Expression Of Newcastle Disease Virus F Gene In The Prokaryotic And Eucaryotic Expression Systems

Newcastle disease(ND) is an acute and highly contiguous avian infectious disease brought by Newcastle disease virus(NDV). Newcastle disease is a violent contagion classified by OIE. Newcastle disease virus is a member of Avian viruses , Paramyxovirinae ,Paramyxoviridae, and a negative RNA virus which has vesica membrane. It exists in all tissues, apparatus, body fluid, secretion and excrement.In the past two decades, an intensive vaccination program against ND has been practiced in both large-scale poultry operations and village poultry farming. Disease outbreaks occur infrequently in some vaccinated focks, however, infections of atypical Newcastle disease in the chicken focks have been more and more frequently reported since the late 1990s. Even some chicken flocks with high antibody level can also suffer ND. Some scholar think that the emergence of new genotypes or subgenotypes could be responsible for ND outbreak in vaccinated focks. Especially when the emergence of variant NDV of goose origin was found, people are more and more interested in mechanism of the atypical Newcastle disease caused by virulent strain.F protein was also called fusion protein, which was in charge of the fusion of virus peplos and susceptible cell. F protein was necessary to NDV's infection and correlated with NDV's pathogenicity. F0 was F protein's inactive precurosor, which had no fusion activity. After F0 protein was splited into two polypeptide chains, F1 and F2, NDV could have infectivity. F1 subunit was important to viral antigenicity, which is also F protein's chief speciesspecific epitope.This study used molecular biology methods to make F gene express in the prokaryotic and eucaryotic expression systems, which provided base materials of the deeper research in this filed. This study maybe summarized as follows:1. The F gene of NDV was amplified by PCR and cloned into pGEM-T Easy vector and sequenced. It was, then, subcloned into high efficient eukaryotic vector pcDNA4/HisMax. The recombinant plasmid pc4F was transfected into COS-7 cells using superfect transfection reagent. The NDV F protein expression in COS-7 cells was detected by immunocytochemistry. The transient expression of NDV-F protein provides the foundation for stable expression, in a cell line, for the future preparation of monoclonal antibodies that can be used to detect NDV and viral antigen variation. The expression will also provide materials for developing a genetically engineered vaccine.2. The gene fragments F1 for the Newcastle disease virus(NDV) fusion protein was amplified from the recombinant plasmid containing NDV F gene by PCR with specific primers. The genes were inserted between BamHI and HindIII sites of pET-28a after cleavage by corresponding enzymes. The recombinant plasmid was named pETF1. An expected protein of 31 ku in size was expressed in E. coli BL21(DE3) induced by IPTG. SDS-PAGE and Western-blotting analyses showed that the expressed protein F1 reacted with NDV positive serum IgG. However it showed no reaction with negative control serum. The F1 was successfully expressed in E. coli and maintained appropriate antigenicity and specificity.