Screening And Validation Of Interacting Proteins Between Haemophilus Parasuis CDT And Host Cells

Haemophilus parasuis is a Gram-negative bacterium of the genus Pasteurellaceae,which is implanted early in the upper respiratory tract of pigs.When the host suffers from environmental persecution or decreased immunity,it spreads to the lungs and causes Pneumonia,even entering the blood circulation and invading multiple tissues,causes systemic Glsser's Disease,mostly fibrous polyserositis,arthritis and meningitis and other symptoms,the outbreak of the disease has a certain impact on global farmers.Cytolethal distending toxin is an important virulence factor of H.parasuis,which consists of three subunits cdt A,cdt B and cdt C.In order to understand the mechanism of interaction between H.parasuis CDT(Hp CDT)and host cells,this study used PK-15 cells as a model to screen host cell-interacting proteins by yeast two-hybrid method.(1)Construction of yeast two-hybrid c DNA library: total RNA of PK-15 cells was extracted,synthesis of double-stranded c DNA by SMART technology and LD-PCR,co-transformation of purified ds c DNA with p GADT7-Rec into Y187 yeast competent cells,in SD/-Leu auxotrophic culture plates were screened for culture,and a yeast c DNA library of PK-15 cells was collected.After detection,the library titer reached 7.5×10~7 CFU/m L,and the inserted double-stranded c DNA fragment ranged from 300 to 2000 bp,and the average fragment size was 500 bp.It is basically compatible with the requirements for constructing libraries and can be used for protein screening for interaction with different subunit proteins of Hp CDT.(2)Construction of bait plasmid: PCR amplification of cdtA,cdtB,cdtC gene fragment,directional cloning into yeast two-hybrid bait vector p GBKT7,transformation of empty vector p GBKT7 and recombinant vector into Y2 H Gold yeast competent cells by PEG/Li Ac method.The toxicity and autoactivation were tested using a screening medium.Double enzyme digestion and sequencing confirmed that p GBKT7-cdt A,p GBKT7-cdt B,p GBKT7-cdt C bait vector were successfully constructed;the transformed product was coated in SD/-Trp,the number of yeast on empty vector p GBKT7 and recombinant vector was not significantly different.The recombinant vector was proved to be non-toxic;the transformed product was coated on SD/-Trp,SD/-Trp/X-?-gal plate,colony-grown,coated on SD/-Trp/X-?-gal/Ab A,and grown aseptically,demonstrating the recombinant vector pair Yeast species have no autoactivation.The yeast two-hybrid vectors p GBKT7-cdt A,p GBKT7-cdt B and p GBKT7-cdt C were successfully constructed,which proved that they have no toxicity and autoactivation,which lays a foundation for further screening of proteins interacting with PK-15 cell library.(3)Mating method screening library and point-to-point verification: the bait vector p GBKT7-cdt A,p GBKT7-cdt B,p GBKT7-cdt C constructed by renal epithelial cell(PK-15)c DNA library were screened by yeast two-hybrid,and the hybridization solution was partially coated onto SD/-Leu,SD/-Trp,SD/-Leu/-Trp auxotrophic plates,the growth state of the colonies was observed;the remaining hybridization solutions were all applied to SD/-Leu/-Trp/X-?-gal/Ab A.Screening positive colonies on auxotrophic plates,and screening positive colonies cloned into SD/-Ade/-His/-Leu/-Trp/X-?-gal/Ab A auxotrophic plates for rescreening.Repeat screening on SD/-Ade/-His/-Leu/-Trp/X-?-gal/Ab A auxotrophic plates,extract the plasmid from the selected positive colonies,and expand the culture,send sequencing and Blast analysis.Finally,five proteins interacting with Cdt A were initially screened,and the mitochondrial ribosomal protein S26(MRPS26),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),and methyl-Cp G binding domain protein 3(MBD3),barrier to autointegration factor 1(BANF1),ribosomal protein L32(RPL32).