Construction Of JEV NS2B/NS4B Expression Celllines And Their Application In Searching Interacting Proteins

Japanese Encephalitis Virus is a member of Flaviviridae family, which is a typical neurotropic virus. JEV is an arbovirus which cause infection in human and domestic animals. The infection in human leads to a high mortality and permanent neurologic and/or psychiatric sequelae in survivors. Pregnant sow may develop abortion, stillbirths or fetal mummies due to JEV infection, while boars develop orchitis and swine develop fever, bringing considerable economy losses.NS2B and NS4 B are two important nonstructural proteins of JEV, for now their functions remain elusive. Our study aimed to construct cell lines that can stably express NS2 B and NS4 B respectively. In addition, we used our cell lines to process Tandem Affinity Purification(TAP) and a subsequent Mass spectrometry analysis(MS) then find proteins that are probably interact with them. Our study thus laid the foundation for exploring the interactions between nonstructural proteins and proteins of JEV resided cells.Below is the overview of our study:1. construction of lentivirus-conducted cell linesNS2B and NS4 B gene was amplified by RT-PCT and then inserted into lentiviral vector Psin-EF2-MCS-Pur(puromycin-resisted) through restrict enzyme sites. Recombinant plasmids were co-transfected with ps PAX2 and p MD2.G to 293 T cells respectively. Packaged virus was harvested to transduct 293 T and puromycin was used to screening the positive cells- Cellline-NS2B/NS4 B. A vector control cell line(Cellline-vector) was constructed at the same time2. identification of cell linesThere are 3 methods used to measure the cell lines, and they are puromycin-resist test, indirect immunofluorescence assay and Western blot. All the 3 way demonstrated that expected proteins can efficiently and stably expressed in cell lines3. ultilization of cell lines for TAPWe harvested protein samples of cell lines and process TAP, final product was separated by SDS-PAGE electrophoresis and stained with silver. Mass spectrometry analysis was conducted to identify the specific bands in treatment group, and we thus found the proteins that probably interact with JEV NS2 B and NS4 B. The results showed possible interaction between NS2 B and ADP/ATP carrier protein(ANT2).