Study On The Screening And Protective Effect In Mice Of Single Chain Fragment Variable Against ? Hemolysin Secreted By Staphylococcus Aureus

Bovine mastitis is one of the diseases with high morbidity and great economic loss in the bovine breeding industry.Staphylococcus aureus is one of the main pathogens of bovine mastitis.?-hemolysin(hlb)is an exotoxin secreted by Staphylococcus aureus,which can assist Staphylococcus aureus to colonize bovine breast cells and cause breast cell injury.Single chain fragment variable(scfv)is one of the new genetic engineering monovalent small molecular antibodies which has been highly valued in recent years,and has broad research and application prospects.Some studies have shown that targeted blocking of secretory virulence factors can provide more effective protection for the body.It is of great scientific significance and application value on preventing bovine mastitis by screening scfv against?-hemolysin and studying its protective effect.In this paper,the screening and protective effects of scfv of?-hemolysin were studied in the following aspects:1.Expression,purification and hemolytic activity of recombinant?hemolysinThe?hemolysin gene was amplified by PCR using the DNA of Staphylococcus aureus ATCC25923 as template,and the prokaryotic expression plasmid p ET32a-hlb was constructed to induce the expression of recombinant hlb protein.After incubated with 2%cow red blood cells,the hemolytic activity of recombinant hlb protein was determined by measuring the OD₄₅₀ of erythrocyte suspension centrifugation.The results showed that recombinant hlb had the activity of dissolving cow red blood cells,and the hemolytic effect increased with the increase of?hemolysin concentration(P<0.05).2.Screening of scfv against?hemolysin and verification of its activity in vitro.The scfv against?hemolysin was screened by recombinant hlb protein from bovine single chain antibody library and obtained by prokaryotic expression.The scfv was incubated with recombinant?hemolysin and Staphylococcus aureus culture respectively and added into 2%cow red blood cells.The activity of scfv in inhibiting?hemolysin hemolysis in vitro was determined by measuring the OD₄₅₀ of erythrocyte suspension centrifugation.The results showed that scfv significantly inhibits the hemolytic activity of recombinant hlb protein(P<0.001).Scfv could inhibit the hemolysis of Staphylococcus aureus culture to a certain extent(P<0.05).3.Scfv has protective effect on mammary gland of mice infected with Staphylococcus aureus.Scfv can inhibit the colonization and amplification of Staphylococcus aureus in the mammary gland of mice,reduce the injury of mammary gland tissue and regulate the local immune response of mammary gland.The results of bacterial count in mammary gland tissue showed that the number of bacteria in mammary gland tissue of negative control group increased after 48 hours infected by Staphylococcus aureus,while the number of bacteria decreased significantly after penicillin and scfv prevention(P<0.05).The results of HE staining showed that the acinar structure of mammary gland was seriously destroyed and inflammatory cells infiltrated in the negative control group after Staphylococcus aureus infection.The injury of mammary gland tissue in penicillin and scfv prevention group was alleviated,the acinar structure was intact,and the number of inflammatory cell infiltrations in the acinar decreased.In the antibody biosafety group,the structure of mammary acinar was complete and a small amount of inflammatory cells infiltrated in acinar.Compared with the blank control group,the number of macrophages in mammary gland tissue of the negative control group increased significantly after Staphylococcus aureus infection(P<0.05).The number of macrophages in mammary gland tissue of penicillin or scfv prevention group was significantly lower than that of negative control group(P<0.05),and concentrated in the site of acinar destruction.The number of macrophages in antibody biosafety group increased significantly(P<0.05)Scfv regulated the expression of cytokines in mammary gland tissue.Compared with the negative control group,the content of IL-18 in mammary gland tissue of mice in penicillin or scfv prevention group decreased significantly(P<0.05),while the content of IL-8 in penicillin prevention group increased significantly(P<0.05),and the IL-8 content in scfv prevention group had a decreasing trend,but there was no significant difference.The contents of IL-8 and IL-18 in antibody biosafety group were significantly higher than those in control group(P<0.05).In conclusion,with targeting blocking the effect of?hemolysin,scfv against?hemolysin can inhibit the spread of Staphylococcus aureus in mammary gland,regulate macrophage infiltration and cytokine expression,and finally protect mammary gland from Staphylococcus aureus from infection to a certain extent.