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Leucine zippers improves protein splicing-mediated coagulation factor Ⅷ gene delivery by dual-vector system
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- Author:
- No author available
- Journal Title:
- Acta Pharmaceutica Sinica
- Issue:
- 1
- DOI:
- No doi available
- Key Word:
- 凝血Ⅷ因子;intein;蛋白质反式剪接;亮氨酸拉链
Abstract： 作者最近基于蛋白内含子(intein)的双载体转B区缺失型凝血Ⅷ因子(BDD-FVIII)基因研究证明,表达后的重、轻链可通过蛋白质反式剪接成为共价连接的完整BDD-FVIII分子并发挥凝血生物活性,但存在不完全剪接的前体蛋白.本文试图通过将具有特异性强相互作用的亮氨酸拉链引入intein序列,提高反式剪接的效率,用双载体系统向培养的COS-7细胞共转融合intein的重链和轻链基因,通过瞬时表达观察了细胞内BDD-FVIII的剪接和分泌至培养上清液的剪接BDD-FVIII量和生物活性.结果显示,Western blotting检测到共转基因细胞内明显增强的BDD-FVIII剪接,表现为前体蛋白的减少；分别用ELISA和Coatest法分析共转基因细胞培养上清液中分泌的剪接BDD-FVIII蛋白量和生物活性为(106±12) ng·mL-1和(0.89±0.11) U·mL-1,明显高于无亮氨酸拉链的intein融合重链和轻链基因共转基因细胞[(72±10) ng·mL-1和(0.62±0.07) U·mL-1],而且不依赖细胞机制的蛋白质剪接反应明显增强,表现为混合培养的转重链和轻链基因细胞上清液中增加的剪接BDD-FVIII蛋白量和生物活性[(36±11) ng·mL-1和(0.28±0.09) U·mL-1].结果表明,亮氨酸拉链可通过增强融合于重链和轻链的intein间的相互作用提高蛋白质反式剪接的效率,改善基于蛋白质剪接的双载体转BDD-FVIII基因的效果,为进一步的动物体内基于蛋白质剪接的双腺相关病毒(AAV)载体转FVIII基因研究提供了实验依据.
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