Font Size: a A A

Screening And Identification Of The Interaction Between Bovine Viral Diarrhea Virus Ns4B And Host Cells

Posted on:2020-03-07Degree:MasterType:Thesis
Country:ChinaCandidate:Z H YaoFull Text:PDF
GTID:2370330599962724Subject:Prevention of Veterinary Medicine
Abstract/Summary:PDF Full Text Request
Bovine viral diarrhea(BVD),caused by Bovine viral diarrhea virus(BVDV),is a viral infection,mainly affecting cattle and with the diseases of respiratory system and digestive system,which has caused great harm to the world?s cattle breeding industry.The pathogenic mechanism of BVD has attracted domestic and foreign scholars?attention.Studies have shown that BVDV Non-structrual protein 4B plays an important role in viral replication.In addition,NS4B,with the RNA-binding activity and NTP enzymatic activity,can induce membrane replication and participate in the process of virus-induced cell membrane rearrangement.In this study,yeast two-hybrid method was used to screen out host proteins that interacts with BVDV NS4B protein.Amplifying the BVDV NS4B gene used PCR and ligating it into the pGBKT7 carrier to construct the bait plasmid pGBKT7-NS4B then we transferred it into Y2H Gold yeast competent cells.It was verified that the bait plasmid of pGBKT7-NS4B had no self-activating activity and toxicity,and the bait plasmid could be used in subsequent yeast two-hybrid experiment.The RNA of Madin-Darby bovine kidney cells(MDBK)was extracted then the ds cDNA was obtained by LD-PCR.The prey was constructed by ligation cDNA library with pGADT7-Rec vector.The library?s capacity was about 3.4×10~6 cfu/ml.It was verified that the library has a good degree of homogenization,and can be used to do the experiment of yeast two-hybrid screening.The pGBKT7-NS4B bait plasmid was two-hybridized with the MDBK cDNA library,and six host proteins(LYZ1,PSMC4,Bak1,MKRN1,TP53BP2 and CKM)which can interact with the NS4B protein were obtained.The pGADT7-Rec-Bak1 prey plasmid was constructed with Bak1protein to do point-to-point hybridization.The pGBKT7-Bak1 bait plasmid and the pGADT7-Rec-NS4B prey plasmid were constructed to do back-cross hybrid.The results were both positive,which proved that Bak1 protein may interacts with BVDV NS4B protein.The host protein Bak1,interacting with the BVDV NS4B protein,was screened from the host cell library and is a positive regulator of apoptosis,which indicats that the NS4B protein may be involved in the process of inducing apoptosis after BVDV-infected cells.This lays a foundation for further elucidating of the molecular mechanism of BVDV in persistent infection in host cells,and provides references for the study of other viruses in the Flaviviridae family.
Keywords/Search Tags:BVDV, NS4B protein, Yeast-two-hybrid system, construction of cDNA library, Bak1
PDF Full Text Request
Related items