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Glycosylation and L303E/F309S mutations improve intein-mediated splicing of the split coagulation factor Ⅷ
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- Author:
- No author available
- Journal Title:
- ACTA PHARMACEUTICA SINICA
- Issue:
- 11
- DOI:
- No doi available
- Key Word:
- 凝血Ⅷ因子;重链变体;分泌;intein;蛋白质剪接
Abstract： 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intdn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.
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