Screening And Functional Research Of Interacting Proteins Of Bovine Viral Diarrhea Virus Ion Channel Protein P7

Bovine viral diarrhea virus(BVDV)mainly infects cattle,sheep,pigs,camels and other animals,causing symptoms such as diarrhea,mucosal erosion,immunosuppression,and reproductive disorders.It also contaminates serum,frozen sperm,and embryos.Such as cattle-source biological products,causing serious economic losses to the breeding industry and other related industries.BVDV p7 is a small peptide composed of hydrophobic amino acids.It forms channel proteins on the lipid bilayer membrane and has ion channel activity.It belongs to the Viroporin family.A large number of studies have shown that viral porins play a key role in many viral replication cycles.At present,there are few studies on BVDV p7 ion channel protein.Which proteins are required to participate in the formation of p7 ion channel? How does this protein participate in the BVDV replication process? It is not clear yet,and urgently needs to be studied and clarified.In this study,the adjacent biotinylated protein labeling system Turbo ID was used to screen the proteins that interact with p7,and CRISPR/Cas9 was used to knock out the interacting proteins,to explore the effect of the interacting proteins on BVDV replication,and to provide evidence for further elucidating the mechanism of p7 forming ion channels,It also provides a target for the establishment of a new method of BVDV prevention and control.The specific content is as follows:1.Preparation and identification of BVDV p7 polypeptide polyclonal antibodyPerform bioinformatics analysis based on the amino acid sequence information of NADL p7 of the BVDV strain in Gen Bank,synthesize surface antigen peptides and couple with Keyhole limpet hemocyanin(KLH)as the carrier protein.Purify by reversed-phase high performance liquid chromatography and analyze the purity.Qualitative identification by mass spectrometry;after emulsification with Freund's adjuvant,New Zealand white rabbits were immunized by subcutaneous injection,and the serum was purified by peptide affinity column to obtain polypeptide polyclonal antibodies.Indirect ELISA and SDS-PAGE were used to detect antibody titer and purity;BVDV NADL infection Bovine kidney cells(Madin-Darby bovine kidney,MDBK)were immunofluorescent stained to detect the reactivity and specificity of the polypeptide polyclonal antibody.Bioinformatics analysis showed that the main antigenic epitopes of p7 protein are distributed at the amino terminal.After coupling KLH,the peptide MSQYGAG EIVMMGN-Cys-KLH is synthesized,and the purity of the purified peptide can reach 80.19%;the indirect ELISA detects the polyclonal antibody titer of the peptide is 1.: 100,000,the purity of the antibody detected by Western blot was 90.2%;immunofluorescence staining showed that the antibody showed positive staining on the cell membrane of MDBK cells infected with BVDV NADL,which was consistent with the location of the p7 ion channel.2.The Turbo ID system screens the interacting proteins of BVDV p7 proteinConstruction of the lentiviral vector p LVML-Myc-p7-(GGGS)3 linker-Turbo ID-IRES-Puro and p LVML-Myc-(GGGS)3 linker-Turbo ID-IRES-Puro;packaging the lentivirus to infect MDBK cells and use puromycin for selection After 3 generations,cells stably expressing p7-Turbo ID and Turbo ID were obtained.Western blot was used to detect the expression of p7;the cells were treated with different concentrations of Biotin for different periods of time.Western blot was used to identify the biotin ligase activity of Turbo ID and determine the optimal treatment of Biotin.Concentration and time: Cells stably expressing p7-Turbo ID and Turbo ID were expanded and cultured and treated with Biotin,and the cells were lysed to extract protein.Dynabeads^TM My One^TM Streptavidin T1 magnetic beads were used for protein profile analysis after affinity chromatography.The test results showed that: Western blot identified p7 expression in cells;50 ?M Biotin treated cells for 18 hours had the best effect;bioinformatics analysis of the screened interacting proteins was performed,and the main enrichment biological processes and pathways were to the endoplasmic reticulum Stress response and endoplasmic reticulum-mediated protein degradation pathways,among which proteins such as SEC22 B,STX5,ANAP29,SRPRA and HSPA5 participate in the protein-protein interaction network that constitutes the above biological processes and pathways.3.Study on the function of BVDV p7 interacting proteinSelect 13 interacting proteins that may participate in the formation of p7 ion channels,design sg RNA and clone it into lenti CRISPR v2 vector,package the lentivirus to infect MDBK cells,select positive cells with puromycin,and use fluorescence quantitative PCR(Real-Time quantitative PCR,qRT-PCR),immunofluorescence(Immunological fluorescence assay,IFA)staining,virus titer determination,cytopathic effect(CPE)observation,etc.to detect BVDV replication,and to study the protein knockout that interacts with p7 Except for the impact on BVDV copying.qRT-PCR detection found that SPCS1,VAPA and SEC22 B knockout significantly reduced the level of BVDV 5'UTR m RNA;12,24,36 and 48 h after BVDV infection,SPCS1,VAPA and SEC22 B gene knockout significantly prevented the green fluorescent labeling BVDV double-stranded RNA(Double strand RNA,ds RNA)accumulation,attenuate the CPE caused by BVDV infection,and reduce the virus titer of progeny.In this study,a rabbit anti-BVDV p7 polypeptide polyclonal antibody with good reactivity and specificity was successfully prepared,which provides a useful tool for further research on the mechanism of p7 formation of ion channels;through Turbo ID adjacent protein labeling technology screening to obtain the interaction with BVDV p7 Protein,CRISPR/Cas9 knocking out SPCS1,VAPA or SEC22 B gene significantly prevents BVDV from replicating,and provides a theoretical basis for elucidating the mechanism of p7 forming ion channels.