Zhu Fu-Xiang, Liu Ze-Long, Qu Hui-Ge, Chi Xiao-Yan
Life Science College of Ludong University, Yantai 264025, China.
Sheng Li Xue Bao. 2009 Dec 25;61(6):526-32.
Coagulation factor VIII (fVIII) is a secretion protein and plays a crucial role in the coagulation cascade. Hemophilia A resulted from deficiency of fVIII is the most common X-linked recessive bleeding disorder. Gene therapy is recognized as an attractive strategy for the eventual cure of this disease. However, the gene therapy is hampered by the big size of fVIII gene when using the most promising gene vectors, adeno-associated virus (AAV) vectors. In this study we explored the intein-mediated protein trans-splicing to deliver a Phe(309)-->Ser mutant full-length fVIII (F309SfVIII) gene by using a dual-vector system. An intein is a protein sequence embedded within a precursor protein and can excise itself through protein splicing. The F309SfVIII is proven to be beneficial to its secretion. The F309SfVIII gene was broken into heavy and light chains before Ser(1239) in B domain and fused with the coding sequences of Ssp DnaB intein respectively to construct a pair of plasmid vectors by inserting them into the pcDNA3.1 vectors. Forty-eight hours after co- or separate transfection of 293 cells, the co-transfected cell lysate showed an obvious ligated F309SfVIII protein band by Western blot with a polyclonal antibody against fVIII. The amounts of secreted F309SfVIII protein in culture supernatants and their bioactivities were (71+/-9) ng/mL and (0.38+/-0.09) IU/mL determined by ELISA and Coatest assay respectively. The supernatant from combined cells with separate transfections also displayed lower levels of F309SfVIII antigen and fVIII activity [(25+/-6) ng/mL and (0.12+/-0.05) IU/mL], indicating the F309SfVIII could be formed by splicing both before and after secretion. The content of F309SfVIII heavy chain protein from co-transfected cell supernatant was higher than that of intein-fused heavy chain transfection alone [(135+/-10) ng/mL vs (37+/-7) ng/mL, P<0.01)]. These data demonstrated that intein could be used as a technical strategy in a dual-vector system delivering F309SfVIII gene with improved secretion of fVIII providing an alternative approach to circumvent the packaging limitation of AAV for F309SfVIII gene transfer, which encourages our continuing study in hemophilia A gene therapy in vivo.
凝血因子VIII（fVIII）是一种分泌蛋白，在凝血级联反应中起关键作用。由fVIII缺乏引起的甲型血友病是最常见的X连锁隐性出血性疾病。基因治疗被认为是最终治愈这种疾病的一种有吸引力的策略。然而，当使用最有前景的基因载体腺相关病毒（AAV）载体时，基因治疗受到fVIII基因大尺寸的阻碍。在本研究中，我们探索了内含肽介导的蛋白质反式剪接，以通过双载体系统递送Phe(309)-->Ser突变全长fVIII（F309SfVIII）基因。内含肽是嵌入前体蛋白中的蛋白质序列，可通过蛋白质剪接切除自身。已证明F309SfVIII对其分泌有益。F309SfVIII基因在B结构域的Ser(1239)之前被分成重链和轻链，并分别与Ssp DnaB内含肽的编码序列融合，通过将它们插入pcDNA3.1载体来构建一对质粒载体。在共转染或分别转染293细胞48小时后，用抗fVIII的多克隆抗体进行蛋白质印迹分析，共转染的细胞裂解物显示出明显的连接的F309SfVIII蛋白条带。通过ELISA和Coatest测定法分别测定培养上清液中分泌的F309SfVIII蛋白的量及其生物活性为(71±9) ng/mL和(0.38±0.09) IU/mL。单独转染的联合细胞的上清液也显示出较低水平的F309SfVIII抗原和fVIII活性[(25±6) ng/mL和(0.12±0.05) IU/mL]，表明F309SfVIII可以在分泌前后通过剪接形成。共转染细胞上清液中F309SfVIII重链蛋白的含量高于单独的内含肽融合重链转染[(135±10) ng/mL对(37±7) ng/mL，P<0.01]。这些数据表明，内含肽可以用作双载体系统中的一种技术策略，用于递送F309SfVIII基因，同时改善fVIII的分泌，为规避AAV对F309SfVIII基因转移包装限制提供了一种替代方法，这鼓励我们在体内甲型血友病基因治疗方面继续研究。
