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. 2010 Nov;45(11):1361-6.
We recently demonstrated that an intein-mediated protein splicing can be used to transfer B-domain-deleted FVIII (BDD-FVIII) gene by a dual-vector. In this study, we observed the effect of a variant heavy chain with six potential glycosylation sites of B domain and L303E/F309S mutations in its A1 domain, which were proven to be beneficial for FVIII secretion, on secretion of spliced BDD-FVIII. By transient co-transfection of cultured 293 cells with intein-fused variant heavy chain (DMN6HCIntN) and light chain (IntCLC) genes, the culture supernatant was analyzed quantitatively by ELISA for secreted spliced BDD-FVIII antigen and by a chromogenic assay for bioactivity. The data showed that the amount of spliced BDD-FVIII protein and coagulation activity in culture supernatant from DMN6HCIntN plus IntCLC co-transfected cells were up to (149 +/- 23) ng x mL(-1) and (1.12 +/- 0.14) u x mL(-1) respectively greater than that of intein-fused wild type heavy (HCIntN) and light chain (IntCLC) co-transfected cells [(99 +/- 14) ng x mL(-1) and (0.77 +/- 0.13) u x mL(-1)] indicating that the variant heavy chain is able to improve the secretion of spliced BDD-FVIII and activity. A cellular mechanism-independent BDD-FVIII splicing was also observed. It provided evidence for ongoing animal experiment using intein-mediated dual-AAV vector technology for delivery of the BDD-FVIII genes.
我们最近证明，内含肽介导的蛋白质剪接可用于通过双载体转移B结构域缺失的FVIII（BDD-FVIII）基因。在本研究中，我们观察了具有B结构域六个潜在糖基化位点以及A1结构域中L303E/F309S突变的变异重链对剪接的BDD-FVIII分泌的影响，这些已被证明有利于FVIII分泌。通过将内含肽融合的变异重链（DMN6HCIntN）和轻链（IntCLC）基因瞬时共转染培养的293细胞，通过ELISA定量分析培养上清液中分泌的剪接BDD-FVIII抗原，并通过显色测定法分析生物活性。数据显示，DMN6HCIntN加IntCLC共转染细胞的培养上清液中剪接的BDD-FVIII蛋白量和凝血活性分别比内含肽融合的野生型重链（HCIntN）和轻链（IntCLC）共转染细胞的[(99±14) ng x mL(-1)和(0.77±0.13) u x mL(-1)]高出(149±23) ng x mL(-1)和(1.12±0.14) u x mL(-1)，表明变异重链能够改善剪接的BDD-FVIII的分泌和活性。还观察到了一种与细胞机制无关的BDD-FVIII剪接。这为正在进行的使用内含肽介导的双AAV载体技术递送BDD-FVIII基因的动物实验提供了证据。
