Construction And Biological Characteristics Of Recombinant Newcastle Disease Virus Expressing Foreign Genes In Tandem Or Fusion

Newcastle disease(ND),caused by the velogenic strains of Newcastle disease virus(NDV),is an acute,severe and highly infectious animal disease,which severely restricts the healthy and green development of poultry industry worldwide.Meanwhile,as an important viral vector,NDV can efficiently express foreign proteins.Accumulating studies focused on different insertion patterns by which the foreign genes are expressed in NDV vector have been reported recently,but the systematic researches between these different insertion patterns are still poorly understood.Therefore,in order to provide theoretical basis for the application of NDV vector,in the first chapter of this study,we systematically compared the biological characteristics and the expression efficiency of foreign protein under three different tandem expression patterns using genotype ?d NDV strain as the backbone.In addition,the interaction between viral and host proteins is a research hotspot in the field of virology,while the preparation of specific monoclonal antibody against viral proteins is time-consuming and laborious,which seriously restricts the research on the pathogenesis of viruses.In the second chapter of this study,the recombinant NDV,of which HN protein was fused with His tag,was obtained by reverse genetics technique.And then the biological characteristics,fusion protein expression and purification of the recombinant NDV were verified,and the feasibility of researching the interaction between virus and host proteins under NDV infection based on His tag antibody was preliminarily discussed by mass spectrometry.The results of this chapter provide an antibody-independent method for studying the interaction between NDV protein and host protein,and opens up a new way for further exploring the function of virus protein.1.Construction of the recombinant NDVs based on different tandem expression patterns and the biological characteristics comparation between themIn this study,two recombinant NDV strains based on the backbone of virulent NDV strain JS-5-05-Go(also known as I4),named rNDVI4-HN-IRES-GFP and rNDVI4-HN-T2A-GFP,were constructed by inserting the enhanced green fluorescent protein(EGFP)gene behind the C-teminal of HN by using the internal ribosome entry site(IRES)or the T2A peptide derived from the insect virus(Ta V).Meanwhile,the recombinant strain rNDV/vI4GFP-HN/L,which was constructed previously by our lab by inserting EGFP between the HN and L,was resuscitated.Subsequently,the biological characteristics and foreign protein expression efficiency of these three recombinant NDV strains were compared.The results showed that these three recombinant viruses replicate well in chicken embryos and cells,while rNDVI4-HNIRES-GFP exhibits considerable advantages,and its virulence and pathogenicity are similar to the parental virus.The GFP expressed by rNDVI4-HN-IRES-GFP shows the strongest fluorescence signal intensity,which is beneficial to the sorting of cells inoculated with fluorescent virus.The fluorescent protein expression efficiency of rNDVI4-HN-T2A-GFP is the weakest,and rNDV/vI4GFP-HN/L is somewhere in between.In conclusion,the virus titer,virulence and pathogenicity of rNDVI4-HN-IRESGFP are similar to the parental virus,and GFP expression efficiency is the highest.This study systematically compares the differences of three recombinant NDV strains constructed by different tandem expression patterns,which may provide crucial theoretical support for the application of NDV vectors.2.Construction of HN-His fused recombinant NDV and preliminary study on the feasibility of simulated interactionA recombinant NDV strain,rNDVI4-HN-His,was successfully constructed and rescued by inserting the 6 × His tag sequence into the C terminal of the HN protein.Compared with the parental strain,the recombinant virus shows similar replication ability either on SPF chicken embryos or cells.The expression of HN protein fused with His tag was verified by western blot and fluorescence co-localization assays,and the fusion expressed protein was purified and detected by nickel column affinity chromatography and SDS-PAGE electrophoresis,respectively.The results showed that the fusion expressed protein HN-His is well purified,and the concentration is 38 ng/?L.Meanwhile,the immune coprecipitation assay was carried out by using antibody against His tag and HN protein respectively,and the capture host proteins were analyzed and compared through mass spectrometry.The results indicated that the host interaction proteins captured by these two antibodies are highly consistent,suggesting that the tag protein antibody could effectively replace the virus protein antibody in the research of virus-host interaction under the condition of fusion expression.To sum up,a recombinant NDV strain,of which HN protein is fused with His tag,is successfully constructed and rescued in this study.The recombinant virus exhibits efficient replication ability and the fusion protein HN-His could be effectively expressed.The fusion protein could be purified by His protein purification kit,and the tag protein antibody could be used to research the interaction between viral protein and host.The results of this chapter provide a new way for the development of ND subunit vaccine and the establishment of ND diagnostic method.Meanwhile,it opens up a new idea to explore the interaction between viral and host proteins under physiological state of NDV particles.