Screening Of Differentially Expressed Proteins Of Plasma From Footrot-affected Dairy Cow And Ex-pression And Antibody Preparation Of Inflamma-tion Biomarker BoHp

The plasma proteome of healthy dairy cattle and those with footrot was investigated using ashotgun LC-MS/MS approach. In total,648proteins were identified in healthy plasma samples,of which234were non-redundant proteins and123were high-confidence proteins;712proteinswere identified from footrot plasma samples, of which272were non-redundant proteins and138were high-confidence proteins. The high-confidence proteins showed significant differencesbetween healthy and footrot plasma samples in molecular weight, isoelectric points and the GeneOntology categories.22proteins were found that may differentiate between the two sets ofplasma proteins, of which16potential differential expression (PDE) proteins from footrotplasma involved in immunoglobulins, innate immune recognition molecules, acute phaseproteins, regulatory proteins, and cell adhesion and cytoskeletal proteins;6PDE proteins fromhealthy plasma involved in regulatory proteins, cytoskeletal proteins and coagulation factors.The IgG concentration in footrot plasma sample was significantly higher than those of healthyplasma sample. Of these PDE proteins, haptoglobin, SERPINA10protein, afamin precursor,haptoglobin precursor, apolipoprotein D, predicted peptidoglycan recognition protein L (PGRP-L)and keratan sulfate proteoglycan (KS-PG) were suggested to be potential footrot-associatedfactors. Bovine haptoglobin (BoHp) has been shown to be a useful biomarker to monitor theoccurrence and severity of inflammatory responses in cattle with foot rot and other inflammatorydiseases. In our study, the nucleotide sequence of the predicted immunodominant region ofbovine haptoglobin (pirBoHp), removing the signal peptide sequence, was synthesized based onthe codon usage bias of Escherichia coli. The synthesized pirBoHp gene was cloned into theprokaryotic expression vector pET-32a (+) containing his-tag. The recombinant pirBoHp proteinwas successfully expressed in E. coli BL21(DE3) cells. Western blot analysis showed that thepurified recombinant pirBoHp protein could be recognized by an anti-His-tag monoclonalantibody. Further investigations indicated that a polyclonal antibody against the recombinantpirBoHp protein could recognize α and β chains of native bovine haptoglobin in a pooled plasmasample from dairy cattle suffering from footrot. Our study may provide valuable information toincrease understanding of plasma protein profiles in cattle and to assist studies of footrot-associated factors.