Cloning, Prokaryotic Expression Of PLRV CP Gene And Preparation Of Antiserum

This research is to clone the coat protein (CP) gene of potato reafroll virus (PLRV), construct prokaryotic expression vector of PLRV-CP gene and prepare antiserum using the fusion protein as antigen for the ELISA detection of PLRV.Total RNA was isolated from diseased potato leaves and used as template, specific DNA band of PLRV-CP gene was achieved by RT-PCR, its length was about 630 bp. PCR product was recovered and cloned into pGEM-T easy vector. PCR amplification and endonuclease digestion results confirmed that PLRV-CP gene had been inserted into the cloning vector, the resulting recombinant plasmid was named pGEM-LRCP. DNA sequencing indicated that the inserted DNA was PLRV-CP gene which contains 627 nucleotides and encodes 208 amino acids. The homology of nucleotide sequences between cloned PLRV-CP gene and other 36 previously published ones is above 96% and the homology of deduced amino acid sequences between them is above 97%.Specific PCR product of gene (without stop codon) was recovered using Ultrafree-DA kit and ligated into expression vector pBAD/Thio-TOPO, then the recombinant plasmid was transformed into E. coli TOP10. Positive clones were selected by PCR amplification and restriction endonuclease digestion analysis. DNA sequencing confirmed that PLRV-CP gene was inserted into the expression vector pBAD/Thio-TOPO in direct orientation and in frame. The recombinant plasmid was named pBAD-LRCP. After induction of the engineered strain with arabinose, there was no expected protein in SDS-PAGE pattern.Using pBAD-LRCP as template, DNA fragment rich in arginine codons was deleted from PLRV-CP gene by further PCR, the yielding recombinant plasmid was named pBAD-LRCP-126. In the presence of 0.2% arabinose, the mutant strain TOP10 (pBAD-LRCP-126) was cultured at 37℃ for 4 hours, specific 34kDa protein band was found in the SDS-PAGE pattern, its size is the same as the expected fusion protein. Western blotting on nitrocellulose film showed that the fusion protein had positive hybridization signal with PLRV-IgG provided by CIP. The result showed that the mutation gene was successfully expressed in TOP10.The fusion protein exists in inclusion bodies. After dissolution of inclusion bodies in binding buffer containing 8M Urea, the fusion protein was purified with metal affinity chromatography column, high purity protein was achieved.The purified protein was used as antigen to immune rabbits. The titer of the antiserum was respectively 1:256 by agarose double-diffusion analysis and it was 1:12800 by indirect ELISA, specific antisera against the fusion protein were prepared. By the use of the prepared antiserum, positive reaction was observed in the indirect ELISA detection of diseased potato leaves, so that the antisera can be used in detection of PLRV.