domain glycosylation The BDD-FVIII heavy chain was introduced to visualize the effect of heavy chain variants on intein spliced BDD-FVIII protein secretion and activity. The cultured 293 cells were co-transfected with the heavy chain variant (DMN6HCIntN) and the light chain (IntCLC) genes respectively fused to Ssp DnaB intein, the amount of spliced BDD-FVIII protein secreted into the culture supernatant was analyzed by ELISA, and the amount of spliced BDD- Method to detect clotting biological activity of culture supernatant. The results showed that the concentration of BDD-FVIII spliced in the supernatants of DMN6HCIntN and IntCLC was (149 ± 23) ng.mL-1 and the activity was (1.12 ± 0.14) u.mL-1, which was significantly higher than that of intein fusion The co-transgenic cells [(99 ± 14) ng.mL -1 and (0.77 ± 0.13) u.mL 1] of wild type heavy chain (HCIntN) and light chain (IntCLC) suggested that the heavy chain variant could significantly promote the splicing of BDD- FVIII protein secretion and activity; in addition, BDD-FVIII splicing was also detected in a cell-free mechanism. This study provides the basis for the further study of BDD-FVIII gene by double AAV vectors in animals.