[Preparation of recombinant Mycobacterium tuberculosis ESAT6-PPE68 fusion protein].
OBJECTIVE
To construct esat6-ppe68 fusion gene and its prokaryotic expression vector for expression in E. coli.


METHODS
With GeneSOEing method, a fusion gene was constructed by splicing esat6 gene and ppe68 gene and cloned into pGEX-4T-1 plasmid to construct the recombinant prokaryotic expression plasmid pGesat6-ppe68. After identification with restriction enzyme analysis, PCR and nucleotide sequencing analysis of the plasmid, E. coli BL21 was transformed with the recombinant plasmid and induced with IPTG to obtain the expression of the fusion protein ESAT6-PPE68 with GST-tag (about 69 kD), which were purified with GST-fusion protein purification kit. The expression of esat6-ppe68 fusion gene was subsequently detected by SDS-PAGE and Western blot analysis.


RESULTS
The sequence of esat6 and ppe68 in the recombinant plasmid was consistent with that in GenBank report. The fusion protein was detected in the cytoplasm in soluble form and represented approximately 40% of the total bacterial protein of E. coli. After purification, the purity of the fusion protein reached 90%, and its antigenicity was confirmed by Western blotting.


CONCLUSIONS
The prokaryotic expression vector pGesat6-ppe68 has been constructed and the fusion protein ESAT6-PPE68 obtained successfully, which provides an experimental basis for potential application of the recombinant ESAT6-PPE68 in the diagnosis of tuberculosis.
Li
Hong-Xia
HX
Department of Parasitology and Lab of Pathogenic Biology, West China Medical Center, Sichuan University, Chengdu 610041, China. lihongxia1118@163.com
Chen
Jian-Ping
JP
Zeng
Lin-Zi
LZ
Liu
Gang
G
Yao
Wei
W
Yang
Jun
J
Liu
Yang-Yi
YY
Wang
Tao
T
Tian
Yu
Y
D000942:Antigens, Bacterial / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; C528814:PPE68 protein, Mycobacterium tuberculosis; D011993:Recombinant Fusion Proteins