| In this study,Hyriopsis schlegelii,a freshwater pearl mussel,was used as the research object.Recombinant vector were constructed by the entire ORF of HsCypD gene and pGEX-4T1,and then transformed them into E.coli BL21(DE3)competent cells for prokaryotic expression.After the small induction,IPTG concentration,temperature and other induction conditions were determined and GST-HsCypD fusion protein with molecular weight of about 67 kDa was successfully obtained.The single GST-HsCypD fusion protein was obtained by purifying the supernatant using GST affinity chromatography resin,and its GST tag was excised by thrombin,then purified by GST affinity chromatography and left the relatively pure HsCypD protein with molecular weight about 41 kDa.The protein concentration and the content were determined by Bradford method.The HsCypD was used as an antigen to prepare its polyclonal antibodies against long-ear rabbits when it meets the antibody preparation concentration requirements.The titer of the antibody was obtained by the indirect ELISA.Result showed,the titer of the antibody was as high as 1:1024000.The results of western blot showed that the polyclonal antibody not only had specific binding to HsCypD which was used as an immune antigen,but also had effective specific binding to the tissue protein extracted from intestines,livers and gonads of H.schlegelii.It indicates that this study obtained the antibody which specifically binds to HsCypD,and provided the experimental basis for the subsequent researches on biological functions of HsCypD.By using the HsCypD antibody,the location of HsCypD in hepatopancreas and female gonad cells of H.schlegelii was investigated by the immunofluorescence localization.The results showed that the fluorescence intensity of HsCypD in the cytoplasm was evident in these tissue cells.In order to further determine the microsurgical localization of HsCypD in cells,the pEGFP-C1-HsCypD eukaryotic vector was successfully constructed and transient transfected into human hepatocellular carcinoma cells(SMMC-7721),and then observed by laser confocal microscopy after expression.It was found that HsCypD was predominantly present inthe cytoplasm.The protein localization was consistent with the previous conclusion of bioinformatics analysis.Hence,we speculate that the protein exercise related functions mainly in the cytoplasm.In order to investigate the effect of HsCypD on the growth of SMMC-7721 cells,the pcDNA3.1(+)-HsCypD and the pcDNA3.1(+)empty plasmids were transient transfected into SMMC-7721 cells.After transfection for 24 hours,the RNA of the untreated control group,the empty control group,and the experimental group were extracted and reverse transcribed into cDNA as templates.Then the effects of HsCypD,p53,Bax,Bcl2 and Caspase-3 gene expression level were detected by real-time fluorescence quantitative PCR.The results showed that,compared with the empty control group,the proapoptotic genes p53,Bax and Caspase-3 were down-regulated and the Bcl2 gene was up-regulated in the HsCypD overexpression group.After that,we use the flow cytometry to detect the cell apoptosis in three groups after 30 hours transfection.The results showed that the apoptosis rate of the experimental group was significantly lower than the empty control group,which indicated that overexpression of HsCypD could attenuate the apoptosis caused by the empty expression of pcDNA3.1(+)and has the certain function of inhibiting apoptosis.After pcDNA3.1(+)-HsCypD and pcDNA3.1(+)were transfected into SMMC-7721 cells for 24 hours,we added different concentrations of adriamycin for8 hours stimulation.Then we used the Caspase-3 spectrophotometry to detect the cells' degree of caspase-3 activation.As a result,the overexpression of HsCypD could inhibited the caspase-3 activation induced by adriamycin,and the higher concentration of adriamycin was able to lead to more pronounced inhibitory effect.Based on the results,Hs CypD has a biological function of inhibiting apoptosis. |
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