前肽缺失体vWF改善FVIII基因断裂转移 细胞的重链和活性分泌

中国生物工程杂志

2012, Vol. 32

Issue (07): 20-25

研究报告

前肽缺失体vWF改善FVIII基因断裂转移细胞的重链和活性分泌

朱甫祥, 刘泽隆, 缪静, 屈慧鸽, 迟晓艳

鲁东大学生命科学学院烟台 264025

Propeptide-deleted von Willebrand Factor Improves Heavy Chain Secretion and Bioactivity by Split FVIII Gene Co-transfected Cells

ZHU Fu-xiang, LIU Ze-long, MIAO Jing, QU Hui-ge, CHI Xiao-yan

Life Science College of Ludong University, Yantai 264025, China

全文: PDF(592 KB) HTML

摘要： 通过转von Willebrand因子(vWF)的前肽缺失突变体(vWF-ΔPro)基因,探讨了vWF-ΔPro对双载体转凝血VIII因子(FVIII)基因的影响。将vWF-ΔPro基因和B-区缺失型FVIII(BDD-FVIII)的重、轻链基因共转染HEK293细胞48 h后,ELISA检测重链的分泌量为(142±29)ng/ml,明显高于未转vWF-ΔPro基因细胞(87±15)ng/ml;未转vWF-ΔPro基因时单独转重链基因的重链分泌水平很低,vWF-ΔPro存在时重链分泌量明显提高,但不如共转基因时的重链分泌,提示轻链可反式促进重链分泌;单独转轻链或与重链共转基因时轻链分泌水平均较高,且不受vWF-ΔPro影响;Coatest法检测分泌的凝血活性显示,转vWF-ΔPro基因可使细胞分泌的凝血活性明显高于未转vWF-ΔPro基因细胞(0.80±0.15 IU/ml vs 0.41±0.08 IU/ml)。另外,转vWF-ΔPro基因条件下,合并培养转重链和转轻链基因细胞的培养上清中,也检测到FVIII凝血活性(0.23±0.09 IU/ml),提示vWF-ΔPro有助于分泌的重、轻链形成功能性异源二聚体。表明转vWF-ΔPro基因可促进双链转FVIII基因,为进一步动物体内实验奠定了基础。

关键词： 前肽缺失型von; Willebrand因子; 凝血VIII因子; 双载体转基因

Abstract: Dual-vector based FVIII gene split delivery has been developped as an altenative strategy to overcome the packaging limitation of adeno-associated virus (AAV) vectors in hemophilia A gene therapy but the efficacy is undesired for the inefficient secretion of heavy chain. A propeptide-deleted mutant of von Willebrand factor(vWF-ΔPro), which functions as a carrier of FVIII was tested for its effect on dual-vector FVIII gene delivery. 48 hours post-transfection of HEK293 cell with vWF-ΔPro, heavy and light chain genes of a B-domain deleted FVIII (BDD-FVIII), a chain-specific ELISA showed high levels of heavy chain secretion of 142?29ng/ml in the culture supernatant, greater than that of cell without vWF-ΔPro co-transfection(87?15ng/ml). The heavy chain transfected cells showed a very low levels of heavy chain secretion in absence of vWF-ΔPro although improved in presence of vWF-ΔPro, but lower than that of heavy and light chain co-transfected cells indicating a pro-secretion effect of light chain on heavy chain in trans. The light chain showed higher efficient secretion in light chain gene transfection alone or co-transfection with heavy chain, which was not affected by vWF-ΔPro. The Coatest assay showed an obviously higher bioactivity (0.80?0.15IU/ml) in supernatant of vWF-ΔPro, heavy and light chain genes co-transfected cell, compared to vWF-ΔPro-free co-transfection cell (0.41?0.08IU/ml). The supernatant from combined cells separately transfected with heavy and light chain displayed FVIII bioactivity of 0.23?0.09IU/ml in presence of vWF-ΔPro, suggesting a function of vWF-ΔPro in assembly of secreted heavy and light chain into a functional hetero-dimer. The data demonstrated that vWF-ΔPro transfection could improve dual-vector mediated FVIII gene delivery forming a basis for ongoing in vivo study.

