Preliminary Study On Biological Functions Of Newcastle Disease Virus Matrix Protein

In this paper, the biological function of Newcastle disease virus matrix protein (M) was studied, the research consists of six parts:1、Bioinformatics analysis of Newcastle disease virus matrix proteinBiological structure of a protein determines its biological function, NDV M protein functions are very few in old researches, and its cognitive structure is also very superficial. In this paper, bioinformatics methods were used to analysis the post-translational modification of the M protein phosphorylation sites, glycosylation sites, ubiquitination sites, amidation sites and myristoylation sites, linear motif and domain, B cells epitopes and T-cell epitopes, secondary structure and subcellular distribution, this will be provide a theoretical basis for future studies about the structures and functions of M protein.2、Polyclonal antibodies preparation and B cell epitopes analysis of Newcastle disease virus matrix proteinNDV M gene prokaryotic expression vectors were constructed and expressed in vitro, mice were immunized after the separation and purification of M protein, real-time monitoring the immune effect and then serum was collected. M protein polyclonal antibody was prepared; meanwhile using bioinformatics methods to predict the linear B cell antigen epitope of M protein, oligopeptide, which got higher composite scores, were synthesized, animals immunized the oligopeptides coupling with BSA, then tested by ELISA and Western blot.3、Establishment of stable DF-1 cell expressing Newcastle disease virus matrix proteinThe M gene random transposition into the host cell genome used PiggyBac transposon system, positive cells with GFP-tagged proteins used to indicate the exogenous gene expression. The M gene was successfully integrated into the host cell genome and stably expressed in the testing. The M protein stably expressing cell model was successfully established, it can reduce the transient transfection system error and improve the efficiency of M protein, increase the level of repeatability of the test, and over-expression analysis of wild-type strain. It also can be repeated for high-throughput test materials.4、Research of matrix protein subcellular localizationBased on bioinformatics method, the nuclear localization signal (NLS) of Newcastle disease virus (NDV) matrix protein (M) was predicted and deleted by an overlap-PCR assay. Subsequently, the plasmids including the full-length M gene and M-△NLS EGFP gene were constructed, respectively, and were transfected into monolayer DF-1 cells growing on the glass slides. The subcellular localization of M protein was measured by a laser confocal microscope. The results showed that the M protein entered into the nuclei with the help of NLS. However, M protein deleted the NLS was not enter into the nuclei. Hence, the NLS is the key of matrix protein in nuclear entry.5% Effection of Newcastle disease virus matrix protein on viral replicationNDV biosynthesis is done in the host cell cytoplasm, it is unnecessary for M protein to enter into the host cell nucleus. The biological function of M protein enters into the host cell nucleus still not known. This article primarily detected the host cell injury, viral titers and viral gene expression in the virus infected host cells to show whether the NDV M protein on the viral proliferation effect or not.6% Proteomics analysis of the host cell proteins interact with NDV M proteinNDV life activities, such as replication, transcription, translation and virion assembly are done in the host cell cytoplasm, but studies have proven its M protein enters the host cell nucleus, in order to find the cause of the occurrence of this phenomenon, this study fusion expression M protein with a small molecule protein FLAG, using co-immunoprecipitation (Co-IP) method to capture and enrichment of host cell protein interactions with the M protein, the captured proteins identified by mass spectrometry. In this study, several chick fibroblast cell proteins were identified directly or indirectly interacting with transient and stable expression of the M proteins.