[Amplification, cloning and expression of 3-phosphoglycerate kinase gene from Clonorchis sinensis].
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.


CONCLUSIONS
The recombination prokaryotic expression vector pGEX-4T-1-PGK has been reconstructed and a fused protein was expressed which contained Sj26 GST.
Wu
De
D
Department of Parasitology, Zhongshan Medical College, Sun Yat-sen University, Guangzhou 510089, China.
Yu
Xin-bing
XB
Xu
Jin
J
Wu
Zhong-dao
ZD
Chen
Shou-yi
SY
He
Dong-gou
DG
D000818:Animals; D003001:Cloning, Molecular; D003004:Clonorchis sinensis / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D010735:Phosphoglycerate Kinase / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D011993:Recombinant Fusion Proteins; D010735:Phosphoglycerate Kinase