Expression And Antibacterial Activity Of The Bacteriophage Lysin Of Clostridium Perfringens In Three Host Bacteria

Clostridium perfringens is a zoonotic pathogen that can cause human disease and a variety of poultry and livestock diseases.Necrotic enteritis(NE)in chickens is an intestinal infectious disease caused by C.perfringens.It can cause intestinal bleeding,necrosis,weight loss and death in chickens.It has caused substantial economic losses to the global poultry industry and has seriously hindered the healthy development of the poultry industry.With the widespread application of antibiotics,bacterial resistance and the drug resistance spectrum are rapidly increasing and expanding,and antibiotic control strategies are facing an unprecedented crisis.In this study,recombinant Lactococcus lactis NZ9000/PNZ8148-Cp51,Bacillus subtilis WB800N/PHT43-Cp51 and Escherichia coli BL21/p MAL-c5X-NVCp51 were constructed.The phage lysin Cp51 gene sequence was optimized for expression in different bacteria and synthesized;the Cp51 gene was then cloned into lactic acid bacteria plasmid PNZ8148 to form a recombinant plasmid and then electrotransformed into electrocompetent L.lactis NZ9000 to form recombinant expression strain NZ9000/PNZ8148-Cp51.Cp51 genes were also cloned into B.subtilis plasmid PHT43 and then electrotransformed into B.subtilis WB800N to form a recombinant expression strain WB800N/PHT43-Cp51,cloned into expression vector p MAL-c5X and transformed into E.coli BL21 to form recombinant expression strain BL21/p MAL-c5X-NVCp51.The results revealed that all three recombinant strains could express phage lysin and have antibacterial activity.The recombinant L.lactis were cultured in large quantities and induced by 1 ng/m L nisin.The expressed recombinant lysin was purified by affinity chromatography column,and a concentration of 124?g/m L lysin was obtained,which was determined by turbidimetry to prove that the purified lysin could decrease the C.perfringens OD_600nmvalue from 0.6 to 0.2,suggesting a strong killing effect on C.perfringens.The recombinant L.lactis and C.perfringens were cocultured in vitro.The number of C.perfringens could be decreased from10⁹ CFU in the control group to 10⁸ CFU by recombinant L.lactis,which proved that the recombinant L.lactis had a certain inhibitory effect on C.perfringens in vitro.The antimicrobial effect of recombinant L.lactis was evaluated using a coccidia and C.perfringens coinfection animal model.The recombinant L.lactis were orally administered for 5 consecutive days with a dose of 10⁹ CFU/day.Compared with the positive control group,the intestinal tract lesions of the recombinant L.lactis group decreased,and the number of C.perfringens and coccidia oocysts also decreased,indicating that recombinant L.lactis had an inhibitory effect on C.perfringens in vivo.The B.subtilis recombinant strain with the Cp51gene was successfully constructed and tested by Western blot.The lysin was successfully expressed,as confirmed by Western blot,but the expression was low,so the inhibitory effect on C.perfringens was weaker than that of recombinant L.lactis.The fusion protein of C.perfringens phage lysin Cp51 and norovirus capsid was expressed in E.coli using the p MAL-c5X vector with maltose tag,and expression of the fusion protein NVCp51 was induced.The cells were then disrupted by ultrasonic wave under optimal conditions,and the recombinant protein was purified by nickel-filled affinity chromatography column.Then,a large amount of soluble recombinant protein was obtained at a concentration of 504?g/m L.Recombinant lysin treated with artificial intestinal juice and artificial gastric juice retained 70%and 74%bactericidal activity,respectively,compared with the untreated recombinant lysine control,indicating that the stability of the recombinant lysin was strong.The calculated results showed that 1 g recombinant lysin protein was equal to 20 U lysin activity.The turbidimetric test results showed that the recombinant lysin,which was diluted to 1?g/m L,still had strong antibacterial activity.The antibacterial activity of the NVCp51 recombinant protein was evaluated by a Coccidia and C.perfringens coinfection animal model.The recombinant protein NVCp51 was orally administered at a high dose of1000 U and a low dose of 100 U.The data for relative weight gain,feed-meat ratio,lesion score and bacteria count were compared and analyzed.The results showed that the high dose of NVCp51 protein had an obvious antibacterial effect.Compared with the positive control group,there were significant differences in each index,similar to the metronidazole group.The low-dose group exhibited a certain antimicrobial effect,but it was not as strong as the effect in the high-dose group.In summary,three recombinant expression strains of C.perfringens bacteriophage lysin were successfully constructed.Proteins expressed in probiotics or isolated from recombinant bacteria had certain inhibitory effects on C.perfringens both in vivo and in vitro.