Screening And Identification Of The Cell Receptors Interacting Protein NS1 Of JEV

Japanese encephalitis virus (JEV) is a member of the flavivirus, which causes Japanese encephalitis (JE).As a mosquito-borne zoonosis, JE has obvious seasonal and infectious.JEV infectionmainly present central nervous system damage. JEV infected pigs mainly causes reproductive failure in female pigs,boars orchitis, piglet encephalitis and other symptoms.The NS1 protein is a nonstructural protein of JEV, participate in the process of virus reproduction, infection, etc. We study JEV NS1 protein as a bait protein, using yeast double hybridization technique and co-immunoprecipitation in the human brain cDNA library screening the protein interact with NS1. This study will be lay a certain foundation to clarify the role of NS1 protein for JEV replication mechanism.1. Construction of Japanese encephalitis virus NS1 protein bait yeastWe extracted the total RNA of JEV SCYA201201 plant transcription cDNA, as the template to amplification NS1 gene sequences by NS1 gene specific primers and connected the NS1 gene with pGBKT7 vector toconstruction of the bait plasmid pGBKT7-NS1.The bait plasmid was 1256 bp from PCR and double enzyme identification. We built the bait plasmid pGBKT7-NS1 to yeast Y2HGold cells, build the JEV NS1 protein bait yeast.We detected the size of about 46 kda NS1 protein from the total protein of bait yeast application by western blot technique, shows that NS1 gene can be expressed in the bait yeast. We detected the toxicity effect and the activation effect of bait yeast:the growing and the number of colonies on the SD/-Trp tabletand was found no obvious difference between Bait yeast and comparison yeast, shows that NS1 fragment insertion to bait yeast have non-toxic.Both Bait yeast and comparison yeast could grow on the SD/-Trp/X-?-Gal tablet and do not become blue. There was no colonies on SD/-Trp/X-a-Gal/AbA plate, proves the bait yeast no activation effect.This study successfully constructed JEV NS1 protein bait yeast, provided research material for further screening of JEV NS1 protein interaction between host receptor protein.2. Screening the interaction protein of NS1 protein by yeast two hybrid systemWe used NS1 protein as the bait protein to screen the interaction protein of NS1 protein in the human brain cDNA library by yeast two hybrid system.We got 72 cloned by SD/-Trp/-Leu/X-a-Gal/AbA culture medium for the first round of screening the product of yeast Y2HGold and the human brain cDNA library. We picked all clones in SD/-Trp/-Leu medium to SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA medium on the screening again, repeated 3 times, get seven positive clones.We got 5 positive monoclonal from the positive clones whichcould be screening by Ampicillin resistanceand identificate by PCR and sequencing. We determinated of sequence BLAST in Genbank, the 5 positive clones correspond a2-macroglobulin(?2M)? Homo sapiens neuronal differentiation 6(NEUROD 6)?DAZ associated protein 2 (DAZAP 2)?TMP metallopeptidase inhibitor 4(TIMP-4) and zinc finger protein 251 (ZNF 251).Furthermore, the five library plasmid were transformated into yeast strain Y187 to construct the prey yeast. The prey yeast and NS1 bait yeast were mixtured to the reply yeast hybrid. The results of the five groups were all positive.This study used yeast two hybrid system successfully screened 5 receptors of JEV NS1 proteinfrom the human brain tissue cDNA library.3. Verification of JEV NSl protein and host protein interactionsWe extracted the total RNA of JEV SCYA201201 plant transcription cDNA, as the template to amplification NS1 gene sequences by NS1 gene specific primers. Connect the NS1 gene to pEGFP vector, construction of the plasmid pEGFP-NS1.The plasmid pEGFP-NS1 was 1256 bp from PCR and double enzyme identification..We extracted the total RNA of human embryonic kidney cells (293 cells) transcription cDNA.The coding region of the five genes were amplified based on cDNA. The five genes were respectively inserted into the eukaryotic expression vector pCMV-HA.Those plasmids were transfected into 293 cells respectively for expression.The results of western blot showed three host protein and NS1 protein hadsuccessfully been expressed. Applying co-immunoprecipitation technology validation NS1 protein interaction with the host proteins inside cells, the results showed host protein DAZAP 2 can interact with the NS1 protein in cells and failed to demonstrate ?2M and TIMP-4 with the NS1 protein interactions in the cells. This study used co-immunoprecipitation technology to verify the host immune proteins interact with NS1 protein in cells,the result showed that host protein DAZAP2 is the interactor of NS1 protein provided research material for further screening of JEV NS1 protein interaction between host receptor proteins.