Screening The Interacting Proteins Of AMA1 And Primary Study On The Function Of MIC2 And 0440 Gene Of Eimeria Tenella

Coccidiosis is one of the most serious diseases in intensive poultry breeding industry,taking risk and hard to control,the losses and the cost of prevention caused by coccidiosis in the world up to 2billion pounds annually.Recently,the main ways to control coccidiosis are using of anticoccidial drugs and live vaccines.But due to the problem of drug resistance,drug residues and potential poison of vaccine,we need to promote new ways to control coccidiosis.Sporozoites are the first stage of infection invading host cell in vitro.Blocking its invasion to host cells is an effective way to prevent coccidiosis.The apical membrane antigen 1（AMA1）is a highly conserved microneme protein in the apicomplexa phylum protozoa Plasmodium and Toxoplasma gondii tachyzoites/merozoite during invasing host cell,AMA1 interact with multiple rhoptry neck proteins（RON2/4/5/8）to form compounds that play an important role in the invasion process.In our previous study,we found that Eimeria tenella AMA1（Eimeria tenella AMA1,EtAMA1）protein expression level in sporozoite was higher than that of unsporulated oocysts,sporulated oocysts and merozoites stage,and its antibody can inhibit invasion of sporozoite,indicating that EtAMA1 is a key protein in sporozoites invasion process,but the mechanism is not clear.The purpose of this study is that to screen the interaction protein of sporozoite with EtAMA1 by using GST pull-down combined with mass spectrometry,and two of the potential interacting proteins were selected to further study their characteristics.These results lay the foundation for elucidating the molecular mechanism of sporozoite invasion of host cells.1.Screening the potential interacting sporozoite proteins of EtAMA1In order to screen the potential sporozoite proteins interacting with Et AMA1,we express and purifying the constructed prokaryotic plasmid of pGEX-6P-1-EtAMA1.The purified recombinant protein EtAMA1-GST was identified by Western blot（WB）.The result of WB showed that a protein about 72 kDa which was in accordance with molecular weight of target protein was recognized by anti-GST antibody,confirming that the obtained protein was EtAMA1-GST fusion protein.Glutathione sepharose beads were incubated with Et AMA1-GST,GST protein or PBS,respectively,and then incubated with sporozoite lysates.Bound proteins were eluted by using sample buffer,and eluants were resolved by SDS-PAGE.The result of SDS-PAGE showed that the bands were significantly different among them.The different bands were digested and analyzed by mass spectrometry.The obtained protein peptides were blasted with Eimeria tenella proteins database deposited in Uniprot and 10potential sporozoite proteins were obtained.These proteins,mainly including microneme proteins,glycolytic enzymes,actin-related proteins and some hypothetical proteins,are involved in many molecular processes,such as invasion,development and energy metabolism.These results provide a basis for further elucidating the molecular mechanism of EtAMA1 playing an important role in Eimeria tenella invasion process.2.Verification of the relationship between the potential protein EtMIC2、Et0440and EtAMA1We select the Eimeria tenella Microneme 2（Microneme protein 2,EtMIC2）and hypothetical protein 0440（Et0440）from the 10 sporozoite proteins that were potentially interacting with EtAMA1for further testing.The ORF for the two genes were obtained by PCR.The ORF of EtMIC2 is 1029 bp,encoding 342 amino acids with predicted protein molecular weight of 38 kDa.The ORF of Et0440 is423 bp,encoding 140 amino acids with predicted protein molecular weight of 15.5 kDa.The eukaryotic expression plasmids pcDNA3.1（+）-flag-Et0440、pcDNA3.1（+）-flag-EtMIC2、p BiFC-VN155-Et0440and p BiFC-VN155-EtMIC2 was transfected into DF-1 cells.The eukaryotic plasmid express succefully by Western Blot and IFA identification.We verified the interaction between proteins by Co-immunoprecipitation（Co-IP）and bimolecular fluorescence complementation experiments（BiFC）.The Co-IP results show that there’s no compound,only a specific band of EtAMA1 appears.The results show that Et0440 and EtAMA1,EtMIC2 and EtAMA1 do not interact with each other.BiFC results showed that the positive control group produced green fluorescence,and the negative control group,p BiFC-VN155-Et0440 and pBi FC-VC155-EtAMA1,pBiFC-VN155-EtMIC2 and pBiFC-VC155-EtAMA1 had no green fluorescence.So we confer Et0440 and EtAMA1,EtMIC2 and Et AMA1 has no interaction.3 Prokaryotic protein expression and preliminary function study of Et0440 and EtMIC2The Et0440 and EtMIC2 gene were cloned into prokaryotic expression vector pET32a and p ET30a respectively,transformed into E.coli BL21.The recombinant protein His-Et0440 and His-EtMIC2 were successfully induced by adding IPTG at 37℃.Two purified proteins were used to immunize the rabbits to obtain polyclonal sera.The results of Western blot showed that both Et0440 and EtMIC2 have good immunogenicity.The indirect immunofluorescence showed that Et0440 mainly distributed in the top ends of sporozoites after incubation with the cell culture medium,and in the front of second generation merozoites,in the both ends of the first generation merozoites;while EtMIC2 is mainly distributed in front of sporozoites and second generation merozoites,in both ends of first generation merozoites.Inhibition experiments were performed using sporozoites preincubated with anti-rEt0440 or anti-rEtMIC2 respectively.We found that the two antibodies pretreatment reduced the capacity of sporozoites to invade the DF-1 cells,and that the inhibitory effects of these two antibodies were dose-dependent.Compared with the same dose of negative control（IgG from naive rabbit serum）,when the antibodies concentration increased to 400μg/mL,anti-rEt0440 can prevent about 40%the parasite invading cells,anti-rEtMIC2 can prevent about 20%the parasite invading cells.These results suggested that two proteins may play an important role during the invasion of host cells.4 Protective efficaccy of Et0440 and EtMIC2 on chickens against Eimeria tenella7 day old chickens were randomly divided into eight groups,and the average weight of each group was basically the same.Recombinant eukaryotic expression plasmid pcDNA3.1（+）-flag-Et 0440 and pcDNA3.1（+）-flag-EtMIC2 and empty plasmid pcDNA3.1（+）–flag were immunized by intramuscular injection.,The infection control group and healthy control group were set.The second immunization conducted at 14-day-old with the same immunization dose and immunizing way as the first immunization.Seven days after the second immunization,all the chickens except the unchallenged control group,were challenged orally with 1×10⁴ sporulated oocysts of E.tenella.Results showed that weight gains（WGs）of all immunized groups during immunization were similar to those of non-immunized group（P>0.05）.The WGs of immunized groups during challenge were generally higher than the control group of infected chickens,but only pcDNA3.1-flag-0440（200μg/bird）group was significantly higher than the weight of infection control group（P<0.05）.The results indicate that E.tenella has obvious inhibitory effect on the development of the growth of chickens and the recombinant plasmid had partial protection against E.tenella challenge.The immunized groups oocysts reduction rate was above 65%,significantly higher than the infection control group and the empty vector group.At the same time,cytokine levels were examined.The results showed that IFN-γof 7days after the second immunization andIL-17 after challenging in the immunized groups were significantly higher than the control group（P<0.05）.Wihle TGF-βwere not significant different before or after challenging（P>0.05）.These above results showed that recombinant plasmid pc DNA3.1（+）-flag-Et0440 and pcDNA3.1（+）-flag-EtMIC2 had partial protective effects against Eimeria tenella challenge.