增强子/启动子组合可增强非病毒表达载体中凝血因子 VIII 的表达。

An enhancer/promoter combination strengthens the expression of blood-coagulation factor VIII in non-viral expression vectors.

作者信息

Picanço-Castro V, Russo-Carbolante E M S, Fontes A M, Fernandes A C, Covas D T

机构信息

Hemocentro de Ribeirão Preto, Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, SP, Brasil.

出版信息

Genet Mol Res. 2008 Apr 8;7(2):314-25. doi: 10.4238/vol7-2gmr447.

DOI:10.4238/vol7-2gmr447

PMID:18551397

Abstract

We explored the potential of fusion of hepatic locus control region 1 (HCR-1) with HCR-2 to express B-domain-deleted human factor VIII (FVIII) in four cell lines. B-domain-deleted human FVIII expression was controlled by HCR-1/HCR-2, followed by liver specific and ubiquitous promoters. Chimera enhancer HCR-1/HCR-2, followed by cytomegalovirus (CMV) promoter, gave 2-fold more FVIII expression in all cell lines (105.6 +/- 2.8 for Hek-293, 68.8 +/- 3.8 for HepG2, 34.8 +/- 1.3 for CHO, and 27.2 +/- 1.6 ng x mL(-1) x 10(6) cells(-1) for L.N.) when compared to the vector with CMV alone (54.8 +/- 3.3 for Hek-293, 32.4 +/- 1.2 for HepG2, 18.6 +/- 1.1 for CHO, and 10.1 +/- 1.7 ng x mL(-1) x 10(6) cells(-1) for L.N.). Elongation factor 1-alpha gene and human CMV promoters were more efficient than the promoters from the human alpha-1-antitrypsin gene, and fviii was less efficient in hepatic cell lines. HCR-1/HCR-2, followed by strong promoters, increases FVIII expression in vitro. Our results underscore the importance of cis sequences for enhancing in vitro FVIII expression; this may be helpful for designing new strategies to improve heterologous expression systems.

摘要

我们探究了肝位点控制区1（HCR-1）与HCR-2融合在四种细胞系中表达B结构域缺失的人凝血因子VIII（FVIII）的潜力。B结构域缺失的人FVIII表达由HCR-1/HCR-2控制，随后是肝脏特异性和遍在性启动子。嵌合增强子HCR-1/HCR-2，随后是巨细胞病毒（CMV）启动子，与单独含有CMV的载体相比，在所有细胞系中使FVIII表达增加了2倍（Hek-293细胞系中为$105.6\pm2.8$，HepG2细胞系中为$68.8\pm3.8$，CHO细胞系中为$34.8\pm1.3$，L.N.细胞系中为$27.2\pm1.6 ng\times mL^{-1}\times10^{6} cells^{-1}$）（单独含CMV的载体在Hek-293细胞系中为$54.8\pm3.3$，HepG2细胞系中为$32.4\pm1.2$，CHO细胞系中为$18.6\pm1.1$，L.N.细胞系中为$10.1\pm1.7 ng\times mL^{-1}\times10^{6} cells^{-1}$）。延伸因子1-α基因和人CMV启动子比人α1-抗胰蛋白酶基因的启动子更有效，并且FVIII在肝细胞系中的效率较低。HCR-1/HCR-2，随后是强启动子，可增加体外FVIII表达。我们的结果强调了顺式序列对增强体外FVIII表达的重要性；这可能有助于设计新策略来改进异源表达系统。