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[Amplification, cloning and expression of 3-phosphoglycerate kinase gene from Clonorchis sinensis]
1Department of Parasitology, Zhongshan Medical College, Sun Yat-sen University, Guangzhou 510089, China.
Objective:
To construct a prokaryotic expression plasmid encoding 3-phosphoglycerate kinase (PGK) gene of Clonorchis sinensis and analyze its expression in E. coli JM109.
Methods:
Total RNA was extracted with Trizol. The gene encoding PGK was amplified by RT-PCR from the total RNA, and was cloned into the pGEX-4T-1 vector. The recombinant plasmids pGEX-4T-1-PGK constructed were transferred into JM109, positive recombinants were screened and identified by tool enzyme digestion, PCR technique and sequencing. The recombinant was induced with IPTG to express the target protein. The expression product was characterized by SDS-PAGE.
Results:
There were three clear bands from extracted total RNA, the PGK gene (1248 bp) was amplified by RT-PCR technique, the sequencing result was consistent with the known sequence. Ten clones were obtained by screening after transferring, six of which were positive recombinants. The positive recombinant was induced to express, and the SDS-PAGE showed the expression products was about Mr 70000.
Conclusion:
The recombination prokaryotic expression vector pGEX-4T-1-PGK has been reconstructed and a fused protein was expressed which contained Sj26 GST.
