Prokaryotic Expression Of Surface Antigens SAG10 And SAG11 Of Eimeria Magna And Preliminary Evaluation Of Their Immune Protection In Rabbits

Eimeria magna is an obligate intracelluar parasite of rabbits,and it is a species with moderate pathogenicity to rabbits.After rabbits are infected with E.magna,in the clincally,it is manifested as depression,anorexia,weight loss and diarrhea,and bloody stool or even death may occur in severe infection.At present,the prevention and treatment of rabbit coccidiosis mainly depends on drugs.Long-term use of drugs will produce drug resistance and drug residues in rabbit meat.Therefore,vaccine prevention is the best way to control rabbit coccidiosis.However,the rabbit coccidiosis vaccine reported so far is a precocious attenuated live vaccine,the vaccine has problems such as high production costs and the risk of virulence reversion,the recombinant subunit vaccine has the advantages of being safe,stable,easy to obtain,and convenient for mass production.To this end,this study found four Em SAG genes(Em SAG1,Em SAG2,Em SAG10,and Em SAG11)from the transcriptome data of Eimeria E.magna determined in the laboratory,two genes(Em SAG10 and Em SAG11)were screened for prokaryotic expression,and the immune protection effect of the recombinant protein was evaluated through animal experiments.This study aimed to provide a reference for the development of recombinant subunit vaccines for E.magna.Four genes,Em SAG1,Em SAG2,Em SAG10 and Em SAG11,were screened from the transcriptome data of E.magna.After cloning and sequencing,real-time quantitative PCR was used to analyze the four genes in four developmental stages of E.magna.The transcription levels of sporulated oocysts,sporulated oocysts,merozoites and gametophytes showed that these four genes were transcribed at all stages,and the transcription levels were different.Among them,Em SAG1 and Em SAG2 were highly expressed at the sporulation stage;Em SAG10 and Em SAG11 were highly expressed at the merozoite stage.At the same time,recombinant proteins Em SAG10 and Em SAG11 were obtained by prokaryotic expression,both of which have good immunoreactivity.By evaluating the immune protection effect of E.magna Em SAG10 and Em SAG11 on rabbits,54 coccidoid-free rabbits were divided into 6 groups(blank contronl group,challenge control group,blank vector control group,Quil-A saponin control group,r Em SAG10 and r Em SAG11 immunization groups,9 rabbits in each group),the 42-days-age of rabbits were immunized for the first time at by subcutaneous injection in the neck,and the blank control(non-immunization and non-challenge),challenge control group were injected 1ml of sterile PBS into each rabbit,blank vector control group were injected 1ml(100 mg/ml)p ET-32a(+)protion,Quil-A saponin control group injected 1ml(2 mg/ml)saponin into each rabbit,r Em SAG10 and r Em SAG11 immunization groups were injected with 1ml(100 mg/ml)r Em SAG10 and r Em SAG11 respectively,and then immunized with the same dose 14 days later,except for the unchallenged control group,each rabbit in each group was orally inoculated with 1×10~5newly collected sporulated oocysts of E.magna.Each experimental rabbit was infected with 100 g of anticoccidial-free feed every day.After infction,the clinical symptoms,weighing,blood and feces samples of each group of rabbits were regularly observed after infection,and necropsy was performed 14 days after infection.By detect the change of Ig G in the serum from before the first immunization to the slaughter,and measure the contents of IL-2,IL-4,IL-10,IL-17,TGF-βand INF-γin the serum befrore challenge.Animal experiments showed that rabbits immunized with r Em SAG10 and r Em SAG11 had a higher average weight gain(82.2%,75.9%),feed;meat ratio(3.8:1,4.5:1),the oocysts decrease rate(82.8%,86%),and the ACI(178.4,168.9)than those in the control group,and also significantly reduced intestinal lesions.The specific Ig G level increased one week after the first r Em SAG10 and r Em SAG11 immunization and was maintained until two weeks after the challenge(p<0.05).The TGF-β,IL-4,and IL-10 levels in the serum increased significantly after the secondary immunization with r Em SAG10 and r Em SAG11,while the IL-2 levels increased significantly after the secondary immunization with r Em SAG11(both p<0.05),suggesting that r Em SAG10 can induce a humoral and cellular immunity,while r Em SAG11 can only induce a humoral immunity.Therefore,r Em SAG10 is a candidate antigen for E.magna recombinant subunit vaccines.