Screening And Identification Of Host Interaction Proteins For VP1 Of E Enterovirus HY12 Strain | Posted on:2021-04-16 | Degree:Master | Type:Thesis | Country:China | Candidate:B W Zheng | Full Text:PDF | GTID:2370330611970639 | Subject:Veterinary Public Health | Abstract/Summary: | PDF Full Text Request | Bovine enterovirus(BEV)infection is an infectious disease clinically characterized by digestive and respiratory disorders causing severe economic losses to the cattle farming.At present,outbreaks of this disease have been reported in many areas in China.As the disease is a new infectious disease in China,research on the pathogenic mechanism of this disease and the interaction between viruses and host cells is lack.This study using the structural protein VP1 of enterovirus HY12 strain that be constructed as recombinant plasmid to express the bait protein to screen and identify host proteins that have interactions with VP1 by yeast two-hybrid technology,in order to make sure the function of VP1 protein in the process of virus invasion into the body that lays a foundation for the ultimate elucidation of enterovirus infection mechanism.In order to screen the host protein interacting with the enterovirus HY12 VP1protein,the VP1 gene was amplified by PCR and cloned into the yeast vector pGBKT7 to construct the bait vector pGBKT7-VP1.The bait recombinant plasmid was transformed into yeast strain AH109 to detect the toxicity to yeast cells by comparing the OD60000 of the recombinant plasmid group and the empty plasmid group at different time points.The yeast containing the bait recombinant plasmid was spreaded on synthetic dropout media to verify its self-activation.AH109 transformed with the bait vector was mixed with the Y187 containing the cDNA of MDBK cell of the yeast two-hybrid to screen the hybridized colonies by synthetic dropout media.Positive colonies of yeast plasmids were verified by DNA sequence analysis and other methods.Extracting the yeast plasmids of positive colonies,then getting the results through PCR and alpha-galactosidase assay.Results showed that twelve potential VP1interacting proteins were identified.Through the Bimolecular Fluorescent Complimentary that the constructed pbJun-VP1 and pbFOS-candidate proteins will be co-transfected into 293T cells and observe the result under a fluorescent microscope to verify the interaction.At the same time,the eukaryotic recombinant plasmids pcDNA3.1B-VP1 and pCMV-Tag 2B-candidate protein were constructed to verify the interaction by Co-Immunoprecipitation.In summary,in this study,the bait vector pGBKT7-VP1 of the yeast two-hybrid system was successfully constructed through PCR and other techniques,and verified that it has no self-activating activity and cytotoxicity through the synthetic dropout media and the OD600.The titer of the library was 108 cfu/mL,and the inserted cDNA fragments of 44 clones were between 200 bp to 2 000 bp,indicating that the library had good polymorphism and high abundance.The AH109 transformed with the bait vector was mixed with the Y187 containing the cDNA of MDBK cell of the yeast two-hybrid,then 12 positive clones that named as candidate proteins were screened by synthetic dropout media and PCR as well as other methods.After DNA sequence analysis,PAK1 and ACTG1 were selected to preliminarily verify the possible interaction between VP1 and PAK1 as well as VP1 and ACTG1 by the alpha-galactosidase assay.VP1,PAK1 and ACTG1 can express in cells verified by Western Blot and construction of eukaryotic recombinant plasmid.Using Co-Immunoprecipitation and Bimolecular Fluorescent Complimentary to further verify the interaction between VP1 protein and two of these candidate proteins,which laid the foundation for the study of BEV infection mechanism and the function of viral structural protein VP1. | Keywords/Search Tags: | HY12 enterovirus, Enterovirus E, Interacting protein, VP1 protein, Yeast twohybrid, Bimolecular Fluorescent Complimentary, Co-Immunoprecipitation | PDF Full Text Request | Related items |
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