Zhu Fu-Xiang, Liu Ze-Long, Qu Hui-Ge, Chi Xiao-Yan
Life Science College of Ludong University, Yantai, China.
Sheng Li Xue Bao. 2010 Aug 25;62(4):373-81.
Low levels of coagulation factor VIII (fVIII) protein expression caused by its inefficient secretion and the over-sized fVIII gene affect the transgene-based gene therapy for hemophilia A adversely. Our previous study demonstrated that intein-mediated protein trans-splicing for delivery of the fVIII gene with a dual-vector system could improve secretion of post-translationally spliced fVIII by light chain in cis. In this study, a human/porcine hybrid fVIII (HP-fVIII) containing replaced A1 and A3 domains of porcine fVIII was investigated for secretion and activity of the spliced HP-fVIII after intein-based dual-vector delivery of the HP-fVIII gene. A pair of expression plasmids comprising intein-fused HP-fVIII heavy and light chains were constructed and transiently co-transfected into COS-7 cells. The spliced HP-fVIII and bio-activity in culture media were quantitatively analyzed by ELISA and Coatest method respectively. The intracellular splicing of HP-fVIII was detected by Western blotting. The results showed that in the culture supernatant of cells co-transfected with HP-fVIII, the amount and activity of spliced HP-fVIII were significantly higher than those of spliced hfVIII secreted from the cells co-transfected with human fVIII [(184+/-34 ng/mL) vs (48+/-12) ng/mL, P<0.01; (1.18+/-0.22) IU/mL vs (0.31+/-0.10) IU/mL, P<0.01], demonstrating the dramatically enhancing effect of porcine A1 and A3 domains on the secretion of intein-spliced HP-fVIII. The spliced HP-fVIII protein and its activity were also detected in the supernatant from combined cells separately transfected with intein-fused HP-fVIII heavy and light chain genes, indicating that the intein-mediated HP-fVIII splicing was independent of cellular mechanism and could occur outside the cell after the secretion of precursor proteins. Additionally, an intracellularly spliced HP-fVIII band was found with a molecular weight similar to human fVIII protein, confirming the HP-fVIII splicing. These results provided experimental basis for ongoing study using intein-based dual adeno-associated virus (AAV) vector to transfer HP-fVIII gene in animal models.
凝血因子VIII（fVIII）蛋白表达水平低是由其分泌效率低下和fVIII基因过大所致，这对基于转基因的A型血友病基因治疗产生了不利影响。我们之前的研究表明，采用双载体系统通过内含肽介导的蛋白质反式剪接来递送fVIII基因，可以提高顺式轻链翻译后剪接的fVIII的分泌水平。在本研究中，对含有猪fVIII的A1和A3结构域被替换的人/猪杂交fVIII（HP-fVIII）进行了研究，以观察基于内含肽的双载体递送HP-fVIII基因后剪接的HP-fVIII的分泌和活性。构建了一对包含内含肽融合的HP-fVIII重链和轻链的表达质粒，并将其瞬时共转染到COS-7细胞中。分别通过ELISA和Coatest方法对培养基中的剪接HP-fVIII和生物活性进行定量分析。通过蛋白质印迹法检测HP-fVIII的细胞内剪接情况。结果显示，在与HP-fVIII共转染的细胞培养上清液中，剪接HP-fVIII的量和活性显著高于与人fVIII共转染的细胞分泌的剪接人fVIII [（184±34 ng/mL）对（48±12）ng/mL，P<0.01；（1.18±0.22）IU/mL对（0.31±0.10）IU/mL，P<0.01]，表明猪A1和A3结构域对内含肽剪接的HP-fVIII的分泌具有显著的增强作用。在分别用内含肽融合的HP-fVIII重链和轻链基因转染的组合细胞的上清液中也检测到了剪接的HP-fVIII蛋白及其活性，这表明内含肽介导的HP-fVIII剪接独立于细胞机制，并且可以在前体蛋白分泌后在细胞外发生。此外，发现了一条细胞内剪接的HP-fVIII条带，其分子量与人fVIII蛋白相似，证实了HP-fVIII的剪接。这些结果为正在进行的使用基于内含肽的双腺相关病毒（AAV）载体在动物模型中转移HP-fVIII基因的研究提供了实验依据。
