Expression Of Newcastle Disease Virus HN Protein In Rice Cell And Applications

Newcastle disease(ND)is a highly contagious avian disease caused by Newcastle disease virus(NDV)and is characterized by high fever,respiratory distress,and mucosal hemorrhage.The HN glycoprotein of NDV is one of the major protective antigens and produces antibodies that neutralize NDV.therefore,the HN protein has been expressed as a target antigen in a variety of systems.The rice expression system can post-translationally modify the exogenous protein to make the structure of the exogenous protein closer to that of the natural protein,making it a more ideal eukaryotic expression system for expressing exogenous proteins.In this study,we used the rice system to express HN protein,obtained high-purity HN protein by SP cation chromatography and gel filtration chromatography,and immunized of HN protein in mice,monoclonal antibodies to recombinant HN protein were obtained by indirect ELISA and IPMA screening.Finally,established a semi-quantitative antibody detection method for NDV.The main experiments and results are as follows.1.Expression and purification of HN recombinant proteinIn this study,the recombinant plasmid pUC57-HN and the intermediate vector p MP3(containing promoter,signal peptide and terminator)were double digested by Eco R Ⅰ and Hind Ⅲ,and the HN gene was ligated to p MP3.Then Eco R Ⅰ and Hind Ⅲ were used to double digest pMP3-HN1(containing promoter,signal peptide and terminator)and the plant vector p CAMBIA1300,and the HN gene was successfully ligated to p CAMBIA1300.The HN gene was successfully ligated into p CAMBIA1300 to form recombinant plasmid p CAMBIA1300-HN1 and transferred into Agrobacterium tumefaciens EHA105.The recombinant plasmid p CAMBIA1300-HN was transferred into rice healing tissue by Agrobacterium mediated method.The HN gene was identified by PCR and inserted into the rice genome.Identification by Western-blot resulted in the successful expression of recombinant HN protein in rice callus.The recombinant HN protein was then purified by SP cation chromatography and gel filtration chromatography,and the purity of HN protein was over 90% by SDS-PAGE.The western-blot showed that the recombinant HN protein reacted well with monoclonal antibody.2.Screening and identification of monoclonal antibodiesTo obtain monoclonal antibodies of HN protein,recombinant HN protein was immunized in BALB/c mice,and four monoclonal antibodies,named 4E6B7,9D5F9,9D5F10 and 9D5E12,were obtained by screening with the IPMA method,and the Western-blot showed that the four monoclonal antibodies were reacted well with HN recombinant protein.The ELISA titer of 9D5E12 was 1:40960.The subtype identification results showed that all four monoclonal antibodies belonged to Ig G1 subclass and the light chains of all four monoclonal antibodies were k chains.3.Establishment of ND antibody semi-quantitative test strip assayIn order to establish a semi-quantitative colloidal gold test strip assay for ND antibody,according to the national diagnostic technical standard for Newcastle disease HI test(HI≥4log2,the detected antibody is positive),the NDV antibody rapid detection test strip was prepared by double antibody sandwich method using colloidal gold labeled HN protein.The results of 308 clinical sera showed that the compliance rate of NDV antibody test paper with and HI test was 97.08%,and the kappa value was 0.942.In conclusion,it has been developed for a simple,rapid,high sensitive and specific semi-quantitative test paper.The test paper can be initially applied to the immune evaluation of Newcastle disease vaccine.In summary,the HN protein of rice was successfully obtained in this study,and the protein purity reached over 90% after purification by SP chromatography and molecular sieve,and four monoclonal antibodies with good activity were obtained.Finally,a semi-quantitative colloidal gold assay for NDV antibody was also established.