[Cloning, expression and activity analysis of full-length gene encoding thioredoxin peroxidase from Oncomelania hupensis].
OBJECTIVE
To clone and express full-length thioredoxin peroxidase (TPx) gene of Oncomelania hupensis and study on the peroxidase activity of the recombinant protein.


METHODS
Total RNA was obtained from the cultivated O. hupensis and a cDNA sequence of the TPx gene was cloned by RT-PCR. The TPx cDNA ends were amplified by the SMARTer RACE cDNA Amplification Kit. After sequencing, blasting and matching, the full-length cDNA of the TPx gene was obtained. The TPx cDNA was ligated with the pGEM-Teasy and transformed into E. coli DH5alpha. After sequencing and blasting, the characteristics of biological information of the TPx gene was analyzed. The positive recombinants with pGEM-Teasy/TPx and expression vector pET-28a were digested by the double restriction enzymes, ligated each other, transformed into E. coli BL21 (DE3), and induced by IPTG for expression. The recombinant TPx was expressed as a histidine fusion protein and was purified with Ni chromatography and NTA cation exchange chromatography. The expressed and purified TPx was analyzed by SDS-PAGE. The different concentrations of TPx recombinant protein (10, 20, 30, 40, and 50 microg/ml) were added into hydrogen peroxide (H2O2) reduction test in vitro to calculate the clearance rate of H2O2, each concentration with parallel control of dithiothreitol sugar alcohol (DTT). In the protection test of super-coiled DNA, the TPx protein was added with a concentration of 2.5, 5.0, and 10 microg/ml, respectively, to observe the protective effect of super-coiled DNA in metal-catalyzed oxidation (MCO).


RESULTS
The complete cDNA encoding TPx was 992 bp. ORF was 747 bp with GenBank accession number of JN831437. The ORF encoded 249 amino acids, and the relative molecular weight (M(r)) of predicted protein was 27 000. The recombinant plasmid pET28a/TPx was built, and the soluble recombinant protein was obtained by induction and purification. The results of SDS-PAGE showed that the M(r) was 27 000. H2O2 reduction test in vitro showed that the H2O2 clearance rate of reaction system containing DTT was significantly higher than the clearance rate without DTT (P < 0.05), the difference among various concentrations was not statistically significant (P > 0.05). The protection of super-coiled DNA showed that the protective effect of 5.0 microg/ml group was better than 2.5 microg/ml group, but there was no difference between the protective effect of 5.0 microg/ml group and 10 microg/ml group.


CONCLUSIONS
The full-length cDNA of the TPx gene of O. hupensis is obtained, and the recombinant TPx protein shows a certain antioxidant activity.
National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Key Laboratory of Parasite and Vector Biology, MOH, WHO Collaborating Centre of Malaria, Schistosomiasis and Filarasis, Shanghai 200025, China.
Xian-Liang
XL
Ma
Qin
Q
Liu
Yi
Y
Zhang
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary; D054464:Peroxiredoxins / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012908:Snails / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D011994:Recombinant Proteins; D054464:Peroxiredoxins