Cloning,Expression,Enzyme Activity and Bioinformatics Analysis of Caspase 3 Gene in Chicken
[Objective]The purpose of this study was to construct expression vectors for chicken Caspase 3(chCaspase 3)gene,identify the protease activity of the expressed protein,and conduct bioinformatics analysis,so as to lay a foundation for further research into the function of chCaspase 3.[Method]The cDNA from chicken bursa of Fabricius tissue was used as template for amplification of the chCaspase 3 gene via PCR.The recombinants of both prokaryotic and eukaryotic expression vectors for chCaspase 3 gene were then constructed.The recombinant prokaryotic expression vector was transformed into Escherichia coli BL21(DE3).Following IPTG induction,prokaryotic expression products of chCaspase 3 were subjected to Western blotting analysis.DF-1 cells were transfected with the eukaryotic expression vector in varying amounts(0,0.5,1.0,1.5 and 2.0 pg).The expression products were then analyzed using Western blotting and indirect immunofluorescence assay(IFA).Enzyme activity of the expressed proteins,both prokaryotic and eukaryotic,was assessed using Caspase 3 activity assay kits.Bioinformatic software was employed to assess the similarity of the chCaspase 3 and to forecast its protein's structural domains and tertiary structure.[Result]The CDS region length of the chCaspase 3 gene was 852 bp,encoding 283 amino acids.Both prokaryotic pET28a(+)-chCaspase 3 and eukaryotic p3 X Flag-CMV7.1-chCaspase 3 recombinant plasmids were constructed successfully.The prokaryotic expression product had a molecular weight of 36 ku,matching expectations.The expression of eukaryotic expression products were increased with the increase of transfected eukaryotic expression vectors.Caspase 3 enzyme activity was observed in both prokaryotic and eukaryotic expression products.Bioinformatics analysis showed the chCaspase 3 had low similarity with other species and distant phylogenetic relationships.The chCaspase 3 protein contained the conserved QACRG pentapeptide and CASc domain,characteristic of the Caspase family,mirroring the three-dimensional structure of human Caspase 3(hCaspase 3).[Conclusion]The chCaspase 3 gene was cloned and expressed successfully,and its recombinant protein demonstrated Caspase 3 enzyme activity.Bioinformatics predictions revealed the three-dimensional structure of the chCaspase 3 protein closely resembled that of hCaspase 3.This similarity provided a basis for uncovering the function of chCaspase 3 and investigating its antiviral innate immunity.
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