Expression And Antibacterial Activity Of The Bacteriophage Lysin Of Clostridium Perfringens

Clostridium perfringens is a major cause of histotoxic and intestinal infections in humansand other animals.Necrotic enteritis?NE?resulting from infection by C.perfringens typeA or type C,can cause intestinal mucosal bleeding and necrosis.Recently,with a stricter controlfor antibiotics and growth promoting agents all over the world,the harm of C.perfringens disease has become increasingly serious,not only causing huge economic losses in animal husbandry but also endangering public security.Samples from clinical outbreaks of NE were collected for isolation of C.perfringens by TSC double agar method,and were typed by multiplex-PCR published in the literatures.Seven strains of C.perfringenstype Awere obtained after separation and identifica tion.The drug sensitivity of the 7 strains of C.perfringens were tested using 12 kinds of drugs that were usually used in farmsaccording to the K irby-Bauer disk diffusion method.The results revealed that C.perfringens was resistant to three or more kinds of drug.None of the strains were sensitive to gentamicin,tetracycline andcompound sulfamethoxazole buthad different resistance to other drugs.Experiments showed that the multi-drug resistance of C.perfringens is serious and urgent,and new treatments are necessary to halt the harm brought about by C.perfringens.Three phage lysin genes were synthesized and inserted into plasmid p ET32a?+?.Then,the recombinant expression vectors p ET32a-Cp51,pET32a-Cp78,and p ET32a-Cpzp were transformed into E.coli BL21?DE3?.After induction with IPTG,the soluble recombinant proteins Cp51,Cp78,and Cpzp were successfully expressed and subsequently purified with Ni²⁺-NTA affinity chromatography.The bactericidal activity of recombinant proteins Cp51,Cp78,and Cpzp for C.perfringenstype Awere detected with a kinetic turbidimetric assay.The results showed that the lytic activity of the three kinds of bacteriophage lytic enzymes was Cp51>Cp78>Cpzp,and the phage lyase had no bactericidal effect on other kinds of bacteria.The results of this study demonstrated that the recombinant protein Cp51 has strong bactericidal activity and specificity against C.perfringens type A,which should have implications for the development of a lysin product for effective control of C.perfringens infection.The animal model of NE using Eimeria spp.and C.perfringenscoinfection was established in this study.Fourteen-day-old C hickens were inoculated with mixed sporulated oocysts of E.acervulina,E.tenella,E.maxima and E.necatrixoocysts,and then were infected with a single dose of C.perfringens?1×10⁹ cfu/bird?at 4 dafter Eimeria infection.Body weight?BW?gain,survival rate,intestinal lesions,bacterial count and other clinical parameters were statistically analyzed at 2 dafter C.perfringens infection.Compared with single coccidian infection or C.perfringens infection,The experimental N E coinfectied withC.perfringens and Eimeria coccidia caused a reduction in BW gain,obvious C.perfringens lesions and bacteria multiplication in the intestines.In conclusion,the NE disease model was preliminarilyestablished.The C.perfringens disease model was appliedto evaluate the treatment effect of C.perfringensphage lysin Cp51and compared with dimeixiaocuoand cytohydrolist.The results showed that BW gain in the Cp51 treatment group wasincreased,and the intestinal lesions and the bacterial count were lower than the positiveanduntreated group.However,the effect of the Cp51 treatment group did not work well as the drug treatment groups.The results of this study showed that C.perfringens phage lyase had a certain effect in the treatment of C.perfringens disease,and had great potential as a bactericide in in vitro experiments,and it has the advantage of being fast and efficient.There is room for the application of phage lysinonthe treatment of bacterial diseases,and more research is needed before use in clinical practice.