Zhong Nan Da Xue Xue Bao Yi Xue Ban 2006 Aug;31(4):458-63
Department of Pathogenic Biology, Xiangya School of Medicine, Central South University, Changsha 410078, China.
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.
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