Cloning,antibody Preparation And Transformation Of RNA-dependent RNA Polymerase Gene Of Odontoglossum Ringspot Virus

Viruses that infect orchids include Potexvirus,Tobamovirus,Cucumovirus,and the like.At present,at least 27 viruses have been reported on orchids,of which Odontoglossum ringpot virus(ORSV)is one of the most widespread and most harmf?L orchid viruses.ORSV is often associated with Cymbidium mosaic virus(Cy MV).)The complex infection of orchids caused great economic losses to the orchids.In addition to retroviruses,all positive-stranded RNA viruses encode Rd Rp,which together with viral-encoded proteases(such as helicases)and host cytokines constitutes the virus' s replicase system and is responsible for viral genome replication mechanisms.Among them,Rd Rp is the core component of the replicative enzyme system,which mediates disease resistance.It not only has a strong degree of resistance,but also lasts for a long time,making it one of the best antiviral genetic engineering strategies.In this study,genetic engineering methods were used to construct prokaryotic expression vectors and the corresponding antibodies were prepared,which laid the foundation for the later study of the structure and function of Rd Rp.The prepared antibodies have high titer and specificity to ORSV Rd Rp,and can be used in immunologic detection and analysis of ORSV Rd Rp.(1)In this experiment,the prokaryotic expression vector p ET32a(+)-ORSV Rd Rp was successf?Lly constructed.The sequencing res?Lts showed that the fragment had 516 amino acid residues and encoded 171 amino acid residues.The recombinant protein was translated from the His-tag tag to about 36 KD.The optimized prokaryotic expression system conditions were as follows: 37°C,0.5mmol/L IPTG,?6h;?Ltrasonically disrupted cells were centrifuged and subjected to SDS-PAGE.The res?Lts showed that the recombinant protein mainly existed in the form of inclusion bodies;Antigens were prepared and polyclonal antibodies were obtained by immunizing BABL/C mice.A reliable,sensitive and specific detection method for ORSV was established using the prepared polyclonal antibody,which laid the foundation for the challenge experiment of transgenic tobacco in the later period.(2)Recombinant plant expression vector p CAMBIA1301-Rd Rp was successf ? Lly constructed and transformed into Agrobacterium tumefaciens LBA4404,then infecting N.benthamiana by injection.Hygromycin(Hyg)was used to preliminarily screen the plants that were transduced with the Rd Rp gene.The detection of DNA,RNA and protein levels showed that the Rd Rp gene was integrated into the tobacco genome.