The Effects Of Four Recombinant Proteins Of Eimeria APP. On Peripheral Blood Mononuclear Cells Of Chickens In Vitro

Chicken coccidiosis,is a class of parasitic diseases seriously endangering the poultry industry,which is caused by nine kinds of Eimeria app.parasiting in intestinal epithelial cells.The diseases are spread worldwide,and they can cause the chickens weight loss,diamhea,bloody stools,even can cause the death of the chickens,that can cause huge economic losses to the poultry industry.At present,the controls of Eimeria infections are still mainly based on the anti-coccidial drugs.But,now with the questions of the increasing number of drug-resistant strains and the more and more serious issues of drug residues,looking for a safe and efficient vaccine have caused more and more attention of the researchers.In recent years,a variety of chicken coccidiosis vaccine research has been made certain achievements.Understanding and mastering the biological characteristics of Eimeria spp.and the anticoccidial immune protection mechanisms have become an important prerequisite to find a better immune protective antigen,it is also an important theoretical basis of the prevention of coccidiosis.A lot of genes which can present protective antigens have been respectively cloned from Eimeria app.in our laboratory,and many prokaryotic expression recombinant plasmids also have been constructed successfully.3-IE、EM6、EM8 and MIF were expressed,and we make further study of these four recombinant proteins in vitro to peripheral blood mononuclear cells(PBMCs).1 The expression of four recombinant proteins and the assay of recombinant proteins binding to PBMCs from chickensThe plasmids were transformed into BL21,then induced by IPTG after digestion with restrict proteins.Blood samples were collected from jugular vein of no Eimeria app.infected chickens.PBMCs were separated and cultured in vitro with a final concentration 5μg/mL of the recombinant proteins.The binding of the recombinant proteins to PBMCs were observed by immunofluorescence antibody technique.The result showed that recombinantenzymes EM8 and 3-1E could bind to PBMCs from chicken in vitro,while the others could not.2 Effects on cytokine expression in chicken peripheral blood lymphocyte induced by four recombinant proteins In order to study the effects of the four recombinant proteins on the cytokines(IL4,IL-17,IFN-y,TGF-P)of chicken peripheral blood mononuclear cells.In this study,we use the recombinant proteins 3-1E,EM6,EM8,and M1F to incubate with chicken PBMCs for 24 hours.Then mRNAs were extracted and quantitied by Real Time PCR to test the expression of cytokines after the incubation.The results showed that the mRNA transcription levels of IL-4,IL-17D,IFN-γ and TGF-β4 were different from control group apparently in;the mRNA transcription levels of IL-10 and IFN-y were different from control group apparently in HC29 group(P<0.01);the mRNA transcription levels remained steady in other groups(P>0.05).3 Effects of four recombinant proteins of Eimeria app.on the proliferation of chicken PBMCsIn order to study the effects of four recombinant proteins on the proliferation from chickens.We use Cell Counting Kit-8 to detect the changes of cells number.Results showed that all of the recombinant proteins had the effect on PBMCs.Especially,in the group of EM8 and MIF,cell proliferation were observed significantly obvious compared to the control group(P<0.01)。4 Effects of four recombinant proteins of Eimeria app.on phagocytosis and NO secretion from chicken macrophageIn order to study the effects of the four recombinant proteins on the macrophage from chickens.The phagocytosis and NO secretion of macrophage were tested after incubation with different groups of the recombinant proteins.The result showed that5 The recombinant proteins EM8 and MIF modulates chicken monocytes and T cells function in different patternsThe peripheral blood mononuclear cells have two major subpopulations:monocytes and T cells.They play an essential role in the innate and adaptive immune systems.Different proteins have different effects on different cell types.In this study,we find the two proteins can bind to both monocytes and T cells.Flow cytometric analyses revealed that the two proteins all can all increase the expression of MHCⅡ and MHCI on monocytes.The moncytes can be activated.