Cloning And Prokaryotic Expressing Of β-defensin Andβ-thymosin From Trachinotus Ovatus

The full-length cDNA sequences of β-defensin and thymosin from Trachinotus ovatus were studied using the homology cloning and RACE technology. The length of these two gene sequences and the speculated encoding amino acid sequences were analysed. The differences of expression quantities in the gill, skin, liver, spleen, head kidney, hind kidney, heart, gonad, intestines, stomach and brain of T.ovatus between β-defensin and thymosin genes were tested by Real-Time Quantitative PCR (RT-PCR). The expression profiles of β-defensin and thymosin from T.ovatus were also detected after infected with Vibrio vulnificus for0h,2h,4h,8h,12h,24h,48h and96h, respectively. The product of amplification was treated by double-endonuclease digestion (BamH I and Sal I) and inserted to pET-32a(+) expression vector. The recombinant plasmid was transformed into BL2-1, and subsequently induced by IPTG. The main results of this study were as follows:1. Cloning and prokaryotic expressing of β-defensin from T.ovatus.The full-length cDNA sequences of defensin from the T.ovatus were reported for the first time in this paper. The cDNA was592bp in total length, within which a192bp open reading frame(ORF) was identified. The ORF was found to encode a putative peptide comprising63amino acids(aa.), containing a20-aa signal peptide and3pairs of disulifide bonds. The putative molecule mass was7.217kD and the theoretical isoelectric point was8.88. Amino acid sequences analysis showed that the sequences contains6cysteine (C),8glycine (G) and9leucine (L), and there was an obvious transmembrane region. BLAST in GenBank database revealed that the polypeptide encoded by this cDNA was highly similar to certain fish defensins. It shared the highest similarily to β-defensin from Siniperca chuatsi was96.8%, and the similarly to β-defensin from Epinephelus coioides was92.1%, so it was predicted to be a member of β-defensins.Results of RT-PCR indicated that the β-defensin gene was expressed in various tissues, with the highest expression levels in the intestine, pronephros, metanephros and other immune-related organs. After infected with V.vulnificus, the β-defensin expressed highest quantity in head kidney after8h-12h, in intestinal after24h, in in spleen after12h. The expression level of control group did not have significantly change after injected with the same amount of sterile saline. It showed that the changes of β-defensin gene expression were caused by V.vulnificus infection.SDS-PAGE showed that pET-32a(+)/β-defensin prokaryotic expression vector was successfully built. The molecular weight of the expression product was consistent with the predicted value. Recombinant fusion protein was efficiently expressed in the supernatant mainly in soluble form. With optimum expression conditions of0.1m mol/L IPTG,16℃,16h, high purity of fusion protein was achieved using His Ni-NTA column purification method.The results of this study would be helpful for better understanding the P-defensin protein activity and non-specific immune mechanisms. It could also play an important role in the development and utilization of marine fish antimicrobial peptides, new fishery medicines and imunoenhaners from or for marine fish.2. Cloning and prokaryotic expression of β-thymosin from T.ovatus.The full-length cDNA sequences of β-thymosin from T.ovatus was also reported for the first time in this paper. The full-length cDNA is601bp, consisting of a72bp5’UTR, a394bp3’UTR which contains the polyadenylation signal (AATAA) and the ploy A tail, and a135bp ORF. The gene encodes44aa. Its presumed molecular weighted was5.02kD and its theoretical isoelectric point is4.93. The predicted protein structures contained the actin binding domain of the β-thymosin. BLAST in GenBank database reveals that the polypeptide encoded by this cDNA was highly similar to certain fish thymosins, the similarly to Tβ12from Oreochromis niloticus and Oryzias latipes were83.7%and76.7%, so it was predicted to be a member of TP12.Results of RT-PCR showed that the β-thymosin gene expressed in various tissues, with the highest expression levels in the spleen and head kidney, followed by stomach, intestine, heart, and with relative low expression in skin, gill, liver, gonad and brain. The β-thymosin expressed the highest quantity in spleen after4h’V.vulnificus injection, in inintestine after24h, in head kidney after12h, respectively. The control group did not change significantly when injected with the same amount of sterile saline. It showed that the changes of β-thymosin gene expression were caused by V.vulnificus infection.SDS-PAGE showed that pET-32a(+)/β-thymosin prokaryotic expression vector was successfully built. The molecular weight of the expression product was consistent with the predicted value. Recombinant fusion protein was efficiently expressed in the supernatant mainly in soluble form. With the optimum expression conditions of0.5m mol/L IPTG,16℃,12h, high purity of fusion protein was obtained using His Ni-NTA column purification method.The results of this study established a good foundation for in-depth study of β-thymosin protein activity and non-specific immune mechanisms for T.ovatus.