Prokaryotic Expression Monoclonal Antibodies Preparation And Cell Localization Of βC1 Genes Of DNAβ Associated With Geminiviruses

Geminiviruses are a group of plant viruses characterized by their unique twinned particles, which encapsidate a circular single-stranded DNA genome. They cause destructive diseases in many economically crops throughout the world. Recent years a novel satellite DNA, refered to as DNAβ, was found to be associated with some monopartite begomoviruses. All reported DNAβpossesses aβC1 gene on the complementary strand that is conserved in position and size and functions as a pathogenicity determinant. In this thesis, theβC1 gene was expressed in Escherichia coli and monoclonal antibodies (MAbs) againstβC1 protein were produced.TheβC1 genes of DNAβassociated with Tomato yellow leaf curl China virus isolate Y10 (TYLCCNV-Y10) and Tobacco curly shoot virus isolate Y35 (TbCSV-Y35) were inserted into prokaryotic expression vectors pET-30a and pET-32a, respectively, and then expressed in Escherichia coli. The recombinant Y10βC1 and Y35βC1 proteins were purified with Ni²⁺ -NTA agarose affinity chromatography. Six hybridoma cell lines secreting MAbs against Y10βC1 protein and nine hybridoma cell lines secreting MAbs against Y35βC1 protein were produced by fusing mouse myeloma cells (SP2/0) with spleen cells from BALB/c mice immunized with the recombinant proteins. The titres of ascitic fluids of MAbs against Y10βC1 protein ranged from 1: 16000 to 1: 128000 in indirect ELISA, and those against Y35βC1 protein ranged from 1: 128000-1: 1024000.Immuno-gold labeling with MAbs against Y10βC1 and Y35βC1 found thatβC1 protein didn't accumulated in healthy tobacco cells, but accumulated mainly in the nuclei and chloroplast of tobacco cells infected with infectious clones of TYLCCNV-Y10 and TbCSV-Y35 DNA-A and DNAβtogether.