Screening Of The Proteins Interacting With NS1 Of TMUV By Yeast Two-Hybrid System And The Identification Of The Function Of The Interacted Protein

From April 2010 onwards,a duck infectious disease,which is characterized by retarded growth,acute anorexia,rhinorrhea,ataxia,diarrhea,paralysis and serious egg production decline,has occurred in some duck farms in some parts provinces of Southeast China.Then the disease quickly spreads to all of the country's duck industry.It accounts for significant economic losses to the duck industry throughout china,so it made a great attention by the majority of clinical veterinary workers.This infection leaded to 90% infection rate and mortality rate varied from 5% to 30%,possibly caused by mixed infections.Initially,the disease was defined as duck hemorrhagic ovaritis(DHO)and further studies show that the causative agent of this disease is Tembusu virus(TMUV).As a member of Ntaya virus(NTAV)group within the family Flaviviridae in genus Flavivirus,Tembusu virus(TMUV)is a positive-sense single-stranded RNA virus,which has a protein envelope.Like other flavivirus virus,the length of the viral genome is 10990 nt with an open reading frame(ORF)of 10278 nt flanked by 5'-and 3'-non-coding regions(NCRs)of 94 and 618 nt,respectively.The only open reading frame encodes three structural proteins: the core(C),membrane(pr M),and envelope(E).In addition,the ORF also encodes seven non-structural(NS)proteins named NS1,NS2 A,NS2B,NS3,NS4 A,NS4B and NS5.Although the NS1 protein does not constitute viral particles,it plays a key role in TMUV replication,infection and pathogenesis.Outside of this,NS1 proteins have multiple epitopes on T cells and B cells,which can induce humoral immunity and cellular immunity.Besides,its structure and gene content are stable and predictable,which has very important research value.The research content was divided into following three parts:1.Screening host proteins interacted with NS1 protein by the yeast two-hybrid(Y2H)systemViral RNA was extracted from the TMUV strain and the NS1 gene was amplified using the RT-PCR method.The rubber recovery product of NS1 and the p GBKT7 vector were digested by restriction endonucleases Eco R I and Bam H I.Ligated with T4-DNA ligase,the recombinant plasmid was transformed into E.coli DH-5? and sequenced.The small-scale Li Ac transformation method was used to prepare competent cells from yeast Y2 H and to transform p GBKT7-NS1 recombinant plasmid,p GBKT7-53 positive control,p GBKT7-Lam negative control and p GBKT7 into yeast which were then grown on SD/-Trp,SD/-Trp/X-?-Gal,SD/-Trp/ X-?-Gal/Aba medium for 3–7 days at 30 ?.Prepare a concentrated overnight culture of the bait strain(Y2HGold [p GBKT7-NS1])with the methods introduced by Matchmaker?Gold Yeast Two-Hybrid System User Manual.Then the bait strain was combined with the c DNA library of duck embryo fibroblasts(DEF)to screen the proteins interacted with the NS1 protein.By restriction enzyme identification,PCR and sequence analysis,the recombinant plasmid of p GBKT7-NS1 was successfully constructed.The results showed that the yeast transfected with plasmids of p GBKT7-NS1 and p GBKT7-53 could only grow on SD/-Trp and SD/-Trp/X-?-Gal medium.It is proved that the recombinant plasmid of p GBKT7-NS1 does not autonomously activate the reporter genes in Y2 H Gold,in the absence of a prey protein.Compared with yeast transferred into the plasmid of p GBKT7,the yeast transferred into the plasmid of p GBKT7-NS1 grown very well on the SD/-Trp medium.By the Y2 H system,the host protein such as MORC,RPS7 and GABARAPL1 were screened.2.GST Pull-Down assayThe Tembusu virus NS1 gene was subcloned in p GEX-6p-1.According to the result of yeast two hybrid test,the gene of Anas platyrhynchos ribosomal protein S7(RPS7)was subcloned in p EGFP plasmid.GST-fusion proteins were expressed in Escherichia coli BL21(DE3)and the Anas platyrhynchos ribosomal protein S7(RPS7)was expressed in 293 T cells.The expression of NS1 and RSP7 was determined by WB analysis.PierceTM GST Protein Interaction Pull-Down Kit was used for the GST pull-down assay.The protein elution was analyzed by SDS-PAGE test with the monoclonal antibody of GFP tag.According to the procedures above,we can analyze whether the prey protein is interacting with the bait protein.The recombinant plasmids named p GEX-6p-1-NS1 was identified by enzyme digestion and sequencing.SDS-PAGE and WBting analysis performed that the molecular weight of the GST-fusion proteins which were expressed in Escherichia coli BL21(DE3)was about 69 k D in silkworm.The results of double-enzyme(Xho I and Kpn I)digestion and sequencing of the recombinant plasmids demonstrated that the gene of RPS7 was inserted in-frame into expression vector p EGFP.The result of SDS-PAGE and WBting analysis showed that recombinant protein about 49.7k D was successfully expressed.Eventually,the monoclonal antibody of GFP tag was used for WB,and the target band of about 120 KD was obtained,which proved that there is a true interaction between the two proteins of TMUV NS1 protein and RPS7 protein.3.Real-time RT-PCR.The Tembusu virus NS1 gene was subcloned in p EGFP plasmid.Then the p EGFP plasmid and the recombinant plasmid of p EGFP-NS1 were transfected into 293 T cells.The 293 T cells were cultured for 24~48h.For real-time RT-PCR,RNA was extracted from 293 T cells by using Trizol reagent.The real-time quantitative RT-PCR analyses were performed following the manufacturer's protocols to measure the m RNA expressions of RPS7,MDM2 and P53.The relative expression values were normalized to the internal control,GADPH.Using real-time quantitative RT-PCR(q RT-PCR),the results showed that the expression of RPS7 and MDM2 m RNA decreased after the protein of NS1 was expressed in 293 T cells compared to the control group.But the expression of P53 m RNA increased compared to the control group.The results show that the expression of NS1 protein indeed affects the expression of RPS7 m RNA and eventually cause the high expression of P53 m RNA.