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内含肽介导F309SfVIII 的连接和重链分泌的增强

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

凝血因子VIII (coagulation factor VIII, fVIII)是一种分泌性血浆蛋白，在凝血级联反应中发挥重要作用，由fVIII 缺 陷导致的甲型血友病是一种最常见的X- 联锁隐性遗传性出血性疾病，基因治疗被认为是该病的理想治疗模式，但在运用最 有希望的腺辅助病毒(adeno-associated virus, AAV)载体转fVIII 基因时受到AAV 的容量限制。本文旨在利用内含肽(intein)的 蛋白质反式剪接，研究双载体真核细胞转具有促分泌作用的F309S 突变体全长fVIII (F309SfVIII)基因。内含肽是一种包埋 在蛋白质前体中的多肽序列，其在蛋白质成熟过程中通过蛋白质剪接作用被切除。将F309SfVIII 的cDNA 于B 结构域的 Ser1239 密码子前断裂为重链(heavy chain, HC)和轻链(light chain, LC)两部分，分别与Ssp DnaB intein 编码序列融合，插入到 pcDNA3.1，构建一对质粒表达载体。共转染或分别单独转染293 细胞，48 h 后用Western blot 观察细胞内转基因的表达 和F309SfVIII 的剪接，用ELISA检测培养上清中分泌的F309SfVIII 蛋白量以及HC 含量，用Coatest 法检测培养上清的fVIII 生物活性。结果显示，共转染细胞有明显的完整F309SfVIII 蛋白条带形成，细胞培养上清中的F309SfVIII 蛋白浓度为(71±9) ng/mL，所产生的fVIII 生物活性为(0.38±0.09) IU/mL，从单独转染后合并培养的细胞上清仍可检测到F309SfVIII 蛋白和fVIII 生物活性，分别为(25±6) ng/mL 和(0.12±0.05) IU/mL，说明上清中的F309SfVIII 蛋白由分泌前、后剪接两部分共同形成。 共转染细胞培养液上清中的HC 含量明显高于单独转染融合内含肽和HC 的细胞培养液上清[(135±10) ng/mL vs (37±7) ng/mL, P<0.01)]。以上结果提示内含肽可作为一种技术手段用于双载体系统真核细胞F309SfVIII 基因转移，并可改善F309SfVIII 的 分泌，为体内甲型血友病基因治疗研究中应用AAV 载体、克服其容量限制转F309SfVIII 基因提供了一种解决方案。

关键词： 凝血因子VI I I ; 分泌; 内含肽; 蛋白质反式剪接

分类号：Q5; Q78

[Intein-mediated F309SfVIII ligation with enhanced secretion of its heavy chain.] [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

Coagulation factor VIII (fVIII) is a secretion protein and plays a crucial role in the coagulation cascade. Hemophilia A resulted from deficiency of fVIII is the most common X-linked recessive bleeding disorder. Gene therapy is recognized as an attractive strategy for the eventual cure of this disease. However, the gene therapy is hampered by the big size of fVIII gene when using the most promising gene vectors, adeno-associated virus (AAV) vectors. In this study we explored the intein-mediated protein trans-splicing to deliver a Phe309→Ser mutant full-length fVIII (F309SfVIII) gene by using a dual-vector system. An intein is a protein sequence embedded within a precursor protein and can excise itself through protein splicing. The F309SfVIII is proven to be beneficial to its secretion. The F309SfVIII gene was broken into heavy and light chains before Ser1239 in B domain and fused with the coding sequences of Ssp DnaB intein respectively to construct a pair of plasmid vectors by inserting them into the pcDNA3.1 vectors. Forty-eight hours after co- or separate transfection of 293 cells, the co-transfected cell lysate showed an obvious ligated F309SfVIII protein band by Western blot with a polyclonal antibody against fVIII. The amounts of secreted F309SfVIII protein in culture supernatants and their bioactivities were (71±9) ng/mL and (0.38±0.09) IU/mL determined by ELISA and Coatest assay respectively. The supernatant from combined cells with separate transfections also displayed lower levels of F309SfVIII antigen and fVIII activity [(25±6) ng/mL and (0.12±0.05) IU/ mL], indicating the F309SfVIII could be formed by splicing both before and after secretion. The content of F309SfVIII heavy chain protein from co-transfected cell supernatant was higher than that of intein-fused heavy chain transfection alone [(135±10) ng/mL vs (37±7) ng/mL, P<0.01)]. These data demonstrated that intein could be used as a technical strategy in a dual-vector system delivering F309SfVIII gene with improved secretion of fVIII providing an alternative approach to circumvent the packaging limitation of AAV for F309SfVIII gene transfer, which encourages our continuing study in hemophilia A gene therapy in vivo.

Key words: coagulation factor VIII; secretion; intein; protein trans-splicing

收稿日期：2009-08-17　　录用日期：2009-10-20

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. 内含肽介导F309SfVIII 的连接和重链分泌的增强[J]. 生理学报 2009; 61 (6): 526-532.

ZHU Fu-Xiang, LIU Ze-Long, QU Hui-Ge, CHI Xiao-Yan. [Intein-mediated F309SfVIII ligation with enhanced secretion of its heavy chain.] [Ariticle in Chinese] . Acta Physiol Sin 2009; 61 (6): 526-532 (in Chinese with English abstract).