Screening And Identification Of Proteins Interacting With Membrane Protein Of Transmissible Gastroenterritis Virus Using Yeast Two-hybrid System

Porcine transmissible gastroenteritis(TGE)is a highly contagious disease caused by transmissible gastroenteritis virus(TGEV),especially in winter and early spring and cold seasons;its clinical symptomes include acute diarrhea,vomiting,and dehydration.Although the virus is capable of infecting swine of all ages,suckling piglets are the most susceptible and have a mortality rate up to 100 %.The inactivated and live attenuated TGEV vaccine are manufactured and have been extensively used in Asia.The emergence of virulent TGEV strains in previously vaccinated farms,it is necessary to develop more effective vaccines and of TGEV.Studying of TGEV interaction with the host to determine virulence genes,since the modification or deletion of these genes may lead to virus attenuation,and to the generation of vaccine candidates to prevent infection by TGEV.Alternatively,analysis of virus-host interactions may teach us the signaling pathways required by the virus for its replication or to cause pathogenesis.TGEV affect many host cell pathways that may have a positive or negative impact on viral replication and virulence.Modulation of a cell signaling pathway affecting TGEV replication may also have an impact on the host,contributing to TGEV virulence.In addition,as all vaccine types would also interact with the same host-cell pathways as the pathogenic TGEV,knowledge on the signaling pathways altered by TGEV may have implications for vaccine development.Once this has been accomplished,the selection of inhibitors of these signaling pathways could result in the identification of antivirals.The M protein is one of the structural proteins of TGEV,mainly embedded in viral lipid capsule,which is the viral assembly scaffold and the most abundant protein in the viral envelope.The M protein interacts with the S,N,and E proteins and plays an essential role in virus assembly.The M proteins also interact other M proteins to form homo-oligomers.At present,the screening and identification of the interaction proteins between M protein and host cells has not been reported.In this paper,M protein and host cell protein interactions as the research object,using yeast two-hyrid system screening porcine intestinal epithelial cells(p IECs)of host cells cDNA library interacting with the M protein;GST pull-down experiment to further verify the interaction of candidate proteins with the M protein.The main results are as follows:(1)Construction and identification of porcine intestinal epithelial cells(p IECs)cDNA libraryThe cDNA of p IECs was extracted by Trizol reagent.The double-stranded cDNA was synthesized by SMART method and remove the short cDNA fragments,the digested cDNA cloned into library vector p GADT7.The primary library was amplified,extracted library plasmid,and then transformed into yeast strain Y187,successfully constructed the yeast two-hybrid cDNA library.(2)Construction and identification of bait vector with TGEV protein in yeast two-hybrid systemBait vector was constructed for screening the interactions between TGEV M protein and host proteins,the gene from the structural proteins M was amplified from p Fast Bac TM-M by PCR and cloned into p MD19-T simple vector.After being verified,it was directional cloning into bait vector p GBKT7 of yeast two-hybrid system.Then the recombinant plasmid was identified by PCR,restriction enzyme digestion and sequence analysis and transform into yeast cells Y2 HGold by PEG/Li Ac method.Toxicity and self-activation of the bait protein were detected using different droupout minimal base.The results showed that the bait vector p GBKT7-M had no toxicity to yeast cells and no self-activation phenomenon to report genes.The results indicated that the constructed bait vector p GBKT7-M can be used for screening the host proteins interacting with TGEV M protein using yeast two-hybrid system.(3)Yeast two-hybrid system screening the M interacting proteinsAccording to the Clontech Matchmaker?Gold Yeast Two-Hybrid System user manual,the bait vector p GBKT7-M contructed by full length of the TGEV M gene to identify host proteins interacting with p IECs yeast two-hybrid cDNA library.After three grounds screening on the different nutritional deficient medium plate,namely SD/-Trp/-Leu/-Ade,SD/-Trp/-Leu/-Ade/-His,SD/-Trp/-Leu/-Ade/-His/X-?-Gal/Ab A,a total of 108 positive clones were identified.The results of cloning and sequencing of these clones showed they encode for 7 proteins.Among them,EIF4A2 protein has a high reproducibility,and according to the literature shows that EIF4A2 involved in promoting virus proliferation and viral proteinsynthesis.Therefore,EIF4A2 will be further studied and validated as a candidate protein.(4)GST pull-down experiment to verify the interaction between M protein and candidate proteinIn this part,the interaction between M protein and candidate protein EIF4A2 was verified by GST pull-down assay.We constructed a prokaryotic expression vector p GEX-4T-M of GST-M fusion protein.Then transformed and expressed the GST-M fusion protein in E.coli strain Rosetta,purified the fusion protein through glutathione-sepharose 4B;the EIF4A2 gene fragment was ligated into prokaryotic vector p ET-28 a to construct the recombinant expression vector p ET-EIF4A2 and the fusion protein was purified by Ni affinity chromatography column.The GST-M fusion proteins were then immobilized on glutathione-sepharose 4B serves as a bait protein,flowed through the purified His-EIF4A2 protein solution.Then the prey protein(His-EIF4A2)was captured,after eluted the bait and prey protein,the interaction between M protein and EIF4A2 was visualized by SDS-PAGE and Western blot.The results showed that there was a specific interaction between the M protein and candidate protein EIF4A2.