| Bovine viral diarrhea virus（BVDV） is the pathogen of Bovine viral diarrhea mucosal disease, which can cause cattle fever, diarrhea, acute and chronic enteritis abortion, abnormal fetus, persistent infection and other clinical symptoms. The disease is a highly contagious disease with high mortality and infectious rate. E2 protein is the envelope protein of BVDV with N-terminal extending envelope surface, which has strong antigenicity and mediates immune response. This protein is the main protective antigen of BVDV, with the characteristics of strong variability, low conservatism etc.At present, the study of gene E2 and E2 protein mainly concentrated in the E2 gene expression, E2 protein antibody preparation and the development of new vaccines, etc. However, in the exact molecular E2 protein and host cell pathogenic mechanisms are few reported. Therefore, this study analyzed the subcellular location of E2 protein in mammals and its effects on the transcription of related genes, in order to lay a foundation for further research on the bioloyical function of the protein. 1. Construction of eukaryotic expression vector pcDNA3.1 / V5 HisA-E2The E2 gene fragments of Changchun184 were amplified by RT-PCR, cloned into eukaryotic expression vector pcDNA3.1/V5 HisA, and then was identified by restriction enzymes digestion and sequence analysis. The correctly identified plasmid was named pcDNA3.1/V5HisA-E2. 2. Subcellular localization of BVDV E2 protein in MDCK cellsThe subcellular location of E2 protein in MDBK cells was observed by confocal laser scanning microscope after the recombinant plasmid pcDNA3.1/V5HisA-E2 was transfected into MDBK cells by lipofectin for 48 h. The results showed that the E2 protein was expressed in both nucleus and cytoplasm, and most of the green fluorescence evenly distributed in a speckled pattern. 3. The effect on cell transcription of BVDV E2 proteinIn this study, pcDNA3.1/V5 HisA-E2 were transfected in cells and overexpressed. The transcription of Beclin-1, Atg12, Atg14, mTORC1, LC3 B and GOPC was analyzed in cells during 2 to 72 h, by real-time quantitative PCR. The result showed that gene E2 played a role in increasing the transcription levels of Beclin-1, Atg12, Atg14, mTORC1, LC3 B and GOPC. The transcription levels of Atg12 and LC3 B were the highest in the groups, followed by Beclin-1 then GOPC, mTORC1 and Atg14.To sum up, This experiment studied the subcellular location of E2 protein and its effect on cell transcription. The result showed that the E2 protein mainly distributed in the cytoplasm and nuclear and its overcexprscion increased the transcription levels of Beclin-1, Atg12, Atg14, mTORC1, LC3 B and GOPC. These results lays a foundation for further research on the biological function of bovine viral diarrhea virus E2 protein. |
