Preparation and biological characteristics study of multiclonal antibodies to influenza virus NS1 protein

Abstract

Objective NS1 protein of influenza virus plays an important regulatory role on the replication and dissemination.It has many functions,such as regulating host cell protein synthesis,inducing apoptosis of infection cell,as well as antagonistic action of interferon,but many functions are still unclear so far.Therefore,the development of a highly specific anti-NS1 antibody plays an important role in NS1 protein for further functional studies.Methods A NS1 gene of influenza virus was amplified by RT-PCR.A plasmid (pET-28a-NS1) was constructed from the expression vector pET-28a( + ).Then NS1 fusion protein was expressed in the prokaryotic cell ( E.coli BL21 ).The purified NS1 protein immunized New Zealand big white rabbit.The specificity of antibody serum was detected by ELISA.The bio-characteristics of the antibody were determined with virus infected cell experiment,Western blotting method and indirect immune-fluorescence test.Results A plasmid (pET-28a-NS1) which can express of NS1 protein was constructed.Then,the NS1 protein by prokaryotic expression was used to immunize rabbits.The anti-NS1 antibody which had a specificity of 1∶256000 was obtained.Experiments showed that the antibody could bind with NS1 protein of influenza A virus independence on subtypes.Therefore,it could detect the expression and location of NS1 protein in the infected cells.For example,NS1 protein could be detected after infected for 6 hours,and did not change significantly with the time of virus replication.After 24 hours,NS1 protein was mainly distributed in the cell nucleus and could locate in the nucleolus,moreover also had a portion located in the cytoplasm.Conclusions In this study,the purified NS1 protein are obtained through prokaryotic expression and higher specificity of polyclonal antibody against NS1 using routine immunization method.The antibody can bind specifically with NS1 proteins of influenza A viruscs independent its subtypes,and can be used to determine the NS1 protein expression effectively in the infected cells.The antibody can be used not only for researching NS1 protein function and its effects on host cells,but also differential diagnosis of viral infection or vaccination of poultry pig and other animals,have good development and application prospect. Key words: Orthomyxoviridae; NS1 gene; Gene expression; Multiclonal antibody; Application research