Experimental Studies On Construction Of EV713D Eukaryotic Expression Vector And Screening Of Interacting Proteins

Objective: Enterovirus71(EV71) has emerged as a major causativeagent of hand, foot and mouth disease. To further study on the function andmechanism of EV713D polymerase, in this present study EV713Deukaryotic expression vector was constructed and host proteins interactingwith EV713D polymerase were screened using tandem affinity purification.Methods: EV71BrCr strain was amplified and RNA of BrCr strainwas extracted. The gene of EV713D polymerase was obtained from EV71BrCr strain genome by reverse transcription-polymerase chain reaction(RT-PCR). Then, it was cloned into the eukaryotic expression vectorpcDNA3.0-Flag-HA by BamH I/EcoRI enzyme digestion and ligation. Afterthe identification of recombinant plasmid pcDNA3.0-3D-Flag-HA byenzyme digestion and DNA sequence analysis, the eukaryotic expressionvector pcDNA3.0-3D-Flag-HA was transfected into RD cells. Thetransfection efficiency of recombinant plasmid was detected byimmunofluorescence, and the expression of3D polymerase in RD cells wasdetermined by Western blot. The host proteins interacting with3Dpolymerase were separated by tandem affinity purification, then LC-MS analysis was performed. And searching in NCBI protein data bank, theproteins and polypeptides interacting with3D polymerase were screened.Finally, the interaction between3D polymerase and candidate proteins wasverified using co-immunoprecipitation and fluorescent colocalizationanalysis.Results: After construction of EV713D eukaryotic expression vectorpcDNA3.0-3D-Flag-HA, agarose gel electrophoresis was performed withtwo samples, the recombinant plasmid pcDNA3.0-3D-Flag-HA withoutdigestion and double digested by BamH I/EcoR I. The later lane showed twobands,1.4Kbp band consistent with the length of3D polymerase product and5.5Kbp band consistent with the length of pcDNA3.0-Flag-HA productrespectively. This can be a preliminary proof of successful construction ofrecombinant plasmid pcDNA3.0-3D-Flag-HA. Moreover, the sequencingresults and the EV71BrCr strain genome retrieved in GeneBank (AB204853.1) were then aligned. The results of the alignment further confirmedthe3D polymerase gene was correctly cloned into the recombinant vector.After transfecting recombinant plasmid into RD cells, immunofluorescenceresult showed that the transfection efficiency of recombinant plasmid was30%, and Western blot result showed the expression of3D polymerase in RDcells. Using tandem affinity purification and LC-MS analysis, then searchingin NCBI protein data bank, a series of3D polymerase-interacting proteinssuch as Cyclin G1were screened. Furthermore, the result of co-immunoprecipitation and fluorescent colocalization analysis proved theinteraction between Cyclin G1and3D polymerase.Conclusion: The EV713D eukaryotic expression vectorpcDNA3.0-3D-Flag-HA was successfully constructed. Then a series ofproteins interacting with EV713D polymerase in host cells were screenedusing tandem affinity purification. This can be the basis of studies on thefunction and mechanism of EV713D polymerase.