Preparation Of Cymbidium Mosaic Virus TGBp1 Antiserum And Research On Self-interaction

Orchids（Cymbidium spp.）,which belong to the Orchidaceae family,has great ornamental value.In recent years,with the development of society and economy,more and more people pay attention to orchids in their lives.At the same time,they have become an important part of the flower industry.However,with the progress of the orchid industry,orchids are extremely vulnerable to virus infection.Cymbidium mosaic virus（Cym MV）is one of the major viruses that may cause major losses of orchids.Specifically,it can seriously affect the quality as it may give rise to chlorotic,deformities and necrosis of orchid plants.Cym MV contains 5 open reading frames（ORFs）.ORF2-4 are three overlapping ORFs,which are called triple gene block（TGB）proteins.And triple gene block protein 1（TGBp1）is the first protein encoded by the TGB ORF.TGBp1 can peform self-interaction,but the specific location of self-interaction is unclear.Therefore our present study carried out the following work on TGBp1.In this study,we utilized the identified Cym MV as template to amplify the TGBp1gene.Then,TGBp1 gene was connected to p ET-28a（+）prokaryotic expression vector.The expression vector was transformed into the Rossetta（DE3）strain that was induced by IPTG sequently to express the fusion protein.SDS-PAGE and Western blot analysis confirmed that the expressed protein was His–fused TGBp1 protein.Polyclonal antibodies against recombinant TGB1 protein were obtained by hypodermal injection of rabbits.The titre of antiserum was 1:2¹⁹ by indirect ELISA method.The antiserum prepared by Western blot detection has good specificity and is a specialized antiserum.The preparation of TGBp1antiserum may lay the foundation for further research on TGBp1 function.In this study,TGBp1 was divided into three truncated mutants TGBp1-1（1-62aa）,TGBp1-2（63-112aa）,TGBp1-3（113-233aa）according to its motifs.TGBp1 could interact with its three truncated mutants by the yeast two-hybrid system assay.Bimolecular Fluorescence Complementary（Bi FC）assay demonstrated that there were interactions between TGBp1 with TGBp1-1 and TGBp1-2.To further verify its protein interactions,GST-pull down technology was applied to detected the interaction between TGBp1 and TGBp1-2 by constructing three truncated mutant prokaryotic expression vectors and expressing GST-TGBp1-1,GST-TGBp1-2,GST-TGBp1-3 fusion proteins.Our present result that TGBp1 mainly interacts with TGBp1-2,may lay a foundation for further research on the movement and replication of the viruses by TGBp1 self-interaction.In this study,TGBp1 is connected with green fluorescent protein by the overlap method,and TGBp1 fused with GFP is connected to the PCB301 vector by homologous recombination.Agrobacterium-mediated tansient expression was carried out to inoculate H2B transgenic nuclear localization tobacco.The confocal microscope will be used to observe the subcellular localization of TGBp1,and TGBp1 was found to be nucleoplasmic colocalization.These results may lay the foundation for the study of the effect of TGBp1nucleoplasmic colocalization on the intercellular movement of viruses and virus infection.