BiP表达下调改善双载体转断裂FVIII基因的功效

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中国生物化学与分子生物学报 ›› 2011, Vol. 27 ›› Issue (12) : 1160-1165.

技术与方法

BiP表达下调改善双载体转断裂FVIII基因的功效

朱甫祥*,刘泽隆,缪静,屈慧鸽,迟晓艳

作者信息 +

Down Regulated BiP Expression Improves Efficacy of
Split FⅧ Gene Delivery by a Dual-Vector

ZHU Fu-Xiang*, LIU Ze-Long, MIAO Jing, QU Hui-Ge, CHI Xiao-Yan

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文章历史 +

摘要

双载体转凝血Ⅷ因子（FⅧ）基因可作为一种转基因策略克服腺相关病毒(AAV)载体容量限制，但重链分泌的低效性影响转基因功效. 为提高重链分泌，本文用RNA干扰技术下调内质网内蛋白伴侣分子免疫球蛋白重链结合蛋白(BiP)的表达，观察对HEK293细胞双载体共转FⅧ基因分泌重链和生物活性的影响. 结果显示，RNA干扰可明显下调BiP表达，但不影响细胞生长； ELISA检测BiP下调细胞单独转重链基因时的重链分泌量为98±38 ng/mL，与轻链共转基因时显著升高到157±32 ng/mL，明显高于对照细胞单独转重链基因和共转重链和轻链基因的重链分泌量(分别为29±8 ng/mL和79±19 ng/mL)；Cotest法检测显示，BiP下调细胞共转重链和轻链基因细胞分泌的凝血生物活性为0.73±0.23 IU/mL，明显高于对照细胞共转重链和轻链基因(0.39±0.07 IU/mL). 结果表明， BiP表达下调通过促进重链分泌，可提高双载体共转FⅧ基因的功效，为进一步动物体内双AAV载体转FⅧ基因的甲型血友病基因治疗研究提供了实验依据.

Abstract

Dual-vector transfer of coagulation factor Ⅷ (FⅧ) gene has been developped as an alternative strategy to overcome the packaging limitation of adeno-associated virus (AAV) vectors but its efficacy was adversely affected by an inefficient heavy chain secretion. In this study, we aimed to improve secretion of FⅧ heavy chain by down-regulating expression of immunoglobulin heavy-chain binding protein (BiP), an ER chaperon protein with RNAi technology, and observed secreted heavy chain and FⅧ bioactivity by HEK293 cells after a dua-vector co-tranduction with split FⅧ gene. The data showed that RNA interference could obviously down-regulate the expression of BiP but no effect was found on cell proliferation. It showed much higher levels of heavy chain secretion from heavy chain gene transfected cell (98±38 ng/mL) and heavy plus light chain gene cotransfected cell (157±32 ng/mL) compared to cotrol cell (29±8 ng/mL and 79±19 ng/mL). The bioactivity secreted by heavy and light chain gene cotransfected cell with BiP down-regulated was 0.73±0.23 IU/mL, greater than that of control cell (0.39±0.07 IU/mL). These data demonstrate that BiP down-regulation could improve efficacy of dual-vector mediated split FⅧ gene delivery forming the basis for ongong study using dual-AAV vector to deliver FⅧ gene for hemophilia A gene therapy.

导出引用

朱甫祥, 刘泽隆, 缪静, 屈慧鸽, 迟晓艳. BiP表达下调改善双载体转断裂FVIII基因的功效[J]. 中国生物化学与分子生物学报, 2011, 27(12): 1160-1165

ZHU Fu-Xiang, LIU Ze-Long, MIAO Jing, QU Hui-Ge, CHI Xiao-Yan. Down Regulated BiP Expression Improves Efficacy of
Split FⅧ Gene Delivery by a Dual-Vector[J]. Chinese Journal of Biochemistry and Molecular Biology, 2011, 27(12): 1160-1165

参考文献

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收稿日期	修回日期	出版日期
2011-09-26	2011-11-02	2011-12-20
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2011-11-29

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