Cloning and expression of the succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum Chinese strain in E. coli.
- Author:
Jun-long YU
1
;
Shi-ping WANG
;
Zhuo HE
;
Gan DAI
;
Xiao-xin JIANG
;
Shao-hua ZENG
;
Xiao-qing XIAO
;
Song-hua ZHOU
;
Wen-kai LI
;
Shao-rui XU
;
Zhi-yue LÜ
;
Xian-chu PENG
Author Information
1. Department of Pathogenic Biology, Xiangya School of Medicine, Central South University, Changsha 410078, China.
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Animals;
Base Sequence;
Cloning, Molecular;
Escherichia coli;
metabolism;
Helminth Proteins;
biosynthesis;
genetics;
Iron-Sulfur Proteins;
biosynthesis;
genetics;
Molecular Sequence Data;
Recombinant Proteins;
biosynthesis;
genetics;
Schistosoma japonicum;
genetics;
metabolism;
Sequence Homology;
Succinate Dehydrogenase;
biosynthesis;
genetics
- From:
Journal of Central South University(Medical Sciences)
2006;31(4):458-463
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To clone the full-length gene encoding succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum (SjSDISP) Chinese strain and express it in Escherichia coli.
METHODS:According to the published incomplete EST (BU804141) of SjSDISP and the sequence of multiclone sites of lambda gt11 vector, 2 pairs of primers were designed and synthesized. Then the 3' and 5'ends of the EST of the SjSDISP from adult Schistosoma japonicum cDNA library were amplified by anchored PCR. After sequencing, a full-length cDNA sequence of the SjSDISP was obtained, and then it was cloned into prokaryotic expression vector pGEX-4T-1. Identified by agarosed gel electrophoresis, endonucleases digestion and PCR, the resultant recombinant plasmid was used for the expression under the temperature-dependent condition and Western blot analysis.
RESULTS:A 1,071 bp sequence was obtained. Sequence analysis showed that the fragment contained a complete open reading frame (ORF), encoding 278 amino acid residues. This target fragment was cloned into the prokaryotic expression vector pGEX-4T-1, and expressed in Escherichia coli. SDS-PAGE revealed that the molecular weight of the expressed fusion recombinant product was 56 kD. Western blot showed that the recombinant protein was recognized by polyclonal rabbit antiserum immunized with Schistosoma japonicum adult worm antigen.
CONCLUSION:Cloning of the full-length gene encoding SjSDISP and its bacterial expression were successfully done.
