[恶性疟原虫谷氨酸脱氢酶在大肠杆菌中的可溶性表达及其纯化与鉴定]

[Soluble expression of Plasmodium falciparum glutamate dehydrogenase in Escherichia coli, and its purification and identification].

作者信息

Li Yan, Ning Yun-shan, Dong Wen-qi, Li Ming

机构信息

Institute of Tropical Medicine, First Military Medical University, Guangzhou 510515, China.

出版信息

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2004 Apr 30;22(2):94-7.

PMID:15281452

Abstract

OBJECTIVE

To make soluble expression of Plasmodium falciparum (FCC1/HN) glutamate dehydrogenase (GDH) in Escherichia coli, purification and immunocompetence identification of the recombinant non-fusion GDH.

METHODS

The GDH gene was cloned into prokaryotic expression vector pET23 (a) to form recombinant expression vector pET23 (a)/GDH. pET23(a)/GDH was transformed into E. coli BL21 (DE3). Induced by IPTG (isopropyl-beta D-thiogalactoside), GDH was highly expressed in the supernatant after sonication. The soluble recombinant GDH was purified by Source-Q and Source-S chromatography. Enzyme-linked immunosorbent assay and Western blotting were carried out to identify the immunocompetence of the purified product.

RESULTS

SDS-PAGE analysis showed that the soluble GDH protein accounted for approximately 15% of the total bacterial protein. By two-step ion-exchange chromatography, the purity of GDH reached more than 90% and the GDH possessed high antigenicity.

CONCLUSION

The soluble expression of GDH results in an integral three-dimensional structure epitope with high biological activity.

摘要

目的

实现恶性疟原虫（FCC1/HN）谷氨酸脱氢酶（GDH）在大肠杆菌中的可溶性表达，并对重组非融合GDH进行纯化及免疫活性鉴定。

方法

将GDH基因克隆至原核表达载体pET23(a) ，构建重组表达载体pET23(a)/GDH。将pET23(a)/GDH转化至大肠杆菌BL21(DE3)。经异丙基-β-D-硫代半乳糖苷（IPTG）诱导，GDH在超声破碎后的上清液中高表达。采用Source-Q和Source-S层析法纯化可溶性重组GDH。通过酶联免疫吸附测定和蛋白质印迹法鉴定纯化产物的免疫活性。