Inducible Expression And Detection Of Caspase-1 In DF-1 Cells And Chick Liver Tissue

Caspase-1 plays a key role in mediating inflammation response and cell pyroptosis.The molecular regulation mechanism of chicken caspase1 in innate immunity was not clear.In our study,we investigated varied expression of caspase-1 in DF-1 cell or liver tissues of chicken following gene cloning,expression and antibody preparation.Our results could provide a better insight into the regulation mechanism of caspase-1 in innate immune response of chick.Firstly,we amplified the coding sequence of caspase-1 gene with a pair of primers designed according to the caspase-1sequence(AF031351.1)from GenBank.The product was cloned into pMD18-T vector,and then subcloned into the vector pET-32a(+).The recombinant plasmid of caspase1/pET-32 a was transformed into the E.coli strain Rosetta and induced expression by IPTG.Then,we prepared anti-casepase1 polyclonal antibody by immunizing a New Zealand rabbit with purified caspase1-His protein.The specificity of the anti-caspase1 antibody was detected by ELISA and western blot methods.The results showed that the caspase-1 coding sequence of chicken was 852 bp.And we successfully constructed caspase1/pET-32 a recombinant plasmid.The recombinant plasmid was expressed mainly in the form of inclusion bodies in E.coli Rosetta.The fusion protein caspase1-His has good antigenicity by detecting with ELISA and western blot.Secondly,the caspase-1 CDS was subcloned into the eukaryotic expression vector pEGFP-N1.The recombinant plasmid caspase1/pEGFP was transfected into DF-1 cell by LipofectamineTM2000 reagent.After transfected 24 h,the location situation of fusion protein was imaged with the fluorescence microscope.Then,we also detected expression of caspase-1 fusion protein in cells by western blot.The results showed that caspase1/pEGFP recombinant plasmid was transfected into and over expression in DF-1 cells.The expression of caspase-1-GFP protein was showed in foci compared with the control GFP protein in cells.Procaspase-1 and active caspase-1 were detected in lysis supernatant of cells treated with LPS-ATP by western blot assay.Finally,SPF postnatal day one chicks were randomLy divided into two groups.Each experiment was performed with nine chicks.Chicks in one group were subcutaneously infected via the neck with the ALV-J strain AJV-HF211.The control chicks were subcutaneously injected with DMEM cell-culture medium.Total RNA was extracted from liver tissues at 1,7 and 17 dpi,respectively and then reverse transcribed into cDNA first-strain.Then,the level of caspase-1mRNA was detected by SYBR Green I relative real-time fluorescence quantitative(RT-q PCR).Prepared paraffin section with the remaining liver tissues and detected expression of caspase-1 protein by immunohistochemistry.The results of RT-qPCR showed that the mRNA levels of of caspase-1 hadn’t significantly variation at 1dpi,then significantly increased at 7 dpi(p<0.01),and decreased at 17dpi(p>0.05).The protein expression level of caspase-1 had an increasing tendency in virus infected livers by immunohistochemistry,compared with the control livers.And the higher expression level was detected at 17 dpi.These indicated that caspase-1 involved in innate immune response at the early stage of ALV-J infection.In summary,we successfully prepared caspase-1 polyclonal antibody following cloning and expressing chicken caspase-1.The mRNA or protein expression of chicken caspase-1 could be effected in DF-1 cells treated with LPS-ATP or in chicken liver infected with the virus ALV-J.Our results could provide a better insight into the regulation mechanism of caspase-1 in innate immune response of chick.