Screening And Preliminary Identification Of MDBK Cellular Proteins Interacting With Bovine Viral Diarrhea Virus

Bovine viral diarrhea virus(BVDV)belongs to the Flaviviridae,the Pestivirus,it is an important pathogen in the world that makes the global cattle industry suffer huge economic losses every year.The biotype of BVDV is divided into cytopathic(CP)and non-cytopathic(NCP).If the fetus is infected with NCP BVDV for the first time in the first three months,it will become a infectious calf(PI),which will produce immune tolerance to BVDV and become poisoned for a lifetime,and it will infect other animals.At present,the main foreign use of vaccine immunization,quarantine and purification to prevent the disease.Because the pathogenesis of the virus is very complex,the mechanism of virus infection and transmission is a hot spot.A large number of studies have shown that E2 protein is a very important membrane protein of BVDV.In order to reveal the pathogenic mechanism of BVDV,it is necessary to study the receptor protein of BVDV and its E2 protein on different host cells,and to determine the interaction between BVDV and host cells,and lay a theoretical foundation for the prevention and treatment of the disease.in this study,eukaryotic plasmids p Fast-E2 and r Bacmid-E2 were successfully constructed by cloning the whole gene of BVDV structural protein E2.The recombinant plasmids r Bacmid-E2 were successfully transfected into sf9 cells,and finally the E2 protein was stably expressed by recombinant baculovirus.After confirmation by Western Blotting,a clear target band appeared at 43.6 k Da,this protein will be a "bait" of the pull-down experiment,the MDBK cell's protein will interact with it.In addition,an NCP-type BVDV was isolated from PI cattle and the experiments of virus overlay protein blot assay and Co-Immunoprecipitation were carried out with it.The results are verified by mass spectrometry,then the results were analyzed by GO.Thirty proteins were screened from the results of mass spectrometry.These proteins were divided into five groups,which were constituent cytoskeletal components,RNA binding function,energy binding function,protein binding function and ion channel binding function.After eliminating the foreign pollution and the protein which were not identified,we make the analysis by GO and read a lot of relevant literature,at last we found 4 proteins,namely,vimentin,?-actinin,actin and tubulin which have the value of research.As the result of mass spectrometry CD46 only had one Unique Peptides,the expression of CD46 molecules which is the only receptor of BVDV was detected by real-time quantitative PCR in MDBK,lymphocytes and mononuclear cells.The results showed that BVDV infection caused MDBK cells and lymphocytes increased the expression of CD46,but the impact on monocytes is not obvious.In a word,we successfully obtained recombinant baculovirus which can express BVDV E2 protein stably,and candidate host proteins that could interact with BVDV were identified.And BVDV was able to induce the upregulation of CD46 gene transcription level in MDBK and bovine peripheral blood lymphocytes.These conclusions have theoretical significance for the pathogenesis of BVDV and the discovery of new receptors.