Expression Of HC-Pro, NIb And CP Genes Of Papaya Ringspot Virus In E.coli And Preparation Of Antibodies

Papaya ringspot virus (PRSV), a species of the genus Potyvirus, is the main pathogen causing severe losses in papaya globally. PRSV particles are flexuous filaments containing single-stranded positive-sense RNA genome of 10326 nucleotides and the genome has a single open reading frame that is translated into a large polypeptide and subsequently cleaved into functional proteins. In order to further study the location and movement of these main proteins in infected plant, and further investigate the protein-protein interaction, protein-nuclear acid interaction, here we study cloning and expression of Helper component protease(HC-Pro), Nuclear inclusion body or RNA-dependent Polymerase (NIb) and Coat protein(CP), then antisera preparation of these fusion proteins.The total RNA of PRSV-infected papaya leaves which contain RNA of PRSV were extracted and purified with Trizol. And three sets of PCR primers were designed according to the genome of PRSV (YK isolate, HA isolate, Thai isolate and Mexico isolate). To enable directional cloning, each of the forward PCR primers contains the sequence CGGGATCC (BamHI), at the 5' end of the primer and each of the backward primers contains the sequence CCCTCGAG (Xhol). HC-Pro, NIb and CP were amplified using M-MuLV Reverse Transcriptase and Taq DNA polymerase by RT-PCR, the sticky-ends PCR products were cloned into pMD18-T. Sequences of the cloned PCR products were analyzed. Comparison of HC-Pro nucleotide and deduced amino acid sequences with other PRSV isolates showed 90.63% and 97.60% identity with China Taiwan YK isolate (X97251) and a Thai isolate, respectively; comparison of NIb nucleotide and deduced amino acid sequences with other PRSV isolates showed 90% identity with south China isolate (X96538 S76059) and 95% identity with a Thai isolate (AAG47346) respectively; While Comparison of CP nucleotide and deduced amino acid sequences with other PRSV isolates showed 95 % identity with a Japan isolate (D50591) and 93% identity with a Thai isolate (P AAO16605), respectively. Phylogenetic trees were drawn based on the HC-Pro, NIb and coat proteins of various isolates of PRSV at amino acid level; it showed that RRSV was much related to Thai isolate.In order to further understand the function of each protein in the virus life cycle, the expression plasmids of PRSV HC-Pro, NIb and CP gene were constructed with pGEX-4T-l and expressed in E.coli (BL21). The fusion protein products were detected by SDS-PAGE respectively. The result of western blot showed that the fusion proteins can react specifically with the antisera produced by the GST fusion proteins. The antisera were prepared and tesed by ELISA. The results show that the antisera can work well with antigens.The fusion proteins (HC-Pro, NIb and CP)and the antisera provide important materials for further studies.