The Polyclonal Antibody Preparation And Application Of Three Papaya RNA Virus Coat Proteins

Papaya (Carica papaya L.) is widely cultivated in tropical and sub-tropical areas, it has the very high economic value and nutritional value, but with the enlargement of planting area, virus diseases have become the biggest obstacle to restrict papaya plant. There are mainly three kinds of papaya virus:respectively is papaya ringspot virus(PRSV), papaya leaf distortion mosaic virus(PLDMV) and papaya mosaic virus(PaMV) respectively, each virus’symptoms of those three viruses infected papaya is very similar, which brings many difficulties to the prevention and control. In order to improve identification accuracy and take targeted measures to control it, their coat protein(CP) genes were amplified CP gene from the genome of PRSV, PLDMV and PaMV preserved in our laboratory, then expressed them in the prokaryotic and prepared their specificity antiserum. The distribution of papaya virus in Hainan island was investigated using ID-ELISA method. The main results and conclusions in this study are as follows:The first, through designing priers being designed according to the PRSV, PLDMV and PaMV sequences(Genbank No.:HQ424465. JX974555and JX524226), the CP genes were amplified by PCR with loci of Nco I and Xho I on both ends, the amplified fragments and the expression vector were digested at the same time, then they were connected together with T4DNA ligase, the recombinant plasmids of pET28-PRSV CP, pET28-PLDMV CP and pET28-PaMV CP were constructed.The second, after transforming the three recombinant plasmid being transformed into expressing bacteria BL21(DE3), the CP genes were expressed efficiently with IPTG inducing by SDS-PAGE analysis. PRSV CP and PLDMV CP existed in the form of inclusion body, while PaMV CP expressed in soluble form. The expressed proteins were purified with Ni2+-NTA agarose affinity chromatography and then dialyzed. The final concentration are0.95mg/mL,0.84mg/mL and1.12mg/mL, the purity are90%,95%and91%respectively.The third, the three purified CP proteins were used to immune rabbits for antiserum preparation. The optimal titer of the three antiserum in indirect enzyme linked immunosorbent assay was determined to512,000. Western blot analysis confirmed that the antiserum only react with the corresponding virus without crossing reaction.The forth,283samples of unhealthy papaya leaves were tested,170samples were found to be infected with PRSV, which accounted for60.2%, they are from various cities and counties of Hainan;12samples (4.9%) infected with PLDMV, mainly from Ledong and Dongfang regions,2samples(0.7%) were infected with PaMV, only from in Haikou. this study has preliminary established the distribution of Papaya virus in Hainan, and provides scientific basis and a effective method for the prevention of papaya virus.