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猪VIII 因子A1和A3结构域对翻译后剪接的人/ 猪杂合VIII 因子的促分泌作用

朱甫祥^*, 刘泽隆, 屈慧鸽, 迟晓艳

鲁东大学生命科学学院，烟台 264025

摘要

凝血VIII 因子(fVIII)蛋白的低效分泌以及基因过大是基于转fVIII 基因的甲型血友病基因治疗的不利因素。本室前期用内含肽(intein)的蛋白质反式剪接实验证明，运用双载体共转fVIII 基因，通过翻译后蛋白质剪接，轻链可顺式促进fVIII蛋白的分泌。本文用猪fVIII 蛋白的A1 和A3 结构域替换人fVIII 蛋白相应的结构，构建人/ 猪杂合fVIII (human/porcine hybridfVIII, HP-fVIII)，研究基于内含肽的双载体转HP-fVIII 基因后剪接HP-fVIII 的分泌和活性。构建一对分别融合Ssp DnaB内含肽的HP-fVIII 重链和轻链基因表达质粒，瞬时共转染COS-7 细胞，用ELISA 和Coatest 法定量分析分泌至培养上清中HP-fVIII 的剪接和生物活性，用Western blotting 检测了细胞内HP-fVIII 的剪接。结果显示，双载体转HP-fVIII 基因细胞上清的剪接HP-fVIII 蛋白量为(184±34) ng/mL，活性为(1.18±0.22) IU/mL，明显高于双载体转人fVIII 基因[蛋白量为(48±12)ng/mL，活性为(0.31±0.10) IU/mL]，提示猪fVIII 的A1 和A3 结构域能显著促进内含肽剪接的HP-fVIII 的分泌和活性；另外，还在混合培养的分别转内含肽融合HP-fVIII 重链和轻链基因细胞上清中检测到剪接的HP-fVIII 蛋白量为(27±5) ng/mL，活性为(0.19±0.07) IU/mL，提示内含肽对HP-fVIII 的剪接可不依赖细胞机制，分泌出胞的前体蛋白仍可进行剪接反应；双载体转HP-fVIII 基因细胞内检测到与转hfVIII 基因阳性对照细胞内hfVIII 条带大小接近的剪接HP-fVIII 蛋白条带，进一步证实HP-fVIII 的剪接。该结果为进一步在动物体内应用内含肽的双腺相关病毒载体转HP-fVIII 基因奠定了实验基础。

关键词： 人/ 猪杂合VII I 因子; 分泌; 内含肽; 蛋白质反式剪接

分类号：Q5,Q78

[Enhancing effect of porcine coagulation factor VIII A1 and A3 domains on secretion of post-translationally spliced human/porcine hybrid coagulation factor VIII.] [Ariticle in Chinese]

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

Life Science College of Ludong University, Yantai 264025, China

Abstract

Low levels of coagulation factor VIII (fVIII) protein expression caused by its inefficient secretion and the over-sized fVIIIgene affect the transgene-based gene therapy for hemophilia A adversely. Our previous study demonstrated that intein-mediatedprotein trans-splicing for delivery of the fVIII gene with a dual-vector system could improve secretion of post-translationally splicedfVIII by light chain in cis. In this study, a human/porcine hybrid fVIII (HP-fVIII) containing replaced A1 and A3 domains of porcinefVIII was investigated for secretion and activity of the spliced HP-fVIII after intein-based dual-vector delivery of the HP-fVIII gene. Apair of expression plasmids comprising intein-fused HP-fVIII heavy and light chains were constructed and transiently co-transfectedinto COS-7 cells. The spliced HP-fVIII and bio-activity in culture media were quantitatively analyzed by ELISA and Coatest method respectively. The intracellular splicing of HP-fVIII was detected by Western blotting. The results showed that in the culture supernatantof cells co-transfected with HP-fVIII, the amount and activity of spliced HP-fVIII were significantly higher than those of splicedhfVIII secreted from the cells co-transfected with human fVIII [(184±34 ng/mL) vs (48±12) ng/mL, P<0.01; (1.18±0.22) IU/mL vs(0.31±0.10) IU/mL, P<0.01], demonstrating the dramatically enhancing effect of porcine A1 and A3 domains on the secretion of inteinsplicedHP-fVIII. The spliced HP-fVIII protein and its activity were also detected in the supernatant from combined cells separatelytransfected with intein-fused HP-fVIII heavy and light chain genes, indicating that the intein-mediated HP-fVIII splicing was independentof cellular mechanism and could occur outside the cell after the secretion of precursor proteins. Additionally, an intracellularlyspliced HP-fVIII band was found with a molecular weight similar to human fVIII protein, confirming the HP-fVIII splicing. Theseresults provided experimental basis for ongoing study using intein-based dual adeno-associated virus (AAV) vector to transfer HP-fVIIIgene in animal models.

Key words: human/porcine hybrid factor VIII; secretion; intein; protein trans-splicing

收稿日期：2010-04-12　　录用日期：2010-05-26

通讯作者：朱甫祥　　E-mail: fuxiangmail@163.com

引用本文：

朱甫祥, 刘泽隆, 屈慧鸽, 迟晓艳. 猪VIII 因子A1和A3结构域对翻译后剪接的人/ 猪杂合VIII 因子的促分泌作用[J]. 生理学报 2010; 62 (4): 373-381.

ZHU Fu-Xiang, LIU Ze-Long, QU Hui-Ge, CHI Xiao-Yan. [Enhancing effect of porcine coagulation factor VIII A1 and A3 domains on secretion of post-translationally spliced human/porcine hybrid coagulation factor VIII.] [Ariticle in Chinese] . Acta Physiol Sin 2010; 62 (4): 373-381 (in Chinese with English abstract).