Bioinformatics Analysis and Prokaryotic Expression of SipD Protein of Salmonella Enteritidis in Chickens
[Objective]This study was aimed to predict the potential biological functions of Salmonella Enteritidis(SE)SipD protein,and express and purify SipD protein,so as to provide a theoretical basis for exploring SipD protein as a candidate antigen for anti-Salmonella nanobodies.[Method]DNAStar software was used to perform similarity alignment on the amino acid sequences of SipD protein among different serotypes of Salmonella strains published in GenBank.Subsequently,the bioinformatics analysis of SipD protein was performed using online software.Primers were designed based on SipD gene sequence in GenBank,using the standard strain of Salmonella Enteritidis(ATCC 13076)as a template.PCR was applied to amplify SipD gene and construct a pET-32a(+)-SipD recombinant plasmid,which was then transformed into Escherichia coli BL21(DE3)competent cells.IPTG was applied to induce the expression of recombinant SipD protein and Ni2+affinity chromatography was used to purify.SDS-PAGE and Western blotting was applied to detect the expression of SipD protein.[Result]SipD protein was highly conserved among different serotypes of Salmonella strains.SipD protein,with the molecular formula C1619H2573N441O540S8,was an extracellular protein without transmembrane domain and signal peptide.The amino acids 1 to 343 of SipD protein had conserved structural domains from the superfamily of invasive plasmid antigen IpaD.The secondary structure prediction of SipD protein showed that alpha helix,extended chain,beta turn and random coil accounted for 59.18％,6.41％,3.21％and 31.20％,respectively.There were 12 antigenic epitopes that bound SipD protein to B cells.The size of SipD gene was 1 029 bp,which was expressed as a soluble protein in Escherichia coli BL21(DE3)competent cells.After purification,dialysis and concentration,the protein concentration of SipD protein was 8.6 mg/mL.SDS-PAGE and Western blotting analysis revealed that SipD protein with high purity could be used to immunize alpacas for the preparation of nanobody.[Conclusion]SipD protein was an extramembrane protein with many antigen binding sites.The recombinant protein of SipD with high purity was obtained by expression and purification,which provided materials for further preparation of nanobody targeting SipD protein of Salmonella Enteritidis in chickens.
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