| This research firstly expressed and purified the recombinant Jian carp Stefin.Secondly,the characterization of its inhibitive activity was conducted.Thirdly,the tissue distribution and expression of Jian carp Stefin at both mRNA and protein levels were investigated by the methods of quantitative real-time PCR and immunohistochemistry respectively.1.Prokaryotic expression and purification of Jian carp StefinExpressed the Jian carp Stefin protein by putting the expression strain of E.coli BL21?DE3?which was linked with the correctly restructured Stefin-pET-30a under the screened expression condition:IPTG concentration 1 mmol/L,inducing temperature 37?,inducing rotate speed 180 r/min,inducing time 6 h.After the recombinant strain was broken,the solution was resuspended by urea splitting solutions whose concentrations were 0.5 mol/L,1.0 mol/L and 5.0 mol/L respectively.The protein was dissolved in 5 mol/L urea splitting solution.Ni2+-NTA agarose affinity chromatography was applied to further purify the recombinant protein.There was a single peak at 135 min and the concentration of imidazole was 374.625 mmol/L.The ultimate acquisition of Stefin with high purity appeared as a single band on SDS-PAGE and the calculated molecular weight was about 11kDa.Recombinant Jian carp Stefin appeared as a single peak on the TSK-GEL G2000SWxl chromatogram and its purity was 97.13%.2.Biological activity characterization of recombinant Jian carp StefinInhibitive activity characterization was conducted.The inhibition type revealed that recombinant Jian carp Stefin could inhibit specifically the activity of Papain,Cathepsin B and Cathepsin L,while not inhibit the activity of Trypsin and Chymotrypsin.The activity inhibition profiles of recombinant Jian carp Stefin against Papain,Cathepsin B and Cathepsin L were detected by using fluorescent synthetic peptide as substrate.The result indicated that the activity inhibition profile of recombinant grass carp Stefin against Papain was reversible,and its Ki value was 7.75×10-11M?0.0775 nM?demonstrated the property of non-competitive inhibitor.Meanwhile,the inhibition effect of Stefin against Cathepsin L was stronger than that against Cathepsin B.Fairly good thermostability and pH stability were observed respectively:recombinant Jian carp Stefin was still stable 70%residual inhibitory activity during pre-set 4?80?,was stable 60%residual inhibitory activity during pre-set pH 3.013.0.3.Tissue distribution and expression level of recombinant Jian carp StefinThe relative gene expression quantification of Stefin in eight grass carp tissues was as follows:from high to low the order of expression amount of Stefin gene were hepatopancreas?14.8941±1.8598?,head kidney?13.7121±0.7358?,small intestine?7.7958±0.0345?,muscle?5.6371±0.3878?,kidney?5.3148±1.8827?,heart?4.3640±1.1050?,spleen?1.6640±0.1079?,gill?1.0350±0.0284?.The mRNA transcription level of Stefin in hepatopancreas and head kidney was extremely significantly higher than those in other tissues?P<0.01?.The mRNA transcription level of Stefin in spleen and gill was extremely significantly lower than those in other tissues?P<0.01?The mRNA transcription level of Stefin in muscle,kidney,and heart was close to each other,with no significant difference?0.01<P<0.05?.Polyclonal antibody was obtained by using the purified Jian carp Stefin to immunize mice.Detected by indirect ELISA assay,the titer of grass carp Stefin polyclonal antibody was higher than 1?128000,which indicated that this antiserum could be used to detect the express distribution of Stefin in grass carp tissues.Subsequently,the polyclonal antibody applied to analyze the tissue distribution and expression of Stefin at protein level by immunohistochemistry.The Jian Stefin presented a ubiquitous distribution in all the tested tissues,and a diffuse distribution was found specifically in the positive cells.IPP 6.0 was applied to analyze the optical density of obtain tissue section and quantify the expression level of Stefin.The result was as follows:from high to low the order of expression amount of Stefin protein were hepatopancreas?0.4279±0.0845?,gill?0.2364±0.0837?,heart?0.1662±0.0602?,head kidney?0.1331±0.0516?,kidney?0.1263±0.0431?,small intestine?0.1102±0.0446?,spleen?0.1058±0.0193?and muscle?0.1017±0.0329?.The protein expression level of Stefin in hepatopancreas was extremely significantly higher than other seven tissues?P<0.01?,gill took the second place and significantly higher than the rest tissues?0.01<P<0.05?,there was no significant difference among heart,small intestine,head kidney,kidney,spleen and muscle?P>0.05?. |
PDF Full Text Request