Key words: Propeptide-deleted von Willebrand factor (vWF-ΔPro) Factor VIII(FVIII) Dual-vector gene delivery

收稿日期: 2011-12-05 出版日期: 2012-07-25

ZTFLH:

基金资助: 山东省自然科学基金(ZR2010CM061);教育部留学回国人员科研启动基金(20071108)资助项目

通讯作者: 朱甫祥 E-mail: fuxiangmail@163.com

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	朱甫祥
	刘泽隆
	缪静
	屈慧鸽
	迟晓艳

引用本文:

朱甫祥, 刘泽隆, 缪静, 屈慧鸽, 迟晓艳. 前肽缺失体vWF改善FVIII基因断裂转移细胞的重链和活性分泌[J]. 中国生物工程杂志, 2012, 32(07): 20-25.

ZHU Fu-xiang, LIU Ze-long, MIAO Jing, QU Hui-ge, CHI Xiao-yan. Propeptide-deleted von Willebrand Factor Improves Heavy Chain Secretion and Bioactivity by Split FVIII Gene Co-transfected Cells. China Biotechnology, 2012, 32(07): 20-25.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I07/20

[1] Arruda V R, Stedman H H, Haurigot V, et al. Peripheral transvenular delivery of adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B. Blood, 2010, 115(23): 4678-4688.
[2] Niemeyer G P, Herzog R W, Mount J, et al. Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy. Blood, 2009, 113(4): 797-806.
[3] Mah C, Sarkar R, Zolotukhin I, et al. Dual vectors expressing murine factor VIII result in sustained correction of hemophilia A mice. Hum Gene Ther, 2003, 14(2): 143-152.
[4] Chen L X, Lu H, Wang J H, et al. Enhanced factor VIII heavy chain for gene therapy of hemophilia A. Mol Ther, 2009, 17(3): 417-424.
[5] Scallan C D, Liu T, Parker A E, et al. Phenotypic correction of a mouse model of hemophilia A using AAV2 vectors encoding the heavy and light chains of FVIII. Blood, 2003, 102(12): 3919-3926.
[6] Burton M, Nakai H, Colosi P, et al. Coexpression of factor VIII heavy and light chain adeno-associated viral vectors produces biologically active protein. Proc Natl Acad Sci USA, 1999, 96(22): 12725-12730.
[7] Zhang K, Shen X, Wu J, et al. Endoplasmic reticulum stress activates cleavage of CREBH to induce a systemic inflammatory response. Cell, 2006, 124(3): 587-599.
[8] De Meyer S F, Deckmyn H, Vanhoorelbeke K. von Willebrand factor to the rescue. Blood, 2009, 113(21): 5049-5057.
[9] Wise R J, Dorner A J, Krane M, et al. The role of von Willebrand factor multimers and propeptide cleavage in binding and stabilization of factor VIII. J Biol Chem, 1991, 266(32): 21948-21955.
[10] 朱甫祥,杨树德,刘泽隆,等. vWF-ΔPro改善基于蛋白质剪接的双载体BDD-FVIII基因转移. 中国生物化学与分子生物学报, 2010; 26(8): 720-726. Zhu F X, Yang S D, Liu Z L, et al. vWF-ΔPro improves protein splicing-based dual vector transfer of B-domain-deleted FVIII gene.Chin J Biochem Mol Biol, 2010, 26(8): 720-726.
[11] Zhu F X, Liu Z L, Chi X Y, et al. Protein trans-splicing based dual-vector delivery of the coagulation factor VIII gene. Sci China Life Sci, 2010, 53(6): 683-689.
[12] Lankhof H, van Hoeij M, Schiphorst M E, et al. A3 domain is essential for interaction of von Willebrand factor with collagen type III. Thromb Haemost, 1996, 75(6): 950-958.
[13] Chen L X, Zhu F X, Li J, et al. The enhancing effects of the light chain on heavy chain secretion in split delivery of factor VIII gene. Mol Ther, 2007, 15(10): 1856-1862.
[14] Sarkar R, Tetreault R, Gao G, et al. Total correction of hemophiliaAmice with canine FVIII using anAAV 8 serotype. Blood, 2004, 103(4): 1253-1260.
[15] Swaroop M, Moussalli M, Pipe S W, et al. Mutagenesis of a potential immunoglobulin-binding protein-binding site enhances secretion of coagulation factor VIII. J Biol Chem, 1997, 272(39): 24121-24124.
[16] Dorner A J, Wasley L C, Kaufman R J. Increased synthesis of secreted proteins induces expression of glucose-regulated proteins in butyrate-treated Chinese hamster ovary cells. J Biol Chem, 1989, 264(34): 20602-20607.
[17] Kaufman R J, Wasley L C, Dorner A J. Synthesis, processing, and secretion of recombinant human factor VI11 expressed in mammalian cell. J Biol Chem, 1988, 263(13): 6352-6362.
[18] Chen L X, Lu H, Wang J H, et al. Enhanced factor VIII heavy chain for gene therapy of hemophilia A. Mol Ther, 2009, 17(3): 417-424.

[1]	陈鑫洁,钱芷兰,刘启,赵清,张元兴,蔡孟浩. 毕赤酵母底盘芳香族氨基酸合成途径改造生产肉桂酸及对香豆酸^*[J]. 中国生物工程杂志, 2021, 41(10): 52-61.
[2]	王伟东,杜加茹,张运尚,樊剑鸣. CRISPR/Cas9在人病毒感染相关疾病治疗研究中的应用^*[J]. 中国生物工程杂志, 2020, 40(12): 18-24.
[3]	徐应永. 基因治疗产品的开发现状与挑战[J]. 中国生物工程杂志, 2020, 40(12): 95-103.
[4]	王玥,牟彦双,刘忠华. 基于CRISPR/Cas系统的单碱基编辑技术研究进展^*[J]. 中国生物工程杂志, 2020, 40(12): 58-66.
[5]	雷海英,赵青松,白凤麟,宋慧芳,王志军. 利用CRISPR/Cas9鉴定玉米发育相关基因ZmCen^*[J]. 中国生物工程杂志, 2020, 40(12): 49-57.
[6]	薛瑞,姚林,王瑞,罗正山,徐虹,李莎. 重组贻贝足蛋白的研究进展与应用^*[J]. 中国生物工程杂志, 2020, 40(11): 82-89.
[7]	陈庆宇,王鲜忠,张姣姣. 基因技术在治疗2型糖尿病中的应用^*[J]. 中国生物工程杂志, 2020, 40(11): 73-81.
[8]	何秀娟,胡凤枝,刘秋丽,刘玉萍,祝玲,郑文云. 乳腺癌细胞QSOX1的CRISPR/Cas9基因编辑及其对增殖侵袭的影响研究^*[J]. 中国生物工程杂志, 2020, 40(11): 1-9.
[9]	吕一凡,李更东,薛楠,吕国梁,时邵辉,王春生. **LbCpf1基因的原核表达、纯化与体外切割检测 ^***[J]. 中国生物工程杂志, 2020, 40(8): 41-48.
[10]	黄昭鸿,黄运红,黄艳梅,龙中儿,山珊. 分型检测致泻性大肠埃希氏菌PCR技术研究进展 ^*[J]. 中国生物工程杂志, 2020, 40(7): 82-90.
[11]	张保惠,熊华龙,张天英,袁权. 基于水疱性口炎病毒(VSV)的溶瘤病毒研究进展 ^*[J]. 中国生物工程杂志, 2020, 40(6): 53-62.
[12]	武瑞君,李治非,张鑫,濮润,敖翼,孙燕荣. 新冠病毒抗体药物研发进展及展望分析[J]. 中国生物工程杂志, 2020, 40(5): 1-6.
[13]	王天柱,吴庆,张宁,王冬杰,许洲,罗伟,杜宗君. 鱼类黑色素合成及信号通路的研究进展^*[J]. 中国生物工程杂志, 2020, 40(5): 84-93.
[14]	刘迪,张洪春. 慢性阻塞性肺疾病基因工程动物模型研究进展 ^*[J]. 中国生物工程杂志, 2020, 40(4): 59-68.
[15]	郭胜楠, 李信晓, 王峰, 刘昆梅, 丁娜, 扈启宽, 孙涛. 海马与新皮质组织特异性GABRG2基因敲除小鼠模型的构建及其在遗传性癫痫伴热性惊厥附加症中的初步研究 ^*[J]. 中国生物工程杂志, 2020, 40(3): 9-20.

Viewed

Full text

Abstract

Cited

Shared

Discussed