Molecular Cloning And Recombinant Expression Of Peroxiredoxin1and Peroxiredoxin4from Sepiella Maindroni

Sepiella maindroni is one of the most commercially important native species for fisheries ofeast china sea, that has important economic value and food value; but with the deterioration ofenvironment and intensive culture development, kinds of diseases were occurred in Sepiellamaindroni populations when exposed to pathogens and kinds of pressure stress. The peroxiredoxinis a superfamily of antioxidative proteins that maintain the balance of intracellular redox, piayimpotant roles in immune system. In the present study, we cloned peroxiredoxin1(SmPrx1) andperoxiredoxin4(SmPrx4) from Sepiella maindroni by the technology of RACE; and themRNAression in different tissues and temporal expression in hepatopancreas after challenged withhydrogen peroxide for SmPrx1and SmPrx4were evaluated by Quantitative PCR Tecnique. At thesame time, SmPrx1was recombined in vitro and the recombined protein was expressed inprokaryctic expression system.The full-length cDNA of SmPrx1was of1062bp, contains a5’ untranslated region (UTR) of79bp, a3’ UTR of359bp, an open reading frame of624bp encoding207amino acids; and thepredicted molecμLar mass and estimated isoelectric point of SmPrx1were24.4kDa and6.63. Theconserved peroxidase catalytic center“FYPLDFTFVCPTEI”and “GEVCPA” were observed in thesequence of SmPrx1, this indicated that it was a member of2-Cys Prx. It is showed thay the aminoacids SmPrx1have highest similarity with Prx10f C.plicata（Identities81%）、Apis florae and（Identities81%）and C.clemensi（Identities78%）by blast and phylogenetic analysis.Expression of SmPrx1in different tissues was determined by RT-PCR and showed the SmPrx1expression in various tissues that we examined; and the expression level in tesits was highest. Theexpression levels of SmPrx1in hepatopancreas increased rapidly after hydrogen peroxidechallenge and reached the hightest at0.25h. As time progressed, the expression level began toreduced and resumed to normal levels at4h. There was significant difference between these twogroups at0.25h、0.5h and1h. SmPrx1was recombined into the vector of pET-21a-d(＋) and the recombined protein was expressed in E.coli BL21(DE3). The antioxidant activity and peroxidaseactivity of SmPrx1were6.17U/mgprot and10.17U/mgprot. The results showed that therecombined protein of SmPrx1has antioxidant activity and was the importance part of antioxidantsystem in Sepiella maindroni.The full-length cDNA of SmPrx4was of991bp, contains a5’UTR of45bp,3’UTR of205bpand an open reading frame of741bp encoding246amino acids. The SmPrx4of predictedmolecular mass and estimated isoelectric point were27.3kDa and6.99. The double of peroxidasecatalytic center of “FYPLDFTFVCPTEI”�'�“GEVCPA”were found in SmPrx4and showedthat SmPrx4was belonged to the subgroup of2-Cys Prx. Through blast and phylogenetic analysis,we know that the amino acids sequence of SmPrx4shares76%、78%and86%identy with thatPrx4of B.glabrata、C.gigas and A.fangsiao. Expression of SmPrx4in different tissues ofSepiella maindroni was determined by RT-PCR; the result showed that we could detected theexpression of SmPrx4in all the examined tissues and the expression in Pancreas was significantlyhigher than other organizations. After challenge by hydrogen peroxide, the expression levels ofSmPrx4in hepatopancreas increased gradually and reached the hightest at0.5h; and then droppeddown gradually, but did not recover to the original level. At0.5h and4h after challenge byhydrogen peroxide, the expression levels of SmPrx4was significant difference betweenexperimental group and control group. All these showed that SmPrx4was an important antioxidantand involved in the responses to challenged with hydrogen peroxide.