采用 Gateway 技术表达和纯化融合组氨酸标签的重组人凝血因子 VII。

Expression and purification of recombinant human coagulation factor VII fused to a histidine tag using Gateway technology.

机构信息

Research Centre, Iranian Blood Transfusion Organisation, Tehran, Iran.

出版信息

Blood Transfus. 2009 Oct;7(4):305-12. doi: 10.2450/2009.0081-08.

DOI:10.2450/2009.0081-08

PMID:20011642

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2782808/

Abstract

BACKGROUND

Factor VII (FVII) is a plasma glycoprotein that participates in the coagulation process leading to the generation of fibrin. The aim of this study was to construct, express and purify recombinant FVII fused to a polyhistidine (his) tag using Gateway technology.

METHODS

To construct the entry clone, blunt-end FVII cDNA and subsequent polymerase chain reaction (PCR) product isolated from a HepG2 cell line was TOPO-cloned into a pENTR TOPO vector. To construct the expression clone, a LR recombination reaction was carried out between the entry clone and destination vector, pDEST26. Chinese hamster ovary (CHO) cells were transfected with 1 microg of DNA of PDEST26-FVII using the FuGENE HD transfection reagent. Two cell lines that permanently expressed recombinant FVII were established. The expression of recombinant FVII was confirmed by reverse transcriptase PCR and enzyme-linked immunosorbent assay. Culture medium containing his-tagged FVII was added to the nickel-nitrilotriacetic acid resin column and bound protein was eluted. The purified protein was detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The biological activity of the recombinant FVII was determined by a prothrombin time assay using FVII-depleted plasma.

RESULTS

The results showed that human recombinant FVII was successfully cloned and the accuracy of the nucleotide sequence of the gene and its frame in the vector were confirmed by DNA sequencing. Stable clones transfected with the construct expressed FVII mRNA and related protein but no expression was detected in the CHO cells containing an empty vector. A protein of about 52 KDa was detected in SDS-PAGE and was further confirmed by western blot analysis. A three-fold decrease in clotting time was observed using this recombinant FVII.

CONCLUSION

As far as we are aware, this is the first report of expression of recombinant FVII fused with a his-tag through Gateway technology. The next steps, including large scale expression, purification, activation and stabilisation, are underway.

摘要

背景

VII 因子（FVII）是一种参与凝血过程导致纤维蛋白生成的血浆糖蛋白。本研究的目的是利用 Gateway 技术构建、表达和纯化与多组氨酸（his）标签融合的重组 FVII。

方法

为了构建入口克隆，钝端 FVII cDNA 及随后从 HepG2 细胞系中分离的聚合酶链反应（PCR）产物被 TOPO 克隆到 pENTR TOPO 载体中。为了构建表达克隆，入口克隆和目的载体 pDEST26 之间进行了 LR 重组反应。使用 FuGENE HD 转染试剂将 1μg 的 PDEST26-FVII DNA 转染到中国仓鼠卵巢（CHO）细胞中。建立了两条永久性表达重组 FVII 的细胞系。通过逆转录 PCR 和酶联免疫吸附试验证实了重组 FVII 的表达。含有 his 标记的 FVII 的培养基被添加到镍-亚氨二乙酸树脂柱中，结合的蛋白被洗脱。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和 Western blot 分析检测纯化的蛋白。通过使用 FVII 耗竭的血浆的凝血酶原时间测定法确定重组 FVII 的生物活性。

结果

结果表明，人重组 FVII 成功克隆，通过 DNA 测序证实了载体中基因及其框的核苷酸序列的准确性。构建体转染的稳定克隆表达 FVII mRNA 和相关蛋白，但在含有空载体的 CHO 细胞中未检测到表达。在 SDS-PAGE 中检测到约 52kDa 的蛋白，并通过 Western blot 分析进一步证实。使用这种重组 FVII 观察到凝血时间减少了三倍。