Construction Of Yeast Two-hybrid CDNA Library Of Eimeria Tenella Sporozoites And Screening Of Interaction Proteins With EtCDPK3

In recent years,due to increasing use of lots of anticoccidiosis drugs,the emergence of drug resistance of Eimeria spp has brought great challenges to the prevention and treatment of coccidiosis.Calcium dependent protein kinases?CDPKs?are belong to a big family of multi gene encoded,it transmit phosphorylation signal to downstream through Ca²⁺pathway which can produce cascade amplification reaction,thus help protein exert related function.In our previous report,the characteristics of EtCDPK3 were studied in our laboratory,the results showed that the Et CDPK3 was highly expressed in the sporozoites and involved in invasion and escape into the host cells.In order to screening the substrates of EtCDPK3,the yeast two hybrid cDNA Library of sporozoite of Eimeria tenella was constructed successfully,and then the recombinant plasmid pGBKT7-EtCDPK3 was constructed as a bait.We screened the yeast two hybrid cDNA library using the bait and obtained 11 putative positive clones which could be interacted with EtCDPK3.Three putative positive clones were be further analyzed by BiFC and Co-IP.1.Construction of yeast two hybrid cDNA Library of sporozoite of E.tenellaThe total RNA was isolated from the sporozoites and used as the template to synthesize the first-strand cDNAs.The dsc DNAs were acquired by Long-distance Polymerase Chain Reaction?LD-PCR?using the first-strand cDNAs as the template and purified by Chroma Spin TE-400 Column to remove less than 200 bp fragments.The dsc DNA,pGADT7-Rec and CarrierDNA were cotransformed into Y187 yeast competent cells.The dscDNA were connected with p GADT7-Rec by homologous recombination reaction.The yeast two-hybrid cDNA library of E.tenella sporozoites was constructed.The results showed that the conversion ratio and titer of the library were 4.33×10⁵/?g pGADT7-Rec and 3.62×10⁷cfu/mL,respectively.PCR amplification revealed that the library contained approximately 93.75%recombinant clones,and the inserted cDNA fragments were between 200bp and2000bp.5 specific genes of E.tenella were obtained by PCR using this yeast two-hybrid cDNA library as template.It concluded that a yeast two-hybrid cDNA library of the sporozoites was constructed and could provide a foundation for screening invasion-related interaction proteins of the sporozoite of the E.tenella by yeast two-hybrid method.2.Construction of recombinant plasmid pGBKT7-Et CDPK3 and screening of interacting proteinsThe ORF fragment of EtCDPK3 was cloned by PCR according to the EtCDPK3 gene sequence.The EtCDPK3 and p GBKT7 empty plasmid were digested with restriction endonuclease BamH I and EcoR I,respectively,then the products were purified after enzyme digestion and ligated to transform E.coli competent cells?TOP10?.After the recombinant plasmid pGBKT7-Et CDPK3 was testedby sequencing and enzyme digestion successfully,the recombinant plasmid pGBKT7-EtCDPK3 was transformed into Y2HGold yeast competent cells,then the transcriptional activity,cytotoxicity and protein expression were analyzed.The results showed that the bait plasmid has no transcriptional activation and no cytotoxicity to the Y2HGold yeast cells and could be expressed in yeast cells successfully.Thus the recombinant plasmid pGBKT7-Et CDPK3could be used to screen the yeast two-hybrid cDNA library of the sporozoites.The constructed yeast two-hybrid cDNA library and the bait plasmid pGBKT7-Et CDPK3 were applied in yeast two-bride system,and the high selective medium?SD/-Ade/-His/-Leu/-Trp?was used to screen the interaction protein.Then all the single clones on the plate were transferred to the medium?SD/-Ade/-His/-Leu/-Trp/X--Gal/Aba?to screen by two times.Eleven positive plasmids were obtained after plasmid sequencing totally.3.Verification of interaction between EtCDPK3 protein and putative positive proteinThree putative positive plasmids were selected randomly to reverse hybridization,the results showed that there were blue monoclones on highly defective medium,thus these three proteins could be interacted with p GBKT7-EtCDPK3.In order to further investigate the relationship between them,the ORF fragments of these three genes were amplified by PCR,and then ligated to the vector pBiFC-VC155 to construct recombinant three eukaryoticexpression plasmids,including pBiFC-VC155-Et12,pBiFC-VC155-Et14,pBiFC-VC155-Et27,at the same time,EtCDPK3 was cloned and ligated to the vector pBiFC-VN155 to construct recombinant eukaryotic expression plasmid pBiFC-VN155-Et CDPK3,successfully.Three positive recombinant plasmids were cotransfected into DF-1 cells with pBiFC-VN155-EtCDPK3 respectively,then the bimolecular fluorescence complementation assays were performed after these proteins could be expressed in DF-1 cells.The result showed that there were no green fluorescence except for positive control.Moreover,in order to test the interaction these three positive plasmids with EtCDPK3,the CO-IP assays were performed.Three genes were ligated to eukaryotic expression vector pcDNA3.1-flag to construct three eukaryotic recombinant plasmids pcDNA3.1-flag-Et12,pcDNA3.1-flag-Et14,pcDNA3.1-flag-Et27,the fragment of EtCDPK3 was also ligated to eukaryotic expression vector pcDNA3.1to construct the eukaryotic recombinant plasmid pc DNA3.1-EtCDPK3,four plasmids were transformed into DF-1 cells,indirect immunofluorescence and Western blot results indicated that the plasmids could be expressed in DF-1cellssuccessfully.ThenthreeeukaryoticrecombinantplasmidspcDNA3.1-flag-Et12,pc DNA3.1-flag-Et14,pcDNA3.1-flag-Et27werecotransformedintoDF-1cellswith pc DNA3.1-EtCDPK3,respectively.The results of Co-IP assay showed that anti-flag monoclonal antibody could precipitate Et12,Et14,Et27 protein,but could not precipitate the protein which EtCDPK3 expressed.These experiments results showed that there could not exist interaction relationship among Et12,Et14,Et27 with EtCDPK3.4.Preliminary study on functional properties of Et CHP12Bioinformatics analysis showed that the ORF of Et12 was 1656 bp,encoding 552 amino acids..In order to further verify the function of Et CHP12,the full-length ORF of Et CHP12 was amplified by PCR and ligated to prokaryotic expression vector pET-28a to construct the recombinant plasmid pET-28a-EtCHP12 successfully.Then the recombinant plasmid was transformed into competent cells E.coli BL21 and induced to express recombinant protein His-EtCHP12?rEtCHP12?.The rEtCHP12protein was soluble protein and purified by the Ni-column affinity chromatography.The purified protein was used to immunize the New Zealand white rabbits to prepare polyclonal antibody.In vitro invasion inhibition assays results showed that the ability of sporozoite invasion into DF-1 cells decreased significantly after the sporozoites incubated with polyclonal antibody compared with normal rabbit serum,with the increasing concentration of antibody,the ability of sporozoite invasion gradually decreased and the inhibition rate increased,when the antibody concentration was 100 ug/mL,the inhibition rate is up to 60%.These results suggested that protein of EtCHP12 played an important role in the invasion of sporozoites.