Researches On The Construction Of Special Vero Cells Stably Expressing NDPK B And The Effect Of Overexpression Of NDPK B On NDV Replication

Newcastle disease is a acute and highly contagious disease caused by Newcastledisease virus, and often sepsis happens. It is listed of ClassⅠanimal infectiousdiseases regulated by Chinese Ministry of Agriculture. Today, Newcastle disease isstill one of the most important infectious diseases which can cause a big loss topoultry industry.At present, it is still unclear for the molecular pathogenesis of Newcastle diseasevirus infection, and there are too limited reports about interaction mechanism betweennewcastle disease virus and host cells. Virus infection is the result of viral genomereplication and gene expressions, and also the comprehensive performance of theinteraction between virus and host. In the study of whole protein proteomics ofchicken embryo fibroblast cells infected newcastle disease virus from differenthomology, we obtained some host protein which changed in quantity, these proteinsmay be related to the infection mechanism of Newcastle disease virus. Consequently,proteomics is the most intuitive strategy in studying on influence on host infectedNewcastle disease virus.Nucleoside diphosphate kinae B (NDPK B) is one of identified proteins in ourstudy. Previous researches indicate that NDPK B play an important role in mediatingcellular apotosis, eliminating impaired cells, such as the cells infected virus, andparticipating in cellular immunity by regulating cellular apotosis. However, there aretoo limited studies on NDPK B protein.In order to explore the function of NDPK B, we constructed the eukaryoticexpression plasmid pEGFP-NDPK B with green fluorescent protein(GFP) and G418sifting locus. Then we identified the expression of NDPK B-GFP fusion protein byRT-PCR、Western blot and Inverted Flurescence Microscope. We also measured theinfluence of NDPK B expression on cell growth activity by MTT tests. The experimental results show that NDPK B can inhibit the cell growth of Vero，Sw1116and A549.Through bioinformatics tools, we have predicted the structure and function ofNDPK B. The results showed the molecular weight of NDPK B is17.29kDa, and153amino acid residue, the isoelectric point is7.73and molecular formula isC777H1216N212O219S8in theory. The half period of NDPK B is30hours incell(mammalian reticulocytes, in vitro). There is a kurtosis located between60and80amino acid residue through average hydrophobicity analysis. The probability oftransmembrane domain and signal peptide forecast is zero. The prediction of activesite showed that No.119amino acid residue is phosphohistidine, the active regionbetween116and124amino acid residue. NDPK B contains ten α-helixs and threeβ-pleated sheets in its forecasting secondary structure.In this study, we have constructed the cell line named Vero/NDPK B, whichcould stably express NDPK B protein. On the basis of Vero/NDPK B cell line, wefound that the overexpressing of NDPK B could inhibit the replication of NA-1orF48E9strain of NDV in Vero cells. This study preliminarily clarifies the function ofNDPK B on NDV replication, and meanwhile lays a solid foundation for furtherstudying on the molecular pathogenesis of NDV infection.