Screening And Validation Of Calcium-dependent Protein Kinase 4 Interacting Proteins Of Eimeria Tenella

Calcium is the second messenger of signal transduction in organisms.Calcium-dependent protein kinases?CDPKs?,as downstream signaling molecules of calcium,play an important role in regulating the metabolism of organisms and their adaptation to the external environment.A preliminary study of the function of CDPK4of Eimeria tenella?EtCDPK4?was conducted in our laboratory.It was found that the protein was highly expressed at the merozoites?and involved into the invasion of host cells by sporozoites.To understand how EtCDPK4 plays a role in the invasion and development of parasites,we screened the interacting proteins of EtCDPK4 and verified two of the proteins that may interact with EtCDPK4.1.Screening interacting proteins of EtCDPK4The total RNA of E.tenella merozoites?was extracted and mRNA was then isolated by PolyA Ttract^?mRNA Isolation Systems III.The first strand of cDNA was synthesized by MMLV-RT reverse transcription.The ds cDNAs were synthesized by LD-PCR and then purified by a purification column to remove cDNAs<200 bp.Purified ds cDNAs and pGADT7-Rec plasmids were co-transformed into Y187 yeast strains and cultured on SD/-Leu deficient medium,then the cDNA library of E.tenella merozoite?was constructed.After detection,the recombinant rate of the library was93.2%,the size of the library was 9.6×10⁹ CFU and the transformation efficiency of the library was 4.1×10⁵ cfu/?g.Then Yeast Two-hybrid technique was performed to screen EtCDPK4 interactive proteins.The pGBKT7-EtCDPK4 strain that has been constructed in our lab was mixed with the E.tenella merozoite?cDNA library,then cultured on SD/-Ade/-His/-Leu/-Trp?QDO?highly deficient medium using Y2H technology.Blue colonies were picked and cultured on the QDO medium for screening repeatedly.At last,the obtained blue clones were sequenced and then blasted with the E.tenella genome?http://www.genedb.org/Homepage?.The finally 30 positive clones were obtained after removed non-E.tenella gene and repeat sequences.The positive plasmids were transformed into Y2H Gold strains with pGBKT7-EtCDPK4 for reverse hybridization after rescued by transformed into E.coli.Totally,8 proteins that may interact with EtCDPK4 were obtained,including 4 unknown conserved proteins?ETH₀0033980,ETH₀0024500,ETH₀0018145,ETH₀0002065?and 1 serine protease inhibitor?ETH₀0011330?,1 DNA-directed RNA polymerase I and III subunit RPAC1?ETH₀0009380?,1 sec63 domain-containing DEAD/DEAH box helicase?ETH₀0021190?,and 1 apical membrane antigen 1 protein?ETH₀0007745?.2.Verification of two interacting proteinsTwo putative interacting proteins:E.tenella serine protease inhibitor Serpin?ETH₀0011330?and DNA-directed RNA polymerase I and III subunit RPAC1?ETH₀0009380?were selected to further verify whether interact with EtCDPK4.We successfully constructed the recombinant plasmids pBiFC-VC155-EtCDPK4,pBiFC-VN155-EtSerpinandpBiFC-VN155-EtRPAC1todoBiFC.pBiFC-VC155-EtCRPK4 co-transfected into DF-1 cells with pBiFC-VN155-EtSerpin and pBi FC-VN155-EtRPAC1,respectively,and observed under fluorescence microscope after 24h.The results showed that the green fluorescence appeared in DF-1cells co-transfected pBi FC-VC155-EtCDPK4 and pBiFC-VN155-EtSerpin as the positive control,but there was no green fluorescence after the co-transfection of pBiFC-VC155-EtCDPK4 and pBiFC-VN155-EtRPAC1.These results showed that there was an interaction between EtCDPK4 and EtSerpin,but there was no interaction between Et CDPK4 and EtRPAC1.At the same time,we constructed the recombinant plasmid pcDNA3.1-flag-EtCDPK4 and pcDNA3.1-flag-EtRPAC1 for Co-IP.Flag-Et CDPK4 co-transfected DF-1 cells with pCAGSS-EtSerpin which has been constructed in our lab and flag-EtRPAC1,respectively.Proteins were collected by lysed cells and incubated in a gel coupling with EtCDPK4 antisera for Co-IP.The results showed that the two proteins were detected in the eluents of DF-1 cells co-transfected flag-EtCDPK4 and pCAGSS-EtSerpin group,while only flag-EtCDPK4 was detected in the eluents of DF-1 cells co-transfected flag-Et CDPK4 and flag-EtRPAC1 group.These results showed that there was an interaction between EtCDPK4 and EtSerpin,and there was no interaction between EtCDPK4 and EtRPAC1.In addition,IFAs were performed to determine the location of EtCDPK4 and EtSerpin.The position of Et Serpin was visualized with a FITC-labeled green fluorescent secondary antibody and the EtCDPK4 position was visualized with a Cy3-labeled red fluorescent secondary antibody.The results showed that when the sporozoites invaded the host cells early,EtCDPK4 and Et Serpin were at the apical end of the parasite,the two proteins were distributed in the cytoplasm in the sporozoites and the second generation of merozoites,The results showed that these two proteins may play an important role together in the process of invasion and development of the parasite in host cells.3.Preliminary analysis of the function of EtRPAC1The ORF sequence of the EtRPAC1 gene was cloned and the prokaryotic recombinant plasmid pGEX-4T-EtRPAC1 was constructed.The recombinant protein was successfully induced to expression,and the polyclonal antibody was prepared after immunized rabbit using purified protein.Bioinformatics analysis revealed that the ORF was 999 bp in length,encoded 332 amino acids with a molecular weight of 37.5kDa.The structural domain found that the protein may contain RNA polymerase Rpb3/RpoA insertion domain,RNA polymerase Rpb3/Rpb11 polymerization domain,DNA-mediated RNA polymerase subunit D and so on.In vitro inhibition experiments showed that the anti-EtRPAC1 polyclonal antibodies reduced the ability of sporozoites to invade DF-1 cells compared to normal rabbit serum IgG.The inhibition rate was positively correlated with the antibody concentration.When the antibody concentration was 400?g/m L,the invasion inhibition rate was about 65%.The result indicated that EtRPAC1 protein may have important functions in the process of E.tenella invading host cells.