To investigate the biological characteristics of Neospora caninum thioredoxin(Trx),the N.caninum Trx(NcTrx)gene was amplified by PCR,and the pET-32a(+)-NcTrx prokaryotic expression vector was constructed and characterized by double-enzyme digestion and sequencing.The NcTrx gene and its deduced amino acid sequences were analyzed using bioinformatics software,and the resulting protein networks were subjected to GO function and KEGG signaling pathway enrichment analysis.The pET-32a(+)-NcTrx was transformed into BL21(DE3)host bacterium.The recombinant NcTrx protein was purified by nickel affinity chromatography after induction with IPTG,and the expression and purification were examined by SDS-PAGE.The concentration of purified protein was determined by Bradford methord.The reactivity of rNcTrx protein was identified by western blot.The results showed that NcTrx gene fragment was 1287bp,encoding 428 amino acids.The protein included a signal peptide and a trans-membrane region,primarily located on the endoplasmic reticulum.Its secondary and tertiary structure were mainly composed of α-helices and random coil,containing 12 B-cell antigenic epitopes.The NcTrx protein belonged to the thioredoxin family and possessed the characteristic redox motif'-CXXC-'.It had 38 phosphorylation sites and 10 O-glycosylation sites.The NcTrx protein exhibited the highest interaction with TKL proteins through hydrogen bonding and electrostatic interactions.The significant enrichment analysis of GO function and KEGG signaling pathway showed that NcTrx and its interacting proteins were mainly involved in biological processes such as protein transport and protein disulfide bond isomerase activity,playing crucial roles in endoplasmic reticulum protein processing,sulfur metabolism,and other signaling pathways.SDS-PAGE results showed that rNcTrx was mainly expressed as inclusion body with a molecular weight of approximately 66ku,and was purified at a concentration of 0.8mg/mL.Western blot results demonstrated that the purified protein was able to react specifically with murine anti-His monoclonal antibody and N.caninum infected mice serum,and a specific band appeared at 66ku.In this study,the NcTrx gene was successfully cloned and the gene and amino acid sequence was analyzed by systemic bioinformatics for the first time,and the NcTrx protein was expressed and identified by prokaryotic system,which would provide reference basis for further study of the biological function of NcTrx.
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