[Cloning and prokaryotic expression of transcriptional co-activator gene of Clonorchis sinensis and functional analysis of the expressed protein].
OBJECTIVE
To construct prokaryotic recombinant plasmids of transcriptional co-activator (TC) gene of Clonorchis sinensis, express and purify the recombinant protein and analyze its biological function.


METHODS
A pair of primers was designed according to the known sequence of TC gene. The TC gene fragment was amplified by PCR. After purification and digestion with BamH I and Sal I, the TC gene was connected to the prokaryotic expression vectors, pGEX-4T-1 and pET30a(+). By cloning target gene into these vectors, pGEX-4T-1 and pET30a(+), prokaryotic recombinant plasmids of TC gene were constructed and transferred into E. coli BL21. The positive expressed recombinants were detected by SDS-PAGE and Western blotting. Immobilized metal (Ni2+) chelation affinity chromatography was used to purify His-TC produced by the expression of the recombinant protein pET30a(+)-TC.


RESULTS
The recombinant plasmids, pGEX-4T-1-TC and pET30a(+)-TC, were constructed successfully. SDS-PAGE testified that the molecular weight of the recombinant protein was correct. Western blot analysis of GST-TC recombinant protein testified that the recombinant protein could be recognized by immunized rabbit serum, which means the protein is GST-immune active and the clone can express recombinant Clonorchis sinensis antigen. After affinity chromatography of the pET-TC protein, there was only one protein band with expected size on the SDS-PAGE gel.


CONCLUSIONS
The TC gene was screened from cDNA library of adult Clonorchis sinensis, cloned, expressed and purified. The purified protein of TC gene will be of importance for further research on the biological function of the gene.
Zhang
Yong-li
YL
Department of Parasitology, Sun Yat-Sen University of Medical Sciences, Guangzhou 510089, China.
Yu
Xin-bing
XB
Wu
De
D
Wu
Zhong-dao
ZD
Bi
Hui-xiang
HX
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D003004:Clonorchis sinensis / Q000235:genetics* / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D015801:Helminth Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology
D050976:Basic-Leucine Zipper Transcription Factors; D015801:Helminth Proteins; D011994:Recombinant Proteins