The Application Of Yeast Two-Hybrid System In Screening Host Proteins Interacting With Spike Protein 1 Of Transmissible Gastroenterritis Virus And Preliminary Validation Of Protein Function

Transmissible gastroenteritis virus(TGEV)causes porcine transmissible gastroenteritis(TGE)which is a pathogenic enteric disease with clinical features of diarrhea,vomiting and severe dehydration.TGE is susceptible to all ages of pigs,especially to two week old piglets at a high fatality rate of 100,which brings a large loss for swine-breeding industry.TGEV is a member of the family Coronaviridae with a positive-stranded RNA genome,and it has four structure proteins which are spike(S),member(M),nucleocapsid(N)and member(M)protein.S protein is resiponsible for TGEV binding receptors and membrane fusion,and it is main inducer in production of neutralizing antibody for TGEV.Currently,it is found in further study that S protein consists of two structure domains,S1 and S2 protein.S protein protrudes the surface of virion in form of trimer structure.S1 protein is connected with viral membrane by S2 protein.S protein contains main B lymphocyte epitopes relate to inducing neutralizing antibody,and the four main antigen sites(A?B?C and D)are all located at Nterminal S2 protein,and site A is main part contributing to production of neutralizing antibody as major antigen.The antigenicity of S1 is maintained via co-translational glycosylation.Moveover,S1 protein contains receptor binding domain,which plays a vital role in the binding of virus and host cells.At present TGEV as crucial pathogen of piglet diarrhea in winter causes huge loss for breeding industry.The research on interaction of TGEV S1 and host cell proteins provides theoretical foundation for clarifying pathogenic mechanism of virus.The main researches are listed as below,(1)The construction of TGEV S1 bait plasmid and test of bait proteinAs template of pMD-19T-S1,the gene sequence of TGEV S1 is amplified by PCR.S1 gene and pGBKT7 are digested with EcoRI and SalI and constructed into pGBKT7-S1,and the recombinant plasmid is transformed into yeast Y2 HGold.The test for bait is in protein expression,autoactivation and toxicity.The result shown that S1 protein was expressed in yeast Y2 HGold detected by Western-blot.The yeasts with pGBKT7-S1 were the same with yeasts with pGBKT7 in size and number,suggesting that bait had no toxicity for yeast.Yeast with pGBKT7-S1 could grow on dropout minimal base SD/-Trp/X-?-Gal/AbA shown as blue clones,indicting that bait protein contributed to autoactivation of yeast.(2)Screening the proteins interacting with S1 by yeast two-hybrid systemAccroding to Matchmaker? Gold Yeast Two-Hybrid System,the hybrid is programmed for yeast Y2 HGold with pGBKT7-S1 and yeast Y187 with IPEC-J2 cDNA.And observe the result of white clones growing on dropout minimal base SD/-Trp/-Leu/-Ade.For strengthening screeing pressure,the white clones are coated on plate SD/-Trp/-Leu/-Ade/-His for second screening,and plate SD/-Trp/-Leu/-Ade/-His/X-?-Gal/AbA for third screening.The blue clones on plates are the positive clones,and BLAST is programed for homology analysis.Twelve candidate proteins are gained.UBX domain-containing protein 1(UBXN1)has the feature of high repetition rate among them and strong inhibitory effect on RNA-virus-induced type I interferon response,which is the reason for interested protein to be further researched on the interaction with TGEV S1.(3)Verificating whether the interaction of UBXN1 and S1 exists by GST-pull downThe recombinant expression plasmid pGEX-4T-S1 and pET-28a-UBXN1 are constructed,and then they are transformed into Rosseta competent cell to induce fusion proteins expression,GST-S1 and His-UBXN1 protein.Finally,the purified GST-S1 is programmed with HisUBXN1 by GST-pull down and elution protein is examinated by Western-blot.The result shows that the interaction exists between TGEV S1 and UBXN1.(4)Preliminary validation whether UBXN1 has protein function for TGEV S1.As target of UBXN1 CDS gene squence,three siRNA fragments are designed.The three interference fragments are transfected into IPEC-J2 cells,respectively.And then the cells are inoculated by TGEV Miller.After TGEV infection,real-time PCR result shows that transcriptional level of TGEV S1 is reduced.The result of western-blot exhibits TGEV S1 expression reducing,and it is found TGEV proliferation is inhibited in MTT plaque assay.Shown from above mentioned,UBXN1 in intestinal epithelial cells has a positive correlation with TGEV S1,impacting virus replication,which offers theoretical basis for further study on candidate target proteins about the effective prevention and control of TGEV infection.