Package And Application Of NDV-pseudotyped HIV-Luc Virus And Prokaryotic Expression And Functional Analysis Of The Single-chain Variable Fragments Against The HN Protein Of NDV

Newcastle disease(ND)caused by Newcastle disease virus(NDV)is a major infectious disease in the poultry industry all around the world.NDV can infect most species of birds and velogenic NDV are List A pathogens that require reporting to the Office of International Epizootics(OIE)if it is epidemic.ND outbreaks will result in strict trade embargoes.NDV is a member of Paramyxovirinae subfamily and has a negative sense single-stranded RNA genome.The infectious pseudotyped HIV-Luciferase(HIV-Luc)viruses with HN and F envelope proteins of NDV have been successfully produced in this study.We further successfully established a novel neutralization assay with HN and F-pseudotyped(NDV-pseudotyped)HIV-Luc viruses,which was used to replace the conventional neutralization assay to detect the neutralizing antibodies against NDV.Besides,the single-chain monoclonal antibody against the HN protein(HN-scFv)of NDV has been produced in bacteria,and its biological activity has been evaluated in this study.1 Package of NDV-pseudotyped HIV-Luc virus and its application in the neutralization assay for NDV infectionIn this research,we successfully produced infectious NDV-pseudotyped HIV-Luc viral particles.Further investigation revealed the cytoplasmic domains of HN and F,especially HN,plays a significant role in the infection efficiency of these pseudotyped HIV-Luc viruses.Replacement of or direct fusion to the cytoplasmic domain of theHN protein by that of Vesicular Stomatitis Virus G(VSV-G)could greatly enhance or destroy the infective potential of NDV-pseudotyped HIV-Luc virus.Here,we further established a novel neutralization assay to evaluate neutralizing antibodies against NDV with NDV-pseudotyped HIV-Luc viruses.Comparative neutralization data indicate that the result determined by NDV-pseudotyped HIV-Luc viruses is as reliable as those by the conventional virus-neutralization assay(VN test)with native NDV.Moreover,the result shows that the novel neutralization assay is more sensitive than the VN test.2 Prokaryotic expression and functional analysis of the single-chain monoclonal antibody against the HN protein of Newcastle Disease VirusTo express the single-chain monoclonal antibody against the HN protein(HN-scFv)of NDV in bacteria and evaluate its specific binding activity,the total RNAs were extracted from one hybridoma producing monoclonal antibody(McAb)against HN of NDV.The variable regions encoding the heavy and light chains of McAb,named VH and VL respectively,were amplified by RT-PCR and inserted into pET-scIG vector to construct the recombinant plasmid of pET-HN-scFv.The recombinant plasmid was transformed into the E.coli(BL21)for HN-scFv expression.In addition,the expression of HN-scFv was induced and analyzed in SDS-PAGE assay,and the results showed that the expressed HN-scFv was mainly in the form of inclusion bodies.Moreover,the testing with flow cytometrey indicated that the renatured HN-scFv was able to specifically recognize the HN expressed on 293T cells.These results revealed that we successfully constructed and functionally expressed the HN-scFv in bacteria which will facilitate the engineering of the McAb in the future